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Probing the interaction between U24 from HHV-6A and Smurf2 WW domains Zhang, Rui 2015

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	  	  	  	  	  	  	  	  	  	  	   	  	  	  	  	  	   	  	  	  	  	  	   	  	  	  	  	  	   	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	   	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	   PROBING THE INTERACTION BETWEEN 	   U24 FROM HHV-6A AND SMURF2 WW DOMAINS 	   by 	   Rui Zhang 	  	  	  	   B.Sc., Nanjing University, 2012 	  	  	  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF 	   	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	   	  	  	  	  	  	   	  	  	  	  	  	  	  	   MASTER OF SCIENCE 	   in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Chemistry) 	   	  	  	  	  	  	  	  	  	  	  	  	  	  	  	   	  	  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) 	   	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	   	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	   June 2015 	  	  	  	  © Rui Zhang, 2015 	   	   ii Abstract  This thesis describes the study of the interaction of the U24 membrane protein, encoded by Human Herpesvirus type-6A (HHV-6A), with WW domains from Smad ubiquitylation regulatory factor 2 (Smurf2) via its PPxY motif. U24 from HHV-6A is of interest because it acts to block endosomal recycling, as mediated by its PPxY motif interacting with WW domain-containing proteins.  We have used a multidisciplinary approach to study the interactions between the Smurf2 WW domains and the PPxY motif-containing region of U24. The GST pull-down experiment demonstrated a difference in affinity between WW domains for the full-length U24 protein, prompting binding studies on interactions between the PPxY motif-containing region of U24 and isolated WW3 domain or WW2 and WW3 domains in tandem (WW23). In Chapter 2, studies were focused on interactions between the third WW domain (WW3) of Smurf2, and the 15-mer U24 peptide containing the PPxY motif. NMR studies demonstrated that the PPxY motif of U24 peptide bound to Smurf2 WW3 domain in a similar way as the interaction between Smurf2 WW3 domain and its cognate ligand Smad7. The dissociation constant was determined to be 123 ± 4 µM at 5°C, reflecting weak binding affinity between WW3 domain and U24 peptide, possibly due to the lack of additional interactions between WW3 domain and the region of U24 peptide beyond the PPxY motif. Circular Dichroism (CD) experiments suggested that isolated WW3 domain was not as stable as other WW domains and binding to U24 peptide enhanced its stability slightly. In Chapter 3, interactions between tandem Smurf2 WW23 and U24 peptide were studied. NMR studies demonstrated that the interaction between Smurf2 WW23 and U24 peptide mainly relied on the U24 peptide binding to its WW3 domain and that the WW2 domain 	   	   iii played only a minor role in the interaction. CD experiments were also carried out to detect the stability of Smurf2 WW23 with and without U24 peptide.    	     	   	   iv Preface   In all chapters, Dr Suzana K. Straus acted in a supervisory role. Experiments were designed by S. K. Straus and myself. I carried out all the experiments and data analysis, except where noted. Yurou Sang and I worked together to synthesize and purify the U24 peptide.   	   	  	   	   v Table of Contents 	  Abstract .......................................................................................................................................... ii	  Preface ........................................................................................................................................... iv	  Table of Contents .......................................................................................................................... v	  List of Tables ................................................................................................................................. x	  List of Figures ............................................................................................................................... xi	  List of Abbreviation ................................................................................................................... xiv	  Acknowledgements .................................................................................................................. xviii	  Dedication ................................................................................................................................... xix	  Chapter 1 Introduction ................................................................................................................ 1	  1.1	   HHV-6 ................................................................................................................................ 1	  1.1.1	   HHV-6A and HHV-6B ............................................................................................... 2	  1.1.1.1	   HHV-6A ............................................................................................................... 3	  1.1.1.2	   HHV-6B ............................................................................................................... 4	  1.1.2	   Basic Biology of HHV-6 ............................................................................................ 4	  1.1.3	   Immune Response by HHV-6 ..................................................................................... 5	  1.1.3.1	   Antibody Response .............................................................................................. 6	  1.1.3.2	   T-cell Responses .................................................................................................. 6	  1.1.3.3	   Targets of the T-cell Response to HHV-6 ........................................................... 7	  1.1.4	   Immunomodulation by HHV-6 ................................................................................... 8	  1.1.4.1	   Possible Mechanism of Immunomodulation by HHV-6 ..................................... 8	  	   	   vi 1.1.4.1.1	   IFNs ............................................................................................................... 8	  1.1.4.1.2	   TNF-α and ILs .............................................................................................. 9	  1.1.4.1.3	   T-cell Activation ........................................................................................... 9	  1.1.4.2	   Proteins with Immune-Modulatory Functions ................................................... 10	  1.1.5	   HHV-6 in Central Nervous System Diseases ........................................................... 11	  1.1.5.1	   Multiple Sclerosis .............................................................................................. 12	  1.1.5.2	   HHV-6 As a Trigger of MS ............................................................................... 13	  1.2	   U24 Protein from HHV-6 ................................................................................................ 15	  1.2.1	   Functions of U24 ...................................................................................................... 15	  1.2.1.1	   Potential Role of U24 in Multiple Sclerosis: Molecular Mimicry ..................... 15	  1.2.1.2	   Potential Role of U24 in Regulation of T-cell Receptor Signaling ................... 16	  1.2.1.2.1	   T-cell Receptor Signaling ........................................................................... 16	  1.2.1.2.2	   TCR/CD3 Complex .................................................................................... 17	  1.2.1.2.3	   Viral Modulation of T-cell Receptor Signaling by U24 Protein ................. 18	  1.2.1.3	   Potential Role of U24 in Multiple Sclerosis: A Block of Endosomal Recycling         ........................................................................................................................... 20	  1.2.1.3.1	   Endosomal Recycling ................................................................................. 20	  1.2.1.3.2	   A General Block of Endosomal Recycling by U24 .................................... 22	  1.2.1.3.3	   Endocytic Trafficking of Myelin Proteins and Its Role in Multiple Sclerosis           ..................................................................................................................... 23	  1.2.1.4	   Conservation of U24 Functions among Human Roseolaviruses ....................... 24	  1.2.2	   Structural Features Critical to U24 Functions .......................................................... 25	  1.2.2.1	   U24 As a Tail-anchored Membrane Protein ...................................................... 26	  	   	   vii 1.2.2.2	   Polyproline Region of U24 ................................................................................ 28	  1.2.2.2.1	   Polyproline Type II Helix ........................................................................... 28	  1.2.2.2.2	   PPxY Motif ................................................................................................. 29	  1.3	   Nedd4 Family of Ubiquitin Protein Ligases .................................................................... 30	  1.3.1	   Introduction to Nedd4 Family of Ubiquitin Protein Ligases .................................... 30	  1.3.1.1	   Structure and Architecture ................................................................................. 31	  1.3.1.1.1	   C2 Domains ................................................................................................ 31	  1.3.1.1.2	   WW Domains .............................................................................................. 31	  1.3.1.1.3	   HECT Domains ........................................................................................... 32	  1.3.1.2	   Functions: Ubiquitination, Endocytosis and Lysosomal Degradation of    Membrane Proteins ........................................................................................................... 32	  1.3.1.2.1	   Nedd4 Family Regulation of Transmembrane Proteins .............................. 32	  1.3.1.2.2	   Endocytic Regulation of the TGF-β Signaling by Smurf ........................... 34	  1.3.2	   Structure, Identification and Ligand Binding of WW Domains ............................... 37	  1.4	   Aim of Thesis ................................................................................................................... 41	  Chapter 2 Probing the Binding of Smurf2 WW3 Domain to U24 Protein from HHV-6A .. 43	  2.1	   Introduction ...................................................................................................................... 43	  2.2	   Materials and Methods ..................................................................................................... 44	  2.2.1	   Smurf2 WW3 Expression ......................................................................................... 44	  2.2.2	   Expression and Isolation of U24 from HHV-6A ...................................................... 45	  2.2.3	   Preparation of a 15-Residue Peptide Representing the Polyproline-Containing N-Terminus of U24 from HHV-6A ...................................................................................... 47	  2.2.4	   GST Pull-Down Assays ............................................................................................ 47	  	   	   viii 2.2.5	   1H-15N HSQC NMR Titrations and Kd Calculations for the Smurf2 WW3 Domain and U24 Peptide Interaction ................................................................................................. 49	  2.2.6	   Circular Dichroism ................................................................................................... 50	  2.3	   Results .............................................................................................................................. 51	  2.3.1	   Smurf2 Interacts with U24 via its WW3 Domain ..................................................... 51	  2.3.2	   Chemical Shifts of Smurf2 WW3 Domain Perturbed by PPxY Motif from U24 at 5°C and 25°C ........................................................................................................................ 52	  2.3.3	   Characterization of Smurf2 WW3 Interaction with U24 Peptide by NMR .............. 56	  2.3.4	   Understanding the Secondary Structure by Circular Dichroism .............................. 63	  2.4	   Discussion ........................................................................................................................ 67	  2.4.1	   Strength of U24-Smurf2 WW3 Interaction .............................................................. 67	  2.4.2	   Comparison with the Smurf2 WW3-Smad7 PY Complex ....................................... 68	  2.4.3	   Comparisons with Other WW Domain-Ligand Complexes ..................................... 72	  2.5	   Conclusion ....................................................................................................................... 77	  Chapter 3 Probing the Binding of Tandem Smurf2 WW Domains to U24 Protein from HHV-6A ....................................................................................................................................... 79	  3.1	   Introduction ...................................................................................................................... 79	  3.2	   Materials and Methods ..................................................................................................... 82	  3.2.1	   Protein and Peptide Preparation ................................................................................ 82	  3.2.2	   1H-15N HSQC NMR Titrations and Kd Calculations for the Smurf2 WW23 Domain and U24 Peptide Interaction ................................................................................................. 83	  3.2.3	   Circular Dichroism ................................................................................................... 85	  3.3	   Results .............................................................................................................................. 85	  	   	   ix 3.3.1	   Studying the Secondary Structure by Circular Dichroism ........................................ 85	  3.3.2	   Characterization of the Tandem Smurf2 WW23-U24 Interactions by NMR ........... 88	  3.3.3	   HSQC Spectrum of Smurf2 WW23 in the U24 Peptide Bound State in Phosphate Buffer  .................................................................................................................................. 92	  3.4	   Discussion ........................................................................................................................ 93	  3.4.1	   Strength of U24-Smurf2 WW23 Interaction ............................................................ 93	  3.4.2	   Comparison with Smurf2 WW3-U24 Interaction ..................................................... 94	  3.4.3	   Comparison with the Smurf2 WW23-Smad7 Complex ........................................... 97	  3.4.4	   Implication for the WW Domains and PY Motifs Interaction ................................ 100	  3.5	   Conclusion ..................................................................................................................... 102	  Chapter 4 Conclusions and Future Work .............................................................................. 104	  4.1	   Summary ........................................................................................................................ 104	  4.2	   Future Work ................................................................................................................... 105	  References .................................................................................................................................. 108	  Appendices ................................................................................................................................. 125	  	  	  	  	  	  	  	  	  	  	  	  	  	   	   x List of Tables 	  Table 2.1 Thermodynamic parameters obtained from ITC measurements for the binding of the Smurf2 WW3 domain to U24 peptide. ......................................................................................... 67	  Table 3.1 Thermodynamic parameters obtained from ITC measurements for the binding of the Smurf2 WW23 domain to U24 peptide. ....................................................................................... 93	                  	   	   xi List of Figures  Figure 1.1 Overview of the disease associations with HHV-6 and HHV-7 and functions of some important proteins from HHV-6. .................................................................................................... 2	  Figure 1.2 Targets of the adaptive immune response to HHV-6 (adapted from [59]). .................... 6	  Figure 1.3 TCR signaling (adapted from [122]). ............................................................................ 17	  Figure 1.4 Pathways of endocytosis and endocytic recycling (adapted from [137]). ..................... 21	  Figure 1.5 Endocytic recycling of myelin proteins and oligodendroglial membrane remodeling (adapted from [144]). ....................................................................................................................... 24	  Figure 1.6 Protein sequences of U24 from HHV-6A (GS strain), HHV-6B (Z29 strain), and HHV-7 (RK strain) include a hydrophobic stretch of amino acids near the carboxy terminus flanked by triplets of positively charged amino acids (indicated by pluses) and a polyproline region near the amine terminus (adapted from [141] with modification). ....................................... 26	  Figure 1.7 Side view and Top view of (A) PPI and (B) PPII. ..................................................... 29	  Figure 1.8 The architecture of some Nedd4 family of E3 ubiquitin-protein ligases (adapted from [170] with modification). ................................................................................................................. 31	  Figure 1.9 The NEDD4 family of proteins regulates diverse cellular processes (adapted from [165]). .............................................................................................................................................. 33	  Figure 1.10 The functions of Smurf2. .......................................................................................... 36	  Figure 1.11 Alignment of a selection of WW domains. .............................................................. 39	  Figure 1.12 Ribbon diagram of the lowest energy structure of the Smurf2 WW3 domain-Smad7 	   	   xii PY peptide complex (adapted from [210]). ..................................................................................... 40	  Figure 2.1 GST pull-down analysis for interaction between U24 and Smurf2 WW domains. ... 51	  Figure 2.2 Comparison of Smurf2 WW3 chemical shifts in the presence and absence of U24 peptide at 5°C. ............................................................................................................................... 54	  Figure 2.3 Comparison of Smurf2 WW3 chemical shifts in the presence and absence of U24 peptide at 25°C. ............................................................................................................................. 55	  Figure 2.4 NMR titration studies of Smurf2 WW3 and U24 peptide. ......................................... 57	  Figure 2.5 1H-15N chemical shift perturbation (CSP) of Smurf2 WW3 when titrated with U24 peptide. .......................................................................................................................................... 59	  Figure 2.6 The surface representation of the structure of Smurf2 WW3, colored as described in Figure 2.5. ..................................................................................................................................... 60	  Figure 2.7 Binding affinity of Smurf2 WW3 and U24 peptide. .................................................. 62	  Figure 2.8 Analysis of the secondary structure by CD spectroscopy. ......................................... 64	  Figure 2.9 Plot of the percentage of beta-strands at different temperatures in Smurf2 WW3 domain with and without U24 peptide. ......................................................................................... 66	  Figure 2.10 Comparison of the Smurf2 WW3-U24 complex and the Smurf2 WW3-Smad complex. ........................................................................................................................................ 70	  Figure 2.11 The charge distribution on the surface of the Smurf2 WW3. ................................... 72	  Figure 2.12 Binding affinity of five WW domains and Smad7 peptide. ..................................... 74	  Figure 2.13 Comparison of various WW domains and their binding partners. ........................... 77	  	   	   xiii Figure 3.1 Ribbon diagram of the Smurf2 WW23-Smad7 complex. .......................................... 81	  Figure 3.2 Analysis of the secondary structure by CD spectroscopy. ......................................... 86	  Figure 3.3 Plot of the percentage of beta-strands at different temperatures in tandem Smurf2 WW23 with and without U24 peptide. ......................................................................................... 88	  Figure 3.4 NMR titration studies of Smurf2 WW23 and U24 peptide. ....................................... 89	  Figure 3.5 Binding affinity of Smurf2 WW3 and U24 peptide. .................................................. 90	  Figure 3.6 1H-15N HSQC spectrum of 15N-labeled Smurf2 WW23 in the presence of U24 peptide at 25°C in phosphate buffer (40 mM phosphate, 20 mM NaCl, 0.05% NaN3, 10% D2O) at pH 7.8........................................................................................................................................................ 92	  Figure 3.7 Comparison of chemical shift perturbations from Smurf2 WW3 and Smurf2 WW23 upon binding to U24 peptide. ........................................................................................................ 96	  Figure 3.8 Superposition of Smurf2 WW3 (blue) and ww23 (red) 1H-15N HSQC spectra recorded in the presence of U24 peptide at 25°C. Peaks are labeled for several residues. ........... 97	  Figure 3.9 Comparison of the Smurf2 WW23-Smad7 complex and the Smurf2 WW3-Smad7 complex. ........................................................................................................................................ 99	     	   	   xiv List of Abbreviation 	                        AIDS        BCA        BCR         BMP        CCR7       CD        CD          CI         CIHHV-6      CNS          CSF          DN         EBV         EDTA      EE           EEA         ENaC       ER          ERC        FISH         GLB       Grb         acquired immunodeficiency syndrome bicinchoninic acid B-cell receptor  bone morphogenetic protein  C-C chemokine receptor type 7 circular dichroism clathrin-dependent clathrin-independent chromosomally integrated HHV-6 infection central nervous system cerebrospinal fluid dominant negative version Epstein-Barr virus ethylenediaminetetraacetic acid  early endosome early endosome antigen epithelial sodium channel  endoplasmic reticulum endocytic recycling compartment fluorescent in situ hybridization Glasgow lysis buffer growth factor receptor-bound 	   	   xv                        GST       HCMV       HEPES     HHV         HIV        HLA         HRV-3C    HSQC  IE            IFNs         IgG         IgM         IL-2          IL-10          IL-1β         IPTG       ITC      LE          LMP2A     MAG        MALDI     MBP          MHC        Glutathione S-transferase  human cytomegalovirus 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid human herpesvirus human immunodeficiency virus human leukocyte antigen human rhinovirus 3C heteronuclear single quantum coherence immediate-early interferons  immunoglobulin G immunoglobulin M interleukin 2 interleukin 10 interleukin-1β isopropyl 1-thio-β-D-galactopyranoside  isothermal titration calorimetry late endosome latent membrane protein 2A myelin-associated glycoprotein  matrix-assisted laser desorption/ionization myelin basic protein major histocompatibility complex  	   	   xvi                        MOG        MS         MTLE       MVBs       NEDD4     NMR     PAGE     PBMCs       PKC        PLP         PP           PPII       PY          RE          RR          SDS     SH3        Smurf       SP          TA        TCR         TfR         TGF-β     myelin-oligodendrocyte glycoprotein multiple sclerosis mesial temporal lobe epilepsy  multivesicular bodies  neuronal precursor cell expressed developmentally downregulated 4 nuclear magnetic resonance polyacrylamide gel electrophoresis peripheral blood mononuclear cells protein kinase C  proteolipid protein  primarily progressive polyproline II  proline-tyrosine recycling endosome relapsing and remitting  sodium dodecyl sulfate SRC homology 3 Smad ubiquitylation regulatory factor secondarily progressive  tail-anchored T-cell receptor transferrin receptor transforming growth factor-β 	   	   xvii       TGFRI      TGFRII      TMD        TNF-α        TOF      TRC       Tregs          WW23      TGF-β receptor type I  TGF-β receptor type II  transmembrane domain  tumor necrosis factor-α time of flight transmembrane domain recognition complex  regulatory T cells WW2 and WW3 domains in tandem 	   	   xviii Acknowledgements 	  First and foremost I would like to give many thanks to my supervisor Suzana Straus for her unyielding support, thoughtful advice and contagious enthusiasm. I am also really grateful to Yurou Sang for her enduring help in the laboratory. Thank you for showing me how to do some biochemical experiments and patiently answering all of my questions. I want to acknowledge all of the members in the Straus research group for their enthusiastic support during my experimental work.  Thanks to all the shops and services people at UBC for their help. In particular, the staff of Bioservices, Elena Polishchuk and Jessie Chen must be acknowledged for their hard work and endless help, ensuring that work could be done efficiently. I must also specially acknowledge Mark Okon for his patience, valuable time, NMR expertise and assistance with my NMR experiments.     I would like to give many thanks to my good friends for their encouragement. Finally, I would like to express my greatest gratitude to my parents. I am grateful for the strength and support of my parents. I love and admire you all and could not have come this far without you.      	   	  	   	   xix Dedication 	  	  	                                          To my parents 	  	  	  	  	  	  	  	  	  	  	  	   	   1 1.1 HHV-6 Herpesviruses are enveloped double-stranded DNA viruses with an icosahedral capsid[1]. They are highly disseminated in nature and can remain latent in their natural host. During latency, the viral genomes take the form of closed circular molecules, and only a small subset of the viral genes is expressed. The latent genomes retain their capacity to replicate, and cause disease upon reactivation. Different herpesviruses have different biological properties. The following herpesviruses are known to primarily infect humans: herpes simplex virus 1, herpes simplex virus 2, human cytomegalovirus (HCMV), varicella-zoster virus, Epstein-Barr virus (EBV) and human herpesviruses 6–8 (HHV-6, HHV-7 and HHV-8)[2]. HHV-6 belongs to the β-herpesvirus subfamily of Herpesviridae, as defined by the Baltimore classification system of viruses. It was first isolated in the mid 1980’s from patients with lymphoproliferative disorders and acquired immunodeficiency syndrome (AIDS)[3]. Two subtypes of HHV-6 have been identified: variant A and B[4]. Both of them share 90% homology of the nucleotide sequence[5]. Their genomes consist of double stranded DNA with approximately 160 kilo base pairs (kbp) (159321 bp for HHV-6A[6] and 162114 bp for HHV-6B[5]). The viral DNA is surrounded by a core, which in turn is enclosed by an icosahedral capsid. Finally, a lipid bilayer constitutes the outermost barrier to the exterior. This bilayer is from a host cell compartment and is acquired during viral replication and budding from the infected cell. As with the other human β-herpesviruses, HHV-6 can also establish persistent latent infection in the host, characterized by highly restricted viral gene expression and the ability to reactivate from latency to produce infectious virus. HHV-6’s reactivation has been Chapter 1 Introduction 	   	   2 established to be associated with transplant complications[7], encephalitis[8], and immune suppression, and further has been proposed to play a role in multiple sclerosis[9-11] and several other diseases such as chronic fatigue syndrome[12] and HIV/AIDS progression[13] (Figure 1.1). In the following part of section 1.1, basic biological properties, immune response, immunomodulation and related diseases of HHV-6 will be explained in detail.       Figure 1.1 Overview of the disease associations with HHV-6 and HHV-7 and functions of some important proteins from HHV-6. Potential functions of U24 protein are: a possible interaction with Fyn-SH3 via PxxP based on mimicry of MBP and with WW domains via PPxY, thereby affecting endocytic recycling (where x is any amino acid).    1.1.1 HHV-6A and HHV-6B In the past, a consensus was reached that HHV-6 had two variants of the same species: HHV-6A and HHV-6B[14]. Recently distinctions between HHV-6A and HHV-6B and relationships to the herpesvirus family overall have been further confirmed by genomic 	   	   3 sequencing[15]. Even though the genomes of these two viruses are co-linear and share an overall 90% identity, divergence of specific sequences [e.g. the immediate-early (IE) region] is higher than 30%, some of which are due to splicing[5, 6, 16]. In addition, there are clear functional differences [e.g. the IE1 gene of HHV-6A and HHV-6B[17, 18]]. Several reports have further demonstrated that the splicing pattern and temporal regulation of transcription of selected genes are different[5, 16, 19, 20]. Overall, HHV-6A and HHV-6B are classified as distinct viruses based on epidemiology, disease associations, biological and immunological properties[4, 21-24]. The two groups also showed different in vitro tropism for selected T cell lines, specific immunological reactivity with monoclonal antibodies, distinct patterns of restriction endonuclease sites, and specific and conserved interstrain variations in their DNA sequences[22, 24, 25].  1.1.1.1 HHV-6A The epidemiology of HHV-6A infection is much less known when compared with HHV-6B. However, it was demonstrated that HHV-6A, but not HHV-6B, has been associated with Hashimoto’s thyroiditis[26] as well as syncytial-giant cell hepatitis[27-29] in liver transplant patients. Additionally, there is evidence suggesting an increased severity of HHV-6A over HHV-6B in cases of clinical neurological disease[30-32]. HHV-6A DNA and mRNA are found more frequently than HHV-6B in patients with neuroinflammatory diseases such as multiple sclerosis (MS)[9, 27, 33] and rhomboencephalitis[31]. HHV-6A has been found predominantly in the central nervous system (CNS) of a subset of patients with MS, and active HHV-6A infection has been detected in blood[27, 34, 35] and in cerebrospinal fluid (CSF)[36] of patients with relapsing/remitting MS[27, 34, 36, 37]. Additionally, HHV-6A has been proposed as a potential accelerating factor in HIV infection, as corroborated by the results of in vivo studies in macaques[15, 38, 39]. 	   	   4 1.1.1.2 HHV-6B In the USA, UK and Japan, 97-100% of primary infections by HHV-6 are caused by HHV-6B and occur between the ages of 6 and 12 months[40-42]. And an overwhelming majority of post-transplant reactivation occurs with HHV-6B. It was shown that HHV-6B, but not HHV-6A, has been associated with mesial temporal lobe epilepsy and status epilepticus[43, 44].  1.1.2 Basic Biology of HHV-6 HHV-6 replicates preferentially in activated CD4+ T lymphocytes, while CD8+ T cells are only efficiently infected with HHV-6A. Besides T lymphocytes, several diverse cell types (e.g. endothelial cells, epithelial cells, astrocytes, B cells) also can be productively infected. Moreover, HHV-6 also has neurotropism and can infect neurons[45], and glial cells[46] including astrocytes[47], oligodendrocytes[48], and microglia[49]. The host tissue range of HHV-6 in vivo is even broader. It includes brain tissue, liver tissue, tonsillar tissue, salivary glands, and endothelium[50]. HHV-6 uses a broadly expressed receptor as the port of entry into cells: the CD46 cell surface molecule, which is a regulator of complement activation receptor expressed on all nucleated cells. HHV-6 integrated into human germ cells at an undetermined time, probably millennia ago. Since that time, HHV-6 has been passed from parent to child, with the viral DNA present in every cell in the body, in some human beings. Since inherited chromosomally integrated HHV-6 infection (CIHHV-6) was initially described[51], several studies using fluorescent in situ hybridization (FISH) have showed that HHV-6 could integrate into human chromosomes[52-54] and also be transmitted vertically through the germ line[55, 56]. In people without CIHHV-6, HHV-6 infection occurs in only a small fraction of the peripheral blood mononuclear cells (PBMCs). However, in people with CIHHV-6 viral DNA is present in every 	   	   5 cell, profoundly affecting the result of viral load measurement[50]. In the United States, approximately 1% of individuals are born with CIHHV-6[57]. Similar or somewhat lower prevalence has been reported in other countries[56]. In the “classical” human herpesvirus replication cycle, latency is achieved following the circularization of the linear double stranded DNA genomes within the nucleus to form a covalently closed circular episome. In contrast, it has been recently reported that HHV-6A reproducibly integrates into the subtelomeric region of the chromosome of in vitro infected cell lines to establish latency[58].  	  1.1.3 Immune Response by HHV-6 One outstanding capacity of HHV-6 is its ability to spread in nearly the entire human population. This is probably, at least to some extent, achieved by the mild clinical phenotype of HHV-6 infection. A virus that immediately kills its host will have difficulties to spread, but a less aggressive virus inducing mild or even subclinical symptoms probably spread more efficiently from one, relatively healthy and mobile individual to another.  In general, HHV-6A and HHV-6B have not been distinguished in studies of the immune response to HHV-6. So far, little is known about the immune mechanisms for HHV-6 that control infection[59], because several aspects of HHV-6 biology interfere with straightforward application of conventional approaches to characterize antiviral immunity. First, antibody titers to HHV-6 and frequencies of T cells recognizing HHV-6 are low, making detection of these responses challenging[60]. Second, HHV-6 infection is restricted to humans and closely related primates, so the lack of a small animal model has inhibited detailed mechanistic studies[61, 62]. Despite these limitations, there have recently been notable advances in defining HHV-6-specific T cell responses, largely based on a few studies and extrapolation from human cytomegalovirus 	   	   6 (HCMV)[63].    1.1.3.1 Antibody Response A few HHV-6 antigens prominently targeted by the antibody response have been identified[59]. These include the major antigenic virion protein U11[64], the late antigen U94[65], the major glycoproteins gH (U48)[66] and gQ (U100)[67], the polymerase processivity factor (U27)[68] and the tail-anchored membrane protein U24[9, 10] (Figure 1.2).   Figure 1.2 Targets of the adaptive immune response to HHV-6 (adapted from [59]). Antibody and T cell responses target the viral surface membrane, tegument, and capsid components of the virion as well as non-structural proteins expressed in infected cells. Inset (left) shows a purified viral particle with components indicated, and cartoon (right) shows intracellular locations for the expected stepwise viral assembly process.   1.1.3.2 T-cell Responses The immunosuppressive properties of this virus were identified shortly after the discovery of HHV-6. Initially, it was reported that HHV-6 arrested interleukin 2 (IL-2) synthesis and T cell proliferation[69]. Subsequent studies recognized immune modulation by effects in both infected 	   	   7 and non-infected cells[70]. In infected CD4+ T cells, HHV-6 induces apoptosis[71], cell cycle arrest[72], inhibition of IL-2 synthesis[73], and T-cell receptor (TCR) and major histocompatibility complex I (MHC-I) down modulation[74]. In antigen-presenting cells, HHV-6 induces MHC-I down-modulation[75] and reduces the capacity of these cells to present antigens and activate T cells[74]. Additionally, interleukin 10 (IL-10), secreted by CD4+ T cells responding to HHV-6, modulates proliferation of other T cell populations[76]. Different subsets of regulatory T cells (Tregs) have been observed in vitro after HHV-6 specific expansion or cloning[60, 77].  1.1.3.3 Targets of the T-cell Response to HHV-6 About 100 proteins are encoded by the HHV-6 genome. Many of them are >1000 amino acids in length, making the identification of immunodominant epitopes a laborious and time consuming task. T cells recognizing information carried by the particular peptide epitopes is essential for identification, characterization and modulation of T-cell responses specific to HHV-6 as compared to closely related viruses. Two approaches were mainly used to screen the number of antigens/peptides: one has focused on HHV-6 proteins present at high levels in virus preparations[60] and the other has focused on HHV-6 homologues of antigens defined for HCMV[77, 78]. Overall, twelve CD8+ T cell epitopes were defined[59]: three from U11, two from U54[78], one from U14 and six from U90, to complement the eleven CD4+ T cell epitopes defined, which are derived from the major capsid protein U57, the tegument proteins U11 and U14, the glycoprotein U48 and DNA polymerase U38 (Figure 1.2). Many of the mapped T cell responses are crossreactive between HHV-6A and HHV-6B, and therefore cannot be used as markers of virus-specific responses. In addition, three putative HHV-6 epitopes have been defined by virtue of cross-reactivity 	   	   8 with human self-antigens. T-cell responses to a HHV-6 U24 peptide that shares homology with the multiple sclerosis autoantigen myelin basic protein (MBP) have been reported. However, the significance of this crossreactive response in multiple sclerosis is controversial[10, 79]. The diabetes-associated glutamic acid decarboxylase islet autoantigen GAD95, which is recognized by CD4+ T cells, was shown to have crossreactive responses with a similar peptide sequence from HHV-6A U95[80], but whether these cells recognize naturally processed antigens is still not known.   1.1.4 Immunomodulation by HHV-6 Modulation of expression of the host’s immune factors is another mechanism of evading the immune response or creating an environment, in which the virus can survive. This is especially significant for herpesviruses, which persist in their host throughout life. For the purpose of immune modulation, HHV-6 exploits its transactivating capacities as well as other mechanisms that are not related to transcription[32].  1.1.4.1 Possible Mechanism of Immunomodulation by HHV-6 1.1.4.1.1 IFNs Interferons (IFNs) are generally part of the cellular innate immune response to viruses. It has been reported that the production of IFN-α increased due to HHV-6 infection of mononuclear cells[81]. In turn, IFN-α was identified to suppress HHV-6 replication[82, 83]. In contrast, IFN-γ release was inhibited by HHV-6 in PBMCs[84], while in continuous T cells IFN-γ levels were unaltered after HHV-6 infection[85].  	   	   9 1.1.4.1.2 TNF-α and ILs Tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in PBMCs were found to be up-regulated by HHV-6, depending solely on virus entry-related events, because viral de novo protein synthesis was not required[86, 87]. Similarly, the transcriptional down-regulation of IL-2 required no active viral replication[69]. Up-regulation of TNF-α release was also found in HHV-6-infected differentiated U937 monocytoma cells[88]. With an immunomicroarray, it was observed that both HHV-6A and HHV-6B increased expression in the Sup-T1 T-cell line for the genes for IL-18, the IL-2 receptor, members of the TNF-α superfamily receptors, mitogen-activated protein kinase, and Janus kinase signaling proteins[85]. The C-C chemokine receptor type 7 (CCR7) was up-regulated when CD4+ T cells were infected with HHV-6[89]. Furthermore, an interesting feature of HHV-6 infection is that, during the course of infection, it down-regulates cell surface expression of its own receptor, CD46, in infected as well as in uninfected cells, both in vitro and in an ex vivo virus propagation system[90].  1.1.4.1.3 T-cell Activation IL-2-induced activation of primary T lymphocytes is an absolute requirement for efficient HHV-6 replication, but at higher concentrations, IL-2 strongly inhibits the virus-induced cytopathic effect[91, 92]. T-cell activation as a stimulus for HHV-6 was required, which was also illustrated by pretreatment of PBMCs with anti-CD3[93]. CD3 is transcriptionally down-regulated by newly formed HHV-6 proteins, leading to reduced surface expression of the CD3/T-cell receptor complex[94, 95]. T-cell activation may be a prerequisite for HHV-6 replication. However, the T-cell proliferative response to mitogen or antigen presentation was seen to be severely impaired after HHV-6 infection, indicating that HHV-6 infection induces a state of immune 	   	   10 suppression[32, 96].  1.1.4.2 Proteins with Immune-Modulatory Functions  As described above, HHV-6 has a number of mechanisms by which it counters the immune system’s ability to eradicate it. In addition, HHV-6 encodes several proteins with immune-modulatory functions[17] (Figure 1.1): 1) The virus encodes a chemotactic U83 protein that binds chemokine receptors, which might have an impact on the cell type recruited to the site of infection. 2) U12 and U51 are another two chemokine receptors. U51 protein plays an essential role for viral growth. The U51 receptor is constitutively active, causing an increase in intracellular levels of the second messenger inositol phosphate[97]. U51 can bind several chemokines in the nanomolar range, and promotes chemotaxis as well as the internalization of chemokines[98]. 3) HHV-6 infected cells that express the IE1 protein have severely impaired IFN-β gene induction in response to several stimuli[99].  4) Expression of U24 causes internalization of the T cell receptor/CD3 complex at the cell surface, resulting in improper T cell activation by antigen-presenting cells[100]. It is presumed that U24 acts by preventing the CD3 complex from reaching the Rab-11 recycling endosomes and thus preventing re-expression at the cell surface. Functionally, by down-modulating the CD3 complex, HHV-6 may ensure that T cells cannot get properly activated, causing reduced secretion of cytokines at the infection site. The role of U24 protein in T-cell signaling and endosomal recycling will be discussed in detail in section 2. 5) The HHV-6 U21 protein causes reduced expression of MHC class I molecules at the cell 	   	   11 surface by binding and targeting MHC class I molecules to endosomal compartments[75].  1.1.5 HHV-6 in Central Nervous System Diseases HHV-6 has been implicated in the development of a diverse array of neurologic conditions, including encephalitis, mesial temporal lobe epilepsy (MTLE), chronic fatigue syndrome and multiple sclerosis (MS) (Figure 1.1). HHV-6 infection is ubiquitous in the general population. Numerous studies have demonstrated HHV-6 DNA sequences in non-pathological brain tissues obtained from autopsies or by surgeries[101-103], suggesting that it can be a commensal virus of the brain. Thus, attributing a pathological role to the virus in diseases of the central nervous system (CNS) can be challenging.  HHV-6 has in vitro tropism for various cells of the CNS and higher frequencies of HHV-6 DNA detection have often been reported in samples taken from patients with neurological diseases, indicating its role in diseases. In addition, HHV-6 viral protein expression has been observed in pathological specimens[45, 103] but not in healthy tissues, suggesting that active viral replication may in part contribute to manifestation of clinical symptoms. Primary childhood infection of HHV-6 is often asymptomatic and self-limiting. However, it also commonly produces febrile illnesses[104]. Furthermore, many studies suggest that the association of HHV-6 with neurological disorders may be related to its ability to enter a state of latency following primary exposure: reactivation later in life could plausibly cause neurological symptoms.  One of the most intriguing characteristics of HHV-6 is that it may be an etiological agent for multiple and quite different pathological conditions of the CNS. Inherent viral properties such as sequence variations and differences in antigenic specificity between the A and B variants of HHV-6 may be responsible for the diverse pathology, as may various host factors. 	   	   12 At present, no antiviral drugs have been approved for the treatment of HHV-6 infection. Currently, anticytomegalovirus drugs such as the nucleoside analog valganciclovir, the nucleotide analog cidofovir or the pyrophosphate analog foscarnet are used to treat HHV-6 infections, because in vitro studies show that they also have activity against HHV-6.  1.1.5.1 Multiple Sclerosis In 1838 Robert Carswell first reported lesions in the dissected brain of a deceased individual. Referring to the histopathological picture of the condition with several (or multiple) scars (or sclerosis) of lesions, the disease was named multiple sclerosis (MS). MS is the most common inflammatory demyelinating autoimmune disease of the CNS of young adults, with onset usually occurring between the ages of 20 and 50. Approximately, 2.1 million individuals are affected by MS worldwide and it affects women more often than men in a ratio of 2:1. The onset and progression of MS are variable; the clinical course of MS is most commonly of a relapsing and remitting (RR) nature but may be primarily (PP) or secondarily (SP) progressive. Sensory and motor disturbances, alterations in vision and cognitive impairment are common clinical features of MS. In the CNS, myelin sheaths surround the axons for insulation to enhance the speed of nerve signals. Myelin sheaths consist of densely packed cell membrane extensions of oligodendrocytes and the two major protein components of CNS myelin are myelin basic protein (MBP) and proteolipid protein (PLP). There are many neurological disorders due to deficiencies in myelin assembly and structure. In MS, oligodendrocytes are thought to be the main target for attack by a CD4+ T helper 1-mediated autoimmune response[105]. However, the predominant lymphocyte found in focal inflammatory demyelinating plaque lesions, which may form within the 	   	   13 periventricular white matter of the brain, brain stem, spinal cord and optic nerve, are CD8+ T-cells[106], which are emerging as important effector cells in MS[107]. In addition to the inflammation, destruction of oligodendrocytes and demylination, axonal loss occurs from disease onset and ultimately results in the development of irreversible neurological disability in the affected individual[108]. Although the etiology of MS is currently unknown, factors that play a role in the development of MS include genetics and environmental insults. The discovery that a large number of immune system-related molecules are genetic risk factors for MS (such as IL-2 receptor α and IL-7 receptor α)[109], in combination with the presence of elevated levels of immunoglobulin (Ig) G and oligoclonal bands in the cerebrospinal fluid (CSF) of MS patients, both of which are characteristic of CNS disorders of infectious origin[110], all support a role for environmental insults in the development of MS. Infectious agents that have been explored as possible etiological agents of MS include bacteria and bacterial superantigens, viruses, as well as protozoa that infect humans. In particular, the mechanisms of viral-induced demyelination[111] include: (1) direct lysis of virus-infected oligodendrocytes by the virus, (2) direct lysis of virus-infected oligodendrocytes by the host immune response to the virus, (3) lysis of uninfected oligodendrocytes by a self-reactive immune response triggered by the virus and (4) lysis of oligodendrocytes by a nonspecific bystander immune response triggered by the virus.   1.1.5.2 HHV-6 As a Trigger of MS HHV-6 has long been considered as a potential candidate virus for the etiology of MS. A relationship between HHV-6A and MS was first suggested by immunohistochemical demonstration of viral antigen in oligodendrocytes of MS white matter lesions but not in control 	   	   14 brains[45]. Since this initial report, several studies have supported an association of HHV-6A/B and MS by the demonstration of elevated antibody titers to HHV-6A/B antigens compared to controls and by amplification of HHV-6A/B DNA from the serum, CSF and brain tissue of MS patients[9, 36, 112, 113]. Exacerbation of relapsing-remitting MS has been linked to higher viral loads in serum and in PBMCs, suggesting association of HHV-6A/B reactivation with disease relapses[9, 114]. Abundant clinical studies have highlighted a correlation between MS and several parameters testing for HHV-6A/B infection. Moreover, the levels of HHV-6A/B-specific IgG and IgM in the serum and in the CSF were reported to be higher in MS patients in several studies, although this phenomenon does not appear to be specific to HHV-6. HHV-6 IgG reactivity was not significantly different among the different patient groups, probably because latent HHV-6 infection is so ubiquitous. It has also been shown that lymphoproliferative responses against HHV-6 antigens were increased in MS patients[9]. The analysis of brain biopsies and postmortem tissues indicated that HHV-6A/B DNA was present more frequently in the brain of MS patients than in control brains and that it was also more frequent in MS lesions than in normal areas of the same brains. Immunohistochemistry analyses confirmed the presence of viral proteins in oligodendrocytes and astrocytes in the brain from MS patients, with a higher frequency in demyelinating plaques[27, 115]. Most interestingly, viral loads were detected more frequently, and levels of HHV-6A/B-specific IgG were increased in MS patients experiencing disease exacerbation, thus suggesting a correlation between HHV-6A/B infection and MS relapses.  Many of the initial studies do not discriminate between the two species, but as mentioned above, HHV-6A and -6B are considered to be two distinct viruses. Based on a few reports, it appears that HHV-6A is found more frequently than HHV-6B in the serum of MS patients[34]. Especially in the case of active infection, only HHV-6A has been found[27]. In contrast, in one 	   	   15 study, intrathecal HHV-6B IgG levels were more abundant than HHV-6A IgG in MS patients, and only HHV-6B-specific IgM levels were found[116]. The potential association between HHV-6A and HHV-6B infection and MS has often been discussed and remains controversial. Some studies provide contradictory results[117, 118], raising methodological and technical questions, especially concerning the choice of control groups and the immunological state of the included patients, who often receive immunosuppressive treatments that may provoke latent herpesvirus reactivation by itself[119].   1.2 U24 Protein from HHV-6 As discussed above, U24 protein not only has several functions, which are essential for HHV-6, but may also play an important role in Multiple Sclerosis, which will be described particularly in section 1.2.1. Inhibition of a basic cellular process by U24 has interesting implications not only for the pathogenicity of roseoloviruses but also for the understanding of the biology of endosomal transport. Therefore, it is worthwhile to further explore the role of the U24 protein and the structural features that are critical for its function.      1.2.1 Functions of U24 1.2.1.1 Potential Role of U24 in Multiple Sclerosis: Molecular Mimicry The molecular mimicry hypothesis propose that this might be elicited with infection with a pathogen that have a peptide that can bind to the MHC molecule in a way that makes the peptide-human leukocyte antigen (HLA) complex structurally similar at the site for TCR binding compared to myelin peptide binding.  Molecular mimicry involving HHV-6A has been proposed as one mechanism by which the 	   	   16 autoimmune process could be triggered. At first, it was reported that 15%–25% of HHV-6-specific T cell clones obtained from healthy donors or MS patients were crossreactive to myelin basic protein (MBP), one of the autoantigens implicated in MS pathology[79]. In fact, MBP and the U24 protein from HHV-6 were later shown to share an identical amino acid sequence of 7 residues. Moreover, T cells directed against an MBP peptide also recognized an HHV-6A peptide, both peptides containing the identical sequence. Interestingly, cross-reactive cells were more frequent in MS patients than in controls[10]. These data were further confirmed by a more recent study, in which the presence of crossreactive CD8+ cytotoxic T cells was found[120]. Altogether, these studies suggest that HHV-6A infection can activate T cell responses, which can simultaneously be directed against myelin sheaths, thus strongly supporting the potential role for HHV-6A in autoimmune diseases affecting the CNS.  1.2.1.2 Potential Role of U24 in Regulation of T-cell Receptor Signaling 1.2.1.2.1 T-cell Receptor Signaling To successfully replicate and spread, a virus must take control of multiple cellular processes. Among the cells of the immune system, T lymphocytes (T cells) are critically significant for the orchestration of the antiviral response and also for the direct killing of infected cells. The T-cell receptor (TCR) is the central signaling pathway regulating T-cell biology. The TCR allows the T cell to recognize antigen presented in the context of major histocompatibility complex (MHC) class I or class II molecules expressed on infected cells or professional antigen-presenting cells. TCR signaling in naive T cells drives their activation and expansion. Ligation of the TCR leads to a cascade of signaling events[121] (Figure 1.3) ultimately resulting in various T-cell functions. In fact, TCR signal transduction is much more complex 	   	   17 than the basic scheme presented below. Signals do not necessarily propagate in a linear pattern; instead, there is significant cross talk between many of the signaling molecules involved.    Figure 1.3 TCR signaling (adapted from [122]). Major activation pathways are depicted by bold green lines; major regulatory mechanisms are depicted by thin red (inhibitory) or green (stimulatory) lines; and alternative LAT-independent TCR signaling is depicted by the dashed green line.  1.2.1.2.2 TCR/CD3 Complex A common feature of many cell surface receptors is their constitutive or ligand-induced endocytosis, via clathrin-coated pits, and subsequent recycling back to the cell surface[123, 124]. The T cell receptor TCR/CD3 complex is one key player in the TCR signaling pathway. It is a large multisubunit complex composed of at least eight polypeptide subunits (TCRαβ, CD3εγ, εδ, and ζζ)[125]. TCR/CD3 complex is very stable and is rapidly internalized and recycled in resting T cells[126]. T-cell activation is self-limited by a rapid internalization and degradation of the TCR/CD3 complex. Cell surface expression of the TCR/CD3 complex is downmodulated upon ligation with MHC/peptide complexes[127], which is considered to be a pivotal event in T cell activation. However, it has also been suggested that surface downmodulation of TCR may not be required for T cell activation[128]. The TCR/CD3 complex is predominantly found in Rab41 	   	   18 vesicles, demonstrating the presence of TCR in early endosomes[129]. Although a small proportion of TCR is associated with Rab71 late endosomes, none was detected in lysosomes. However, evidence was obtained for the degradation of TCR/CD3 complexes in lysosomes of T cell-antigen presenting cell conjugates [130]. The data further suggests that there are multiple mechanisms by which ligated TCR/CD3 complexes are prevented from returning to the cell surface. For instance, some studies have shown that unpaired TCRα and CD3δ chains are translocated from the endoplasmic reticulum (ER) into the cytosol, where they are ubiquitinated and degraded by proteasomes[131, 132]. It has also been shown that the CD3ζ and CD3δ chains are ubiquitinated in a tyrosine kinase–dependent manner following TCR ligation[133].   1.2.1.2.3 Viral Modulation of T-cell Receptor Signaling by U24 Protein Since T cells pose a threat to the successful replication of viruses, and since TCR signaling is central to the development and function of T cells, it is not surprising that many viruses have evolved mechanisms to modulate TCR signaling. For T lymphotropic viruses, the T cell itself is the major site of viral infection and replication; therefore, the virus may stimulate TCR signaling to drive T-cell proliferation such that the T cell is permissive for viral replication. For other viruses, which predominantly infect nonlymphoid cells, the ability to modulate TCR signaling constitutes an immune evasion mechanism, in which the virus inhibits the ability of T cells to respond to infected cells[122]. HHV-6 preferentially infects CD4+ T cells, on which the TCR/CD3 complex signaling receptor is present. One key player in the generation of an adaptive immune response is the TCR/CD3 complex signaling receptor. Infection of T cells with HHV-6 results in downmodulation of surface CD3 and the αβ TCR heterodimer[134]. This downmodulation may 	   	   19 result in part from decreased transcription of CD3[95, 134]. However, while surface levels of TCR components are markedly decreased by HHV-6 infection, their intracellular levels remain relatively normal, suggesting that redistribution of the TCR complex may play a more important role than transcriptional regulation[100]. Redistribution of the TCR complex has been ascribed to the U24 protein of HHV-6. A library of HHV-6 candidate genes was generated and tested, and it was concluded that only U24 significantly downregulated both CD3 and αβ TCR from the surfaces of Jurkat cells[100]. Importantly, the expression of several other cell surface molecules involved in immune recognition was not affected by U24 expression, indicating that U24 does not massively modify the cell surface proteome[100]. Furthermore, it was found that U24 protein from HHV-6 caused a block in CD3 access to recycling endosomes[100] and thus CD3 accumulated in early and late endosomes and cannot be recycled back to the cell surface, resulting in redistribution and downregulation of TCR complex.  It was also indicated that U24 downregulated the CD3/TCR complex independently of the T-cell activation pathway[100]. In the sense that it is not associated with T-cell activation, U24-mediated downregulation of CD3 is quite unique. Moreover, the cells become resistant to activation upon stimulation by antigen-presenting cells. It is likely that the downmodulation of the TCR complex by HHV-6 results in hyporesponsiveness of T cells to external antigenic stimulation. CD3 downregulation by U24 might thus represent a novel mechanism of immune evasion by blocking T-cell activation. Inhibiting T-cell activation will affect the release of cytokines and potentially dampen the generation of an adaptive immune response. In addition, lymphocyte activation often creates an environment favorable to the reactivation and replication of lymphotropic herpesviruses. By controlling the level of CD3, HHV-6 might prevent its own 	   	   20 reactivation, thereby keeping HHV-6 in a latent state, which is less prone to immune recognition[100].  1.2.1.3 Potential Role of U24 in Multiple Sclerosis: A Block of Endosomal Recycling 1.2.1.3.1 Endosomal Recycling Cells internalize extracellular material, ligands, and plasma membrane proteins and lipids by endocytosis. This removal of membrane from the cell surface is balanced by endosomal recycling pathways that return much of the endocytosed proteins and lipids back to the plasma membrane. The balance between endocytic uptake and recycling controls the composition of the plasma membrane and contributes to diverse cellular processes, including nutrient uptake, cell adhesion and junction formation, cell migration, cytokinesis, cell polarity and signal transduction. Endocytosis occurs by various mechanisms, which can be divided into those that are clathrin dependent and those that are clathrin independent[135, 136] (Figure 1.4).      	   	   21  Figure 1.4 Pathways of endocytosis and endocytic recycling (adapted from [137]). Itinerary of cargo proteins enter cells by clathrin-dependent (blue cargo) and clathrin-independent (red cargo) endocytosis. Subsequent routing of cargo to the early endosome (EE), juxtanuclear endocytic recycling compartment (ERC) and the recycling endosome (RE) is shown. RAB5 and early endosome antigen 1 (EEA) function in the generation and maintenance of the early endosome. Some cargos are selected in the early endosome to the multivesicular body pathway and to be transported on to late endosomes (LE).  The most well-understood endocytic process — receptor-mediated endocytosis — involves the internalization of receptors and their ligands by clathrin-coated pits, as already described above for the TCR/CD3 complex. Many of the ligands are subsequently degraded in late endosomes or lysosomes, whereas many of the receptors are re-used up to several hundred times. This recycling of receptors back to the plasma membrane was one of the first characterized examples of recycling in a membrane trafficking pathway. In addition to maintaining the homeostatic regulation of molecules in each of the compartments, the transport rates of membrane trafficking can be altered in response to signaling mechanisms to increase or decrease the surface expression of molecules. Examples of this regulation include a decrease in the surface expression of many signaling receptors in response to stimulation by their ligands (receptor downregulation), or an increase in the surface expression of glucose transporters in response to insulin. 	   	   22 1.2.1.3.2 A General Block of Endosomal Recycling by U24 The transferrin receptor (TfR), which appears to be expressed in all nucleated cells in the body, continuously recycles between the plasma membrane and recycling endosomes through early endosomes and thus it has been extensively studied as a canonical early endosomal recycling receptor[138, 139]. TfR also has been observed to traffic in a manner similar to the TCR complex[126, 140].  Thus the effect of U24 on TfR surface expression was detected to further test the hypothesis that U24 may alter the early endocytic recycling. The obtained data supported that U24 did mediate a downregulation of TfR. As discussed above, U24 had been already characterized as a protein that downregulates the TCR/CD3 complex from the surface of T cells (discussed in section 1.2.1.2.3) and did not colocalize with CD3ε. Therefore taken together, the downregulation of TfR by U24, as well as the inability to detect any physical or spatial interaction between U24 and TCR/CD3 complex, suggests that U24 mediates a block in early endosomal recycling[141] (Figure 1.1). Furthermore, this block in recycling seems to be specific to early endosomal recycling, as surface levels of receptors that recycle through different endosomes, MHC-I[142] and CD1d[143], did not change upon transient transfection of U24. U24 not only mediates the downregulation of the TCR/CD3 complex through causing a block in CD3 access to recycling endosomes, but also mediates the downregulation of the TfR for a block in early endosomal recycling. Furthermore, a PPxY motif (discussed in detail in section 1.2.2.2.2) near the amino terminus of U24 is identified to downregulate both the TCR complex and TfR, suggesting a general block in endosomal recycling.  	   	   23 1.2.1.3.3 Endocytic Trafficking of Myelin Proteins and Its Role in Multiple Sclerosis As discussed in the section 1.5, multiple sclerosis is an immune-mediated degeneration of the myelin sheath. The onset of myelin biogenesis requires the setup of an intricate membrane sorting and trafficking machinery, following axon recognition and positioning of myelination-competent oligodendrocytes[144]. The highly specialized myelin membrane has a unique composition of proteins and lipids, which need to be sorted and directed to the sites of myelin membrane growth. Sorting of myelin components has been indicated to occur in the Golgi-apparatus, by endocytic recycling and at the level of the plasma membrane. Newly synthesized myelin components including proteins (such as the major myelin protein PLP) and lipids, after initial lipid-raft mediated self-assembly in the Golgi-apparatus, reach the plasma membrane by non-targeted vesicular transport. The coalescence of these myelin components at the plasma membrane and the segregation of compacted versus non-compacted membranes are reinforced by MBP, which acts as a diffusion barrier. Local MBP translation is necessary for avoiding the premature clustering and compaction of membranes at inappropriate intracellular sites. In addition, myelin biogenesis requires the operation of directed vesicular trafficking (Figure 1.5). PLP is endocytosed by clathrin-independent (CI) endocytosis and recycled through late endosomes (LE). In a distinct pathway, myelin-associated glycoprotein (MAG) and myelin-oligodendrocyte glycoprotein (MOG) utilize the clathrin-dependent (CD) endocytosis and are targeted to late endosomes and recycling endosomes (RE)[145], respectively. Differential endocytic sorting of MAG, MOG, and PLP may be linked to their distinct localization to the adaxonal loop, abaxonal loop, and compact domain of myelin[146]. It is thus indicated that endocytic recycling assists membrane remodeling regulating the precise spatiotemporal targeting 	   	   24 of myelin proteins essential for polarized myelin membrane growth and subdomain formation[144].    Figure 1.5 Endocytic recycling of myelin proteins and oligodendroglial membrane remodeling (adapted from [144]). MAG (yellow) and MOG (red) are internalized by CD endocytosis, while PLP (grey) utilize a CI pathway. PLP and MAG are sorted to LE/Lys, while MOG resides in RE.   Therefore, for myelinating cells of the CNS, errors in membrane traffic probably result in dysmyelination and/or inefficient remyelination responsible for the irreversible clinical course of myelin diseases such as MS. U24 from HHV-6 is reported to mediate a general block in endosomal recycling and thus it can be assumed that U24 may block the endocytic recycling of myelin proteins resulting in degeneration of the myelin sheath, reflecting the potential role of U24 in MS.  1.2.1.4 Conservation of U24 Functions among Human Roseolaviruses Both variants of HHV-6 as well as HHV-7 encode the ability to downregulate the TCR complex. It has been further found that U24 proteins from other roseoloviruses have a similar genetic organization and a conserved function that is dependent on a proline-rich motif. While 	   	   25 the U24 protein seems to be unique to HHV-6A, HHV-6B and HHV-7, other herpesviruses have the ability to modulate immune receptor signaling.  For instance, Epstein-Barr virus (EBV) is a human herpesvirus that predominantly infects B-lymphocytes, in which it establishes latency and induces alterations in B-cell receptor (BCR) signaling. However, EBV can also infect T cells[147]. BCR and TCR signaling in EBV-infected T cells are mediated via the action of the latent membrane protein 2A (LMP2A). LMP2A consists of a large cytoplasmic amino-terminal domain, 12 hydrophobic transmembrane domains, and a small cytoplasmic carboxyl-terminal domain[148]. LMP2A localizes to membrane lipid rafts[149]. Through specific tyrosine phosphorylation motifs within its amino-terminal domain[150], LMP2A can bind the TCR pathway-associated kinases Lck, Fyn, and ZAP-70 via specific tyrosine phosphorylation motifs within its amino-terminal domain[151]. In addition, like U24, LMP2A has the PPxY motif as well. Two PPxY motifs from LMP2A allow binding to the NEDD4 family E3 ubiquitin ligases AIP4[152]. Thus, LMP2A mediates downregulation of the TCR, presumably by facilitating ubiquitin-mediated degradation of LMP2A-associated kinases. Stable expression of LMP2A in Jurkat cells leads to TCR downregulation and attenuation of TCR signaling[122].  1.2.2 Structural Features Critical to U24 Functions  The HHV-6A U24 ORF encodes a small protein with no known similarity to any cellular proteins. At the C terminus, there are a series of hydrophobic residues indicating a transmembrane region and triplets of positively charged amino acids flanking both ends of its putative transmembrane domain, which represent the basic architecture of tail-anchored (TA) proteins; besides, at the N-terminus, there is a polyproline region.  	   	   26  Figure 1.6 Protein sequences of U24 from HHV-6A (GS strain), HHV-6B (Z29 strain), and HHV-7 (RK strain) include a hydrophobic stretch of amino acids near the carboxy terminus flanked by triplets of positively charged amino acids (indicated by pluses) and a polyproline region near the amine terminus (adapted from [141] with modification).   HHV-6B encodes a highly homologous protein with 84% amino acid identity and thus is likely to behave similarly to the U24 in variant A. HHV-7 encodes a positional homologue with a predicted C terminal transmembrane region, but it has only 28% identity with HHV-6A U24 protein[100] (Figure 1.6).  1.2.2.1 U24 As a Tail-anchored Membrane Protein Tail-anchored (TA) proteins belong to a special class of membrane proteins that insert post-translationally into the membrane of the endoplasmic reticulum (ER) but also into the mitochondrial outer membrane[153]. They participate in cellular processes such as protein translocation, apoptosis, vesicular traffic or lipid biosynthesis[154, 155]. TA proteins are anchored in the lipid bilayer by a single transmembrane domain (TMD) located near their C termini. U24 protein has a hydrophobic region at the carboxy terminus preceded by an unstructured amino-terminal tail lacking a signal sequence. The TMD often functions as a targeting signal for membrane insertion. In addition, U24 encodes triplets of positively charged amino acids flanking both ends of its putative transmembrane domain like other TA proteins, which is critical to its downregulation of CD3ε[141].  	   	   27 The C-terminal location of this signal poses a unique challenge to membrane targeting of the completed protein, since hydrophobic TMDs are prone to aggregation[156]. To cope with this problem, cells have evolved diverse mechanisms for chaperoning the newly synthesized membrane protein and targeting it to its destination membrane[157]. TA proteins of the secretory pathway are initially inserted into the ER membrane and from there sorted to their ultimate destination. Membrane insertion of TA proteins in vitro requires ATP or GTP[158] but in some cases can proceed even in the absence of nucleotides[158, 159]. Nucleotide dependence could reflect the involvement of molecular chaperones, such as members of the Hsp70 family and SRP[158, 160]. A more recent pathway for the membrane insertion of TA proteins was discovered in mammalian cells, which involves the ATPase Asna1, also called transmembrane domain recognition complex (TRC) 40[161, 162]. Asna1/TRC40 is the mammalian ortholog of Arr4/Get3 in yeast.  The presence of a dominant negative version (DN) of Asna1/TRC40 decreased the ability of U24 to downregulate both the TCR complex and TfR from the cell surface[141]. Therefore, Asna1/TRC40 was found to be essential to the function of U24. The reported data also suggested that there was no inherent defect in early endosomal trafficking and surface receptor expression in Asna1/TRC40 DN-expressing cells, indicating that Asna1/TRC40 may not affect other TA proteins in endosomal pathways but may affect U24 membrane insertion directly to inhibit the functions of U24[141].  TA proteins have been increasingly identified as essential in many basic cellular processes including protein trafficking and cell survival[163]. The ability of U24 to disrupt endocytic recycling is consistent with the role of other TA proteins, such as the syntaxins[163], to facilitate vesicular traffic and fusion. While the molecular mechanism of U24 function has not yet been 	   	   28 identified, the putative cytoplasmic domain of U24 may be acting to recruit factors that disrupt membrane fusion[141].     1.2.2.2 Polyproline Region of U24 At the N-terminus of U24 there is a proline-rich region (PPxY motif), which was demonstrated to be essential for an U24-mediated block of endosomal recycling (Figure 1.1).  1.2.2.2.1 Polyproline Type II Helix  Proteins and peptides rich in repeating proline residues typically adopt a polyproline II helix (PPII) secondary structure. The PPII helix (Figure 1.7) is a left-handed helix with an axial translation of 3.20Å composed of 3.0 residues per turn, and its all peptide bonds are in trans configuration with (φ, ψ, ω)=(-75°, +145°, 180°). The PPII helix is relatively open and has no internal hydrogen binding, as opposed to the more common helical secondary structures. In contrast, the PPI helix is a right-handed helix with an axial translation of 1.90Å composed of 3.3 residues per turn, and its all peptide bonds are in cis configuration with (φ, ψ, ω)=(-75°, +160°, 0°). The PPII helix is characterized by the circular dichroism (CD) spectrum with a strong negative band at 202-206nm and a weak positive band at 225-229nm. Proline-rich peptides, which bind WW domains, usually adopt PPII helical conformation (see section 3.2 below).   	   	   29                                                                     Figure 1.7 Side view and Top view of (A) PPI and (B) PPII.  1.2.2.2.2 PPxY Motif As discussed above, U24 can downregulate both CD3 and TfR[100, 141], through a common mechanism closely related to its PPxY motif[141]. The PPxY motif was thus suggested to be essential for a U24-mediated block of endosomal recycling[141]. Additionally, the sequence and function of the PPxY motif are highly conserved in U24-6A, U24-6B and U24-7.  Two main domain classes known to bind to polyproline regions are SH3 domains and WW domains[164]. SH3 domains primarily bind a PxxP consensus sequence (where x is any amino acid) while type 1 WW domains bind a PPxY consensus sequence. PPxY motifs recruit WW domain-containing proteins such as E3 ubiquitin ligases for a diverse array of cellular processes[165]. Posttranslational addition of ubiquitin to proteins normally signals a protein for transport to lysosomes, where the ubiquitinated protein is then degraded. There were experiments demonstrating that U24 can be coimmuneprecipitated with overexpressed WW domain-containing proteins and that this association is dependent on a complete PPxY motif[141]. This suggested that U24 function is dependent on a WW domain-containing protein; however, its binding partners have yet to be identified. (A) (B) 	   	   30 As mentioned above, LMP2A from EBV is involved in mediating TCR signaling. LMP2A also has two PPPY motifs allowing it to bind to the Nedd4 family E3 ubiquitin ligase AIP4[152]. Thus, LMP2A mediates downregulation of the TCR through its PPPY motifs, presumably by facilitating ubiquitin-mediated degradation of LMP2A-associated kinases. Nedd4 family E3 ubiquitin ligases are found throughout eukaryotes and regulate diverse biological processes through the targeted degradation of proteins that generally have a PPxY motif for WW domain recognition, and are found in the nucleus and at the plasma membrane[166]. The members of Nedd4 family E3 ubiquitin ligases include rNedd4, hNedd4, hSmurf2, hSmurf1 and so on. They all have multiple WW domains. In particular, hSmurf2 can act in the endosomal recycling[167] (discussed in detail in section 1.3.1.2.2) and thus it has potential role in mediating U24 function through the interaction between PPxY motif from U24 and its WW domains (Figure 1.1). The structure and function of Nedd4 family E3 ubiquitin ligases, especially Smurf2, will be discussed in detail in the following sections.   1.3 Nedd4 Family of Ubiquitin Protein Ligases 1.3.1 Introduction to Nedd4 Family of Ubiquitin Protein Ligases Nedd4 (neuronal precursor cell expressed developmentally downregulated 4)[168] is a member of the HECT domain superfamily of E3 ligases. Nedd4 and Nedd4-like proteins have been identified in budding and fission yeast, Drosophila, mouse, rat and human[169], and are involved in ubiquitination and degradation of many substrates. These proteins share a similar architecture, composed of an N-terminal C2 domain, multiple WW domains, an E2 binding region and the HECT domain at the C-terminus (Figure 1.8).  	   	   31  Figure 1.8 The architecture of some Nedd4 family of E3 ubiquitin-protein ligases (adapted from [170] with modification).  1.3.1.1 Structure and Architecture 1.3.1.1.1 C2 Domains In Ca2+-responsive isoforms of protein kinase C (PKC), C2 domains were first identified[171]. This 120 residue domain consists of a β-sandwich formed from two four-stranded β-sheets[172]. Two Ca2+ ions are coordinated by conserved aspartate residues (or other oxygen-containing residues such as asparagine and glutamine) located in loops[173]. The majority of C2 domains, including the C2 domain of Smurf2, binds to phospholipids and mediates intracellular targeting to the plasma membrane[174], endosomes and multivesicular bodies (MVBs). It can also bind to specific proteins, such as annexin A13b, the growth factor receptor-bound (Grb) Grb7–Grb10–Grb14 family of adaptors[175] and, in some cases (for example, Smad ubiquitylation regulatory factor 2 (Smurf2)), the HECT domain of the same Nedd4 family protein[176].  1.3.1.1.2 WW Domains WW domains, discussed in much greater detail below, are small protein-protein interaction domains of approximately 40 residues that derive their name from two highly conserved 	   	   32 tryptophan residues, usually spaced 22-23 amino-acids apart, and an invariant proline[177, 178]. WW domains mediate associations primarily by binding proline-rich sequences. The first target for WW domains identified was the PY motif, which has the consensus sequence XPPXY[179]. The WW domains of Smurf2 are able to bind a number of proteins that contain this core motif, leading to ubiquitination and degradation of these interacting molecules.  1.3.1.1.3 HECT Domains The HECT is located at the C-terminus of all proteins that harbor it, including the Nedd4 family of E3 ligase. HECT is a 350-residue domain in which the conserved Cys residue that forms thioester complexes with ubiquitin is towards the C terminus (the C lobe) of the protein, whereas the N-terminal (the N lobe) region binds the E2 enzyme[180]. Regions outside of the HECT domain determine substrate binding. The structure of the HECT domain of Smurf2 indicates a more open architecture[180] compared with other Nedd4 family members.  The HECT domain has four known biological functions: direct binding to E2 enzymes, formation of thioester with ubiquitin via an active site cysteine residue, transfer of ubiquitin to the ε-amino group of lysine residues on substrate protein and transfer of additional ubiquitin moieties to the growing polyubiquitin chain[181]. 	  1.3.1.2 Functions: Ubiquitination, Endocytosis and Lysosomal Degradation of    Membrane Proteins 1.3.1.2.1 Nedd4 Family Regulation of Transmembrane Proteins Ubiquitin-mediated processes are involved not only in the targeting of proteins for degradation by the 26S proteasome but also in the sorting of proteins at different steps in the 	   	   33 biosynthetic and endocytic pathways[182, 183]. Proteins at the plasma membrane are tagged with monoubiquitin to direct internalization. Within vesicles, the ubiquitinated proteins are then transported to the late endosome and further sorted, in the multi-vesicular body, for recycling to the plasma membrane or destruction at the lysosome. The Nedd4 proteins participate in these processes through ubiquitination of target proteins and several Nedd4 proteins from eukaryotes seem to have developed both redundant and specialized functions (Figure 1.9).       Figure 1.9 The NEDD4 family of proteins regulates diverse cellular processes (adapted from [165]).  For example, the latent membrane protein 2A (LMP2A) from EBV is able to regulate the transmembrane proteins. The Epstein-Barr virus (EBV) is the causative agent of infectious mononucleosis and is associated with a number of cancers. EBV infection can persist throughout life and the viral protein LMP2A is required to maintain this latency[184]. LMP2A is predicted to span the membrane 12 times, leading to both the N and C-terminal tails being located in the cytoplasm. At the N-terminus, there are two PPPY motifs that have been shown to recruit the Nedd4 family proteins Nedd4-1, Nedd4-2, AIP4, and WWP2[152, 185].  	   	   34 In addition to mediating TCR signaling mentioned in section 2.1.4, it has been suggested that LMP2A has a dual function in maintaining B-cell survival while blocking B-cell receptor (BCR) signaling. LMP2A and the LMP2A-associated kinase Lyn and Syk can be ubiquitinated by Nedd4 proteins as well[186, 187]. These functions depend on the PPPY motifs of LMP2A and can be blocked by dominant negative AIP4 and WWP2 mutants where the catalytic cysteine has been mutated[152], which suggests that LMP2A may prevent viral reactivation to maintain viral latency not only by sequestering kinase away from the BCR but by degrading it as well.  Overall, it is shown that one specific protein from Nedd4 family can participate in different cellular processes and several different proteins from Nedd4 family can participate in one cellular process as well (Figure 1.9). For example, Nedd4-1 is able to modulate epithelial sodium channel (ENaC) stability, interfere with BCR signaling and facilitate viral budding and Nedd4-2 and WWP2, besides Nedd4-1, all have the ability to modulate ENaC stability. Therefore, identifying the role of Nedd4 proteins in one specific cellular process is a significant but laborious task.  1.3.1.2.2 Endocytic Regulation of the TGF-β Signaling by Smurf Smad ubiquitin regulatory factor (Smurf) 1 and 2 are Nedd4 family members that carry out distinct functions in the regulation of signaling pathways triggered by the transforming growth factor-β (TGF-β) superfamily. These pathways control cellular responses leading to growth, differentiation, morphogenesis and apoptosis[188, 189]. The TGF-β receptor family of Ser/Thr kinases comprises type I (TGFRI) and type II (TGFRII) receptors[190], which are localized to the membrane and play an essential role in the TGF-β signaling. Upon ligand binding at the cell surface, TGFRII phosphorylates and activates TGFRI, leading to signal transduction through the 	   	   35 Smad proteins. Receptor (R-) Smads (Smad2 and Smad3 for TGF-β signaling and Smad1, Smad5 and Smad8 for bone morphogenetic protein (BMP) signaling) then become phosphorylated and associate with common mediator (Co-) Smad, Smad4, which leads to nuclear translocation to regulate the transcription of target genes. Inhibitory (I-) Smads (Smad6 and Smad7) can then disrupt the pathway through interaction with the activated receptor[170, 191, 192]. Most Smads can bind to the WW domains of the Smurfs through their proline-tyrosine (PY) motifs. The Smurfs inhibit TGF-β receptor family signaling by targeting Smads, the receptors themselves or TGF-β responsive transcription factors for ubiquitylation-mediated degradation. Smurf1 and Smurf2 not only have distinct modes of regulation, but they also have distinct substrate specificities despite high sequence homology. Unlike Smurf1, Smurf2 is localized to the nucleus. Smurf2 is able to regulate the activity of Smad2 and Smad3 via different mechanisms. Smurf2 regulates the steady state level of Smad2 through polyubiquitination and proteasomal degradation[193], but induces multiple mono-ubiquitination on the MH2 domain of Smad3[194]. The multi-ubiquitinated Smad3 has a lower binding affinity for its partner Smad4, resulting in its being unable to form transcriptionally active Smad3–Smad4 complex. Therefore, Smurf2 regulates Smad3 not by ubiquitin-dependent degradation but by preventing the formation of Smad3-containing protein complexes[167] (Figure 1.10(A)).   	   	   36     Figure 1.10 The functions of Smurf2. (A) Smurf2 regulation of the TGF-β signaling (adapted from [167] with modification); (B) Endocytosis/internalization of TGF-β receptor (TGFR) after addition of ubiquitin moieties (adapted from [195] with modification).  In addition, TGF receptors are localized to the membrane and thus receptor-mediated endocytosis has important functions in signal transduction. Smad7 recruits Smurf2 to the TGFRI kinase to facilitate the proteasomal and lysosomal degradation of TGFRI. Upon ligand-bound (A) (B) 	   	   37 activation of the TGF-β receptor, the negative regulator Smad7 is synthesized and accumulates in the nucleus where it interacts with Smurf2[196-198]. This Smurf2-Smad7 complex then translocates to the cytoplasm enabling interaction with the TGF-β receptors, thereby targeting them to lipid rafts. Lipid rafts-mediated endocytosis of TGFR enables downregulation of the signaling pathway[175]. In this instance, Smad7 facilitates both a change in subcellular localization and recruitment to the substrate of Smurf2. An overview of the internalization of the TGF receptor and interaction with Smurf2 is shown (Figure 1.10(B)). Smurf2 is constitutively associated with Smad7 and this interaction requires the second and third WW domains of Smurf2 and the PY motif of Smad7[166]. Consistent with the idea, a Smad7 mutant with a disruption in the PY motif does not interact with Smurf2 and is compromised in its ability to inhibit TGF-β signaling. Therefore, Smurf2 is proposed to have the potential to bind to the PPxY motif of U24 protein to function in the endosomal pathway. The structure of Smurf2 WW domains critical for binding to PPxY motif will be discussed in the following section.  1.3.2 Structure, Identification and Ligand Binding of WW Domains WW domains, as mentioned previously, are small modular binding domains that were named for their two highly conserved tryptophans and an invariant proline[199]. WW domains were first identified by three separate groups on the basis of sequence similarity between repeated sequences in Rsp5p and Nedd4 regions found in dystrophin and the nematode protein Yo61[177, 178, 200]. Since then, WW domains have been identified in a variety of proteins, in single or multiple copies, including enzymes, adaptor molecules, cytoskeletal proteins and signaling molecules, often alongside other types of domains. Therefore, these domains are part of a growing list of protein-protein interaction domains. 	   	   38 WW domains recognize various polyproline motifs and can be divided into four groups based on their ligand binding specificity. Group I binds to PY motifs with the sequence PPxY[179, 201]. Group II binds to ligands with a PPLP motif, usually within the context of multiple Pro residues[202]. Groups III and IV recognize polyproline motifs flanked by Arg or Lys[203] and phospho-(Ser/Thr)-Pro sequences[204], respectively. The WW domains from Nedd4 family of E3 ubiquitin ligases belong to group I. Several structures of these group I WW domain-PY motif complexes have been solved. These structures clearly delineate the molecular determinants involved in recognition of the PPxY sequence. However, the prevalence of group I WW domain-type interactions in cellular protein-protein interactions makes it difficult to understand how specificity is maintained.  WW domain structure consists of a compact three-stranded antiparallel β-sheet, such that the N- and C-termini of the sheet are close in space, typical of modular binding domains. WW domains are named for two conserved Trp residues. The first conserved Trp is on one side of the sheet in a hydrophobic cluster that is important for domain stability, with mutation of this residue destabilizing the structure[205, 206]. The second Trp is found on the opposite face of the sheet in a hydrophobic pocket and is integrally involved in binding to ligands with polyproline sequences[206, 207]. Mutation of either the binding site Trp or ligand Pro residues can disrupt ligand binding, demonstrating the importance of the Trp in the recognition of target ligands[179, 208]. Additional conserved residues in the WW domain are partitioned in the structure between the two sides of sheet and participate either in the packing interaction in the hydrophobic cluster or form the binding site. Taken together, WW domains adopt a similar structure, with structurally equivalent residues in all WW domains involved in packing of the hydrophobic cluster.   	   	   39 However, neither the WW2 nor the WW3 of Smurf2 is canonical[209]. Smurf2 WW2 has a tyrosine at the site normally occupied by the conserved core tryptophan, which likely destabilizes these domains. Smurf2 WW3 has the highly conserved binding site tryptophan, which interacts with prolines of target PY motifs (Figure 1.11).                                Figure 1.11 Alignment of a selection of WW domains. Smurf2 sequences are shown in bold and two conserved Trp residues are highlighted in yellow.  NMR studies performed on Smurf2 WW3 domain in complex with Smad7 PY motif provided some information for Smurf2 WW3-PY motif binding[210]. The interaction between the Smurf2 WW3 domain and the Smad7 PY motif is the first example of PY motif recognition by a WW domain with a Phe substitution at the usual binding site. In the Smurf2 WW3 domain-Smad7 peptide complex (Figure 1.12), the conserved core Trp-303 and the conserved Pro-328 form part of the hydrophobic cluster together with Phe-315 and Gln-324 as seen in other WW domain structures. Tyr-314 on the β2-strand and Thr-323 and Phe-325 on the β3-strand form the XP groove, binding the first and second Pro residues of the PY motif, which are in a PPII helical conformation. The Tyr is bound by a large hydrophobic residue (Val-316) on the β2-strand and a neutral His-318 and the aliphatic portion of an Arg-321 on the β2-β3 loop. In general, the XP grooves and Tyr binding pockets of group I WW domains are very similar and are not sufficient for discrimination among group I ligands. In addition, the Phe decreases the hsmurf2 WW2 hsmurf2 WW3 rNedd4  WW2 rNedd4  WW4 hNedd4  WW3 hNedd4  WW4 hNedd4l WW3 hNedd4l WW4   DLPEGYEQRTTQQGQVYFLHTQTGVSTWHDPRV PLPPGWEIRNTATGRVYFVDHNNRTTQFTDPRL GLPPGWEEKQDDRGRSYYVDHNSKTTTWSKPTM  PLPPGWEERTHTDGRVFFINHNIKKTQWEDPRM FLPKGWEVRHAPNGRPFFIDHNTKTTTWEDPRL PLPPGWEERTHTDGRIFYINHNIKRTQWEDPRL FLPPGWEMRIAPNGRPFFIDHNTKTTTWEDPRL PLPPGWEERIHLDGRTFYIDHNSKITQWEDPRL      	   	   40 affinity of the WW3 domain for the PY motif, requiring additional interactions that are provided by the PY-tail, which adopts a loop conformation and binds the β1-strand and the β1-β2 loop of the WW3 domain[210]. Intrapeptide interactions stabilize the loop structure and are essential for high affinity binding. These unique features likely contribute to the specific recognition of Smad7 by Smurf2. Residues within the PY motif and the PY motif binding pockets are well conserved, therefore, regions flanking the PY motif, which exhibit more sequence variability than the PY motif binding pocket and may provide specificity required for differentiating group I ligands.    Figure 1.12 Ribbon diagram of the lowest energy structure of the Smurf2 WW3 domain-Smad7 PY peptide complex (adapted from [210]). The WW3 domain backbone is shown in blue, loop regions in gray and side chains in turquoise, except for positions typically occupied by conserved Trp residues, which are in green. The PY peptide backbone is red with side chains shown in gold. Residues designated with a prime symbol (‘) are Smad7 residues. 	   In most cases, the PY motif portion of the peptide was modeled as a polyproline type II helix in the ligand-binding site of the WW domains, a conformation adopted by PxxP motif-containing ligands bound to SRC homology 3 (SH3) domains[211]. The peptide binding site 	  	   	   41 of SH3 domains, like that of WW domains, also consists of a flat surface composed of aromatic residues, suggesting that a polyproline type II helix is a reasonable model for the conformation of a PY motif-containing peptide in the WW domain binding site.   1.4 Aim of Thesis So far, it has been demonstrated that U24 protein from HHV-6A can downregulate both CD3 and TfR, and thus U24 acts to block early endosomal recycling. Furthermore, a PPxY motif at the N-terminus of U24 has been identified as essential for those functions of U24. Although U24 functions were suggested to be dependent on a WW domain-containing protein, the cognate ligand has not yet been identified. Recognition of the cognate ligand for the U24-PY motif, as well as obtaining the structural and biochemical data on the WW domain-PY motif complex, is thus critical for understanding the function and mechanism of U24 and its effects on potential diseases, in particular in the context of MS, as outlined above. In addition, Smurf2 is a common E3 ubiquitin protease ligase, involved in ubiquitination and endocytosis of transmembrane proteins. Therefore, Smurf2 WW domains play a possible role in regulating U24 function through WW domain-PY motif interactions (Figure 1.1).  Through the thesis project, two specific aims were addressed:     (1) To investigate the interaction between Smurf2 WW3 domain and PPxY motif of U24 from HHV-6A based on structural data by using GST pull-down assay, NMR spectroscopy and circular dichroism (CD) spectroscopy. The possible role of Smurf2 WW3 domain as the binding domain will be discussed (in Chapter 2);     (2) To investigate the interaction between Smurf2 WW2 and WW3 domains in tandem and PPxY motif of U24 from a structural perspective and to compare with the interaction between 	   	   42 Smurf2 WW3 domain and PPxY motif of U24. Methods including NMR spectroscopy and CD spectroscopy are used (in Chapter 3).                              	   	   43 2.1 Introduction As discussed in Chapter 1, U24 protein from HHV-6A can downregulate both CD3 and TfR, and thus acts to block early endosomal recycling. The PPxY motif, at the N-terminus of U24, has been identified as being essential for those functions of U24. Nedd4 family of ubiquitin protein ligases shares a common architecture comprising an N-terminal C2 domain required for proper localization, multiple WW domains involved in target recognition, and a C-terminal HECT domain, which catalyzes the transfer of ubiquitin onto target proteins. In addition, Nedd4 family of ubiquitin protein ligases can ubiquitinate specific proteins, targeting them for degradation or sorting in the endosomal pathway. Those proteins may therefore play a possible role in regulating the function of U24 through interaction of their WW domains with the PPxY motif in U24.  Smurf2 is a member of Nedd4 family of ubiquitin protein ligases that has been demonstrated to drive degradation of the transforming growth factor-β (TGF-β) receptors and other targets. For example, Smurf2 has been implicated in ubiquitination of Smad1 and Smad2. Smad7, which binds to the TGF-β receptors and Smurf2, acts as an adaptor allowing Smurf2 to bind to and ubiquitinate the TGF-β receptors, targeting them for degradation. Smurf2 WW3 domain in complex with Smad7 has been studied[210], expanding the understanding of the molecular determinants involved in this regulation. U24 and Smurf2 functions may be linked, because both of them act in the endosomal pathway. To examine target binding by the Smurf2 protein, we studied the recognition of U24 by Chapter 2 Probing the Binding of Smurf2 WW3 Domain to U24 Protein from HHV-6A  	   	   44 the Smurf2 WW domains. NMR spectroscopy was used since this method is most suitable for investigating potential molecular interactions. Titration experiments yield accurate values of the interaction constant and also allow a mapping of the residues implicated in the interaction. Finally, each experiments result in a structural model of the interaction complex. Our results on the interaction between the WW3 domain of Smurf2 and U24 from HHV-6A will be presented in this chapter. And our results will be compared to those obtained for the Smad7 ligands.  2.2 Materials and Methods 2.2.1 Smurf2 WW3 Expression The plasmid expressing the GST-Smurf2 WW3 protein was a kind gift from Dr. Julie D. Forman-Kay (Hospital for Sick Children, Toronto). The vector pGET 6P1 with DNA encoding the Smurf2 WW3 (amino acids 297-333) domain was cloned into the BamH1 and XhoI restriction sites. Glutathione S-transferase fusions were expressed in Escherichia coli BL21(DE3) cells at 25°C with a 16-h isopropyl 1-thio-β-D-galactopyranoside (IPTG) induction period. WW3 was labeled with 15N by expressing the protein in M9 minimal medium containing 15NH4Cl as the sole nitrogen source. Cells were lysed by sonication in 10 mL binding buffer (PBS with 1% Triton X-100, pH 7.3), supplemented with lysozyme, EDTA-free protease inhibitor cocktail (Sigma, USA) and DNase I (Boehringer, CA). The resulting cell lysate was centrifuged for 1 hour and the supernatant was syringe-filtered through a 0.45 µM membrane filter. Approximately 1 mL of Glutathione-Sepharose 4B beads (GE healthcare, USA) were placed in a 15 mL falcon tube and equilibrated by adding 5 mL binding buffer. The beads were pelleted by centrifugation at 500 ×g for 5 minutes, the buffer removed and the procedure was repeated twice more. The cell lysate 	   	   45 was added into the equilibrated beads and incubated for at least 30 minutes with end-over-end rotation at ambient temperature. After this period, the beads were sedimented by centrifugation and the supernatant was carefully decanted. The beads were washed by addition of 5 mL binding buffer. The washing process was then repeated twice for a total of three washes. Afterwards, Triton X-100 was removed by three more successive washing steps using Human Rhinovirus 3C (HRV-3C) protease reaction buffer. After the addition of 2 mM dithiothreitol, WW3 domain was cleaved from glutathione S-transferase (GST) by using Human Rhinovirus 3C protease (Thermo Scientific, USA) with end-over-end rotation at 4°C for 16 hours. The suspension was centrifuged to pellet the beads and the supernatant was carefully collected. After the HRV 3C protease reaction buffer was added, the beads were suspended and immediately centrifuged. The supernatant was collected and the process was repeated twice. A total volume of 6 mL supernatant containing purified WW3 domain was finally collected. The still-bound GST protein was removed from the beads with elution buffer, which consisted of 50 mM Tris, 10 mM reduced glutathione, 1% Triton X-100, pH 8.0. The purified WW3 domain was dialyzed against a solution of 40 mM HEPES, 10 mM NaCl, pH 7.2, using a dialysis membrane with MWCO of 2000 (Spectrum Laboratories, USA).       2.2.2 Expression and Isolation of U24 from HHV-6A The full-length gene of U24 from HHV-6A was fused to a hexahistidine tag and a thrombin cleavage site and then cloned into pMAL-c2x using BamHI-HindIII sites[212]. The construct was expressed in Origami 2 (Novagen, USA) Escherichia coli at 18°C in LB and induced using 0.3 mM IPTG. The cells continued to grow for 16 hours, after which they were harvested by centrifugation.  	   	   46 The cells were lysed by sonication in 30 mL of lysis buffer (20 mM KH2PO4, 0.5 M NaCl, 1% Triton X-100, 10 mM imidazole, pH 7.4), supplemented with lysozyme, EDTA-free protease inhibitor cocktail and DNase I. the lysate was stirred for 3h at 4°C and then centrifuged at 25000g for 1 hour. The supernatant was filtered through a 0.45 µM membrane filter and loaded onto a column containing 5 mL of Ni-NTA agarose (Qiagen, USA), equilibrated with lysis buffer. The column was washed with 100 mL of lysis buffer. The imidazole concentration of the lysis buffer was then raised to 25 mM, and the column was further washed with another 25 mL. The construct was eluted using 25 mL of elution buffer (20 mM KH2PO4, 0.5 M NaCl, 1% Triton X-100, 500 mM imidazole, pH 7.4). The elution fraction was dialyzed against a solution of 20 mM KH2PO4, 62.5 mM NaCl, 0.5% Triton X-100, pH 7.4, using a dialysis membrane with MWCO of 2000. The dialyzed fraction was transferred to a falcon tube and bovine thrombin (GE Healthcare, USA) was added. The digestion mixture was inverted end over end at ambient temperature for 16 hours. Afterward, the solution was filtered through a 0.45 µM membrane filter and re-loaded on the column containing 5 mL of Ni-NTA agarose equilibrated with the dialysis buffer. The cleaved U24 from HHV-6A was collected in the flow-through fraction. To remove the thrombin and any remaining trace contaminants, the solution containing U24 was loaded on a Q-Sepharose column (GE Healthcare, USA) equilibrated with dialysis buffer, and purified U24 was collected in the flow-through fraction. Purity was assessed by MALDI-TOF mass spectrometry and SDS-PAGE. The purified U24 from HHV-6A was dialyzed against pull-down buffer (20mM KH2PO4, 75 mM NaCl, 0.5% Triton X-100, pH 7.4) at 4°C.  	   	   47 2.2.3 Preparation of a 15-Residue Peptide Representing the Polyproline-Containing N-Terminus of U24 from HHV-6A  A synthetic 15-mer peptide representing the N-terminus of U24 from HHV-6A (MDPPRTPPPSYSEVL) was obtained using standard solid-phase peptide synthesis protocols, employing Fmoc protected amino acids[213]. Double coupling was required for the last nine residues (MDPPRTPPP) of the peptide representing the N-terminal sequence of U24 from HHV-6A.  The synthetic peptide was purified by preparative gradient RP-HPLC on a Waters 600 system with a Waters 2996 photodiode array detector with 229 nm and 278 nm UV detection using a Phenomenex C18 column (10 µm, 2.12 cm × 25 cm) at a flow rate of 10 mL/min, with an acetonitrile gradient of 0 to 35 % buffer B (10% deionized water and 90% acetonitrile containing 0.1% trifluoroacetic acid) in buffer A (90% deionized water and 10% acetonitrile containing 0.1% trifluoroacetic acid). The solution containing purified peptide was lyophilized, and the final mass of the purified product was confirmed by MALDI-TOF mass spectrometry.  2.2.4 GST Pull-Down Assays DNA encoding the Smurf2 WW2 (amino acids 250-288), WW3 (amino acids 297-333), and Smurf2 WW2 and WW3 domains in tandem (Smurf2 WW23, amino acids 250-333) were included in pGEX 6P1 vector. Glutathione S-transferase fusions were expressed in Escherichia coli BL21(DE3) cells at 25°C with a 16-h isopropyl 1-thio-β-D-galactopyranoside (IPTG) induction period. To initiate purification of the GST-fusion protein, the cells were lysed by sonication in 10 mL binding buffer (PBS with 1% Triton X-100, pH 7.3), supplemented with lysozyme, EDTA-free protease inhibitor cocktail and DNase I. The resulting cell lysate was 	   	   48 centrifuged for 1 hour and the supernatant was syringe-filtered through a 0.45 µM membrane filter. Approximately 1 mL of Glutathione-Sepharose 4B beads was placed in a 15 mL falcon tube and equilibrated by adding 5 mL binding buffer. The beads were pelleted by centrifugation at 500 ×g for 5 minutes, the buffer removed and the procedure was repeated twice more. The cell lysate was added into the equilibrated beads and incubated for at least 30 minutes with end-over-end rotation at ambient temperature. The beads were sedimented by centrifugation, and the supernatant was carefully decanted. The beads were washed by addition of 5 mL binding buffer. The washing process was then repeated twice for a total of three washes. Afterwards, elution buffer (50 mM Tris, 10 mM reduced glutathione, 1% Triton X-100, pH 8.0) was added and the resulting slurry was rotated end over end overnight at ambient temperature. The beads were sedimented by centrifugation and the supernatant was collected and dialyzed against the pull-down buffer (20 mM KH2PO4, 75 mM NaCl, 0.5% Triton X-100, pH 7.4) at 4°C. In separate microcentrifuge tubes, 20 µL of Glutathione-Sepharose 4B beads were added and equilibrated by adding 1 mL of pull-down buffer (20 mM KH2PO4, 75 mM NaCl, 0.5% Triton X-100, pH 7.4) via centrifugation at 2000 rpm in a microcentrifuge (Fisher Scientific, USA) for 5 minutes at 4°C. The steps were repeated twice for a total of three times. The concentrations of GST, GST-tagged WW domains and U24 from HHV-6A were determined by BCA assay. The 500-µL stock solutions of GST and GST-tagged WW domains were mixed with the equilibrated beads. After incubation for 1 hour, GST and GST-tagged WW domains were immobilized on the beads. The beads were then pelleted by centrifugation and washed three times with 0.5 mL of fresh pull-down buffer and intermittent centrifugation. Afterwards, the coated beads were incubated with 500-µL stock solution of U24 and the slurry was mixed again with end-over-end rotation for 30 min at 4°C. The beads were sedimented and the supernatant 	   	   49 was removed. The beads were subsequently washed with three successive rounds of 0.5 mL of Glasgow Lysis Buffer (GLB), which consists of 10 mM PIPES-NaOH, 120 mM KCl, 30 mM NaCl, 5 mM MgCl2, 1% Triton X-100, and 10 % glycerol. Then the coated-beads were mixed with 20 µL of 2× Novex dye (Invitrogen, USA) supplemented with 5% β-mercaptoethanol and heated for 5 min at 95°C. Bound proteins were resolved on denaturing sodium dodecyl sulfate-polycrylamide gel electrophoresis (SDS-PAGE) in a Tris-Tricine buffer system, and then the gel was silver-stained for analysis.   2.2.5 1H-15N HSQC NMR Titrations and Kd Calculations for the Smurf2 WW3 Domain and U24 Peptide Interaction All spectra were measured on Bruker NMR spectrometers operating at proton resonance frequencies of either 600 MHz or 850 MHz and equipped with cryogenic probes. Experiments were performed in buffer containing 40 mM HEPES, pH 7.2, 10 mM NaCl, 0.05% NaN3, 10% D2O, unless otherwise stated. The final concentration of [15N]Smurf2 WW3 domain was calculated from the method of Edelhoch [214] with the extinction coefficients (ε280) of Trp and Tyr determined by the protparam algorithm (http://www.expasy.ch). Binding during the titration series was monitored with 1H-15N HSQC experiments, with a spectral width of 16 ppm in the 1H dimension and 29 ppm in the 15N dimension. Titrations were carried out in eleven consecutive steps, each one an addition of the U24 peptide from HHV-6A to result in the following protein/peptide molar ratio: 1:0, 1:0.18, 1:0.55, 1:1.22, 1:1.40, 1:1.83, 1:2.71, 1:3.77, 1:4.65, 1:5.70, and 1:6.58. Combined chemical shift perturbations (Δδ [HN,N]) were calculated using the equation:                Δδ [HN,N] = ([ΔδHNWHN]2 + [ΔδNWN]2)1/2                    (2.1) 	   	   50 where WHN and WN are weighting factors for the HN and N shifts, respectively (WHN=1; WN=0.154[215]), and ΔδHN/N = δbound – δfree. Subsequently, the effective dissociation constant Kd, was determined independently for each residue by fitting the titration data, and the concentrations of WW3 domain and U24 peptide using the equation[216]:      Δδobs = Δδmax{([P]t + [L]t + Kd) – [([P]t + [L]t + Kd)2 - 4[P]t[L]t]1/2}/2[P]t      (2.2) where Δδobs is the change in the combined chemical shift from the free state of Smurf2 WW3 domain, Δδmax is the maximum combined chemical shift change on saturation, [P]t is the concentration of Smurf2 WW3 domain and [L]t is the concentration of U24 peptide.  Smurf2 WW3 domain in complex with U24 peptide was assigned using three-dimensional (3D) gradient-enhanced [1H-15N]-NOESY-HSQC (τm=150 ms) spectra and [1H-15N]-TOCSY-HSQC (τm=60 ms) spectra.    2.2.6 Circular Dichroism Far-UV CD measurements were conducted on a Jasco J-815 spectropolarimeter equipped with a circulating temperature-controlled water bath. Experiments were conducted on a 200-µL sample of WW3 domain alone, U24 peptide alone or in the context of the complex with WW3/U24 peptide ratio (1:5.4) in a solution of 10 mM HEPES, 2.5 mM NaCl, pH 7.2. Data were collected using a quartz cuvette with a 1-mm pathlength in the 200-250 nm wavelength range. Starting at 5°C, the temperature was incremented to 65°C in steps of 5°C. Data were recorded at a rate of 100 nm/min in 0.1-nm intervals. Each data set represents an average of four scans. The raw data were corrected by removing the contribution from the buffer and were converted to mean residue ellipticity [θ] as a function of wavelength of electromagnetic radiation using the equation:  	   	   51                 [θ] = [(106 Δε)/ncl] deg · cm2 /dmol                     (2.3) where Δε is the observed ellipticity in millidegrees, c is the concentration in micromolar, n is the number of residues and l is the cuvette pathlength in millimeters.  2.3 Results 2.3.1 Smurf2 Interacts with U24 via its WW3 Domain Smurf2 protein is comprised of three WW domains. One or more among these WW domains might be responsible for the interaction with U24. Of note, WW1 has been identified to be not required for Smurf2 to bind PY motifs. To determine which WW domain binds the U24 protein, GST pull-down assays were performed with GST-tagged WW2, GST-tagged WW3 and GST-tagged WW23, incubated with full-length U24. Background interaction was measured in control experiments performed with GST alone.                                            Figure 2.1 GST pull-down analysis for interaction between U24 and Smurf2 WW domains. The resolved bands were detected by silver stain. Full-length U24 protein was loaded as a reference in lane 1. U24 appears to bind Smurf2 WW3 and Smurf2 WW23 domains via its PPxY motif, but there is no interaction between U24 and Smurf2 WW2 domain. GST alone does not interact with U24 in the control experiment.   1     2    3    4    5    6    7    8 	  GST-WW23 GST-WW2/WW3 GST U24 U24 GST+U24 GST-Smurf2 WW2 GST-Smurf2 WW2+U24 GST-Smurf2 WW3+U24 GST-Smurf2 WW3 GST-Smurf2 WW23 GST-Smurf2 WW23+U24 	   	   52 As shown in Figure 2.1, full-length U24 from HHV-6A is able to bind to GST-tagged WW3 domain (lane 6) and GST-tagged WW23 domains (lane 8), in contrast to GST alone (lane 2). These results indicate that U24 could bind to Smurf2 WW3 domain and Smurf2 WW23 domains. The interaction between GST-tagged WW2 domain and U24, however, is not observed (lane 4). Taken together, the assay demonstrates that U24 binds to WW3 alone and WW23, a construct containing both the WW2 and WW3 domains of Smurf2 in tandem, and does so via its PPxY motif.  2.3.2  Chemical Shifts of Smurf2 WW3 Domain Perturbed by PPxY Motif from U24 at 5°C and 25°C  The pull-down results show that U24 protein from HHV-6 binds preferentially to Smurf2 WW3 domain via its PPxY motif, which is located at the N-terminal region of U24. Thus, a 15-residue construct containing the PPxY motif is expected to be efficient for studying the interaction of Smurf2 WW3 domain-PPxY motif, although the full-length U24 spans residues 1-87. The peptide (MDPPRTPPPSYSEVL) was synthesized to probe the interactions of the Smurf2 WW3 domain and U24. To investigate the interaction, 1H-15N Heteronuclear Single Quantum Coherence (HSQC) experiments were performed. Only the backbone and nitrogen-containing side chains of Smurf2 WW3 are observed in the HSQC spectra because the protein is labeled with 15N. When Smurf2 WW3 binds to a partner ligand, residues at the binding interface experience a change in their chemical environment; consequently, chemical shifts can be altered. The peaks in the HSQC spectra of the free and bound Smurf2 WW3 domain at 5°C (Figure 2.2(A)) are well dispersed, indicating that Smurf2 WW3 domain is well folded in both states. At 25°C, however, 6 residues 	   	   53 (Thr-308, Ala-309, Thr-310, Val-316, Gln-324 and Phe-325) are broadened beyond the limits of detection in the apo spectrum (Figure 2.3(A)) due to the conformational heterogeneity and/or amide exchange with solvent, and most of them reappear as saturation with U24 peptide is reached. The normalized chemical shift changes of all peaks observed at both 5°C and 25°C (Figure 2.2(B) and Figure 2.3(B)) are virtually identical, demonstrating that Smurf2 WW3 experiences a similar binding process when binding to U24 peptide at 5°C and 25°C.             	   	   54    Figure 2.2 Comparison of Smurf2 WW3 chemical shifts in the presence and absence of U24 peptide at 5°C. (A) The HSQC spectrum of [15N]Smurf2 WW3 free (black) and bound to U24 peptide (red) at 5°C. (B) Plot of the normalized chemical shift changes versus the residue number of Smurf2 WW3. The normalization factor was 3.77 for the nitrogen shifts (blue bars) and 0.343 for the proton shifts (red bars). (A) (B) 	   	   55    Figure 2.3 Comparison of Smurf2 WW3 chemical shifts in the presence and absence of U24 peptide at 25°C. (A) The HSQC spectrum of [15N]Smurf2 WW3 free (black) and bound to U24 peptide (red) at 25°C. (B) Plot of the normalized chemical shift changes versus the residue number of Smurf2 WW3 domain. The normalization factor was 3.218 for the nitrogen shifts (blue bars) and 0.343 for the proton shifts (red bars).   (A) (B) 	   	   56 2.3.3  Characterization of Smurf2 WW3 Interaction with U24 Peptide by NMR  Titration experiments by Nuclear Magnetic Resonance (NMR) are commonly used to characterize the interaction of a protein and its ligand. To further obtain a structural explanation for the interaction between Smurf2 WW3 domain and U24 peptide containing PPxY motif, an exploration of the interactions was carried out by adding U24 peptide into a 15N-labelled Smurf2 WW3 protein sample at 5°C, while monitoring the chemical shift perturbations for backbone residues in 1H-15N HSQC spectra (Figure 2.4(B)). The sequences of Smurf2 WW3 domain and U24 peptide corresponding to the N-terminal region of U24 are shown in Figure 2.4(A). Almost all the resonances of apo Smurf2 WW3 domain, which were observed at 5°C, are virtually identical with previous data[210], confirming that both of them share a common three-dimensional structure.  During the titration, a set of peaks was consistently perturbed, which indicated binding to U24 peptide. For nuclei that are not involved directly in the interaction, we expect a relatively small chemical shift perturbation. The Smurf2 WW3 amide 1HN and/or 15N chemical shift values were affected by peptide addition in either of the following ways. Gradual, be it large, chemical shift changes were observed upon binding for some residues as follows: Tyr-314, Val-316, Asp-317, His-318, Asn-320, Thr-326, Asp-327 showed chemical shift perturbation above 0.16 ppm. Other signals of amide groups disappeared from the spectra during the titration (Arg-321, Thr-323, Gln-324, Phe-325, lateral chain of Asn-319 and Gln-324) until an excess of ligand was added. Upon saturation with ligand, all binding sites become constitutively occupied, leading to reappearance of the peaks.    	   	   57   Figure 2.4 NMR titration studies of Smurf2 WW3 and U24 peptide. (A) Sequences of U24 peptide and Smurf2 WW3. The sequence of PPxY motif in U24 peptide is highlighted in grey. (B) HSQC spectrum of 15N-labeled Smurf2 WW3 free (red), following addition of U24 peptide at molar ratio of 1:0.17 (orange), 1:0.55 (yellow), 1:1.22 (green), 1:2.71 (cyan), 1:6.58 (magenta) at 5°C. The shifts caused by U24 peptide binding are highlighted by black arrows and crosspeaks are labeled on the spectrum.  	  U24 (HHV-6A)  MDPPRTPPPSYSEVL                 hSmurf2 WW3   PLPPGWEIRNTATGRVYFVDHNNRTTQFTDPRLSAN  298  333  320  310 (A) (B) 	  T308 F325 E304 V316 D327 Y314 H318 R321 W303 N320 T322 T326 T323 Q324 F315 D317 	   	   58 Although for one interacting system the thermodynamic (Kd) and kinetic (kon and koff; Kd= koff/ kon) parameters are constant, the NMR behavior of the resonances can be quite different according to the change in chemical shift that a nucleus undergoes upon interaction[217]. Some peaks move gradually, indicating the fast exchange between the unbound and bound forms. Some peaks can be attenuated and/or disappear due to line broadening, indicating specific binding with intermediate exchange kinetics, which are typical of ligand-protein interactions. In our case, the interaction is in the intermediate-fast exchange regime. A set of peaks corresponding to Arg-321, Thr-323, Phe-325, and Gln-324 undergoes extensive line broadening with increasing ligand concentration, indicating specific binding of the Smurf2 WW3 and U24 peptide. Such behavior indicates that these residues are at the binding sites or near the binding interface. In addition, as residues Tyr-314, Val-316, Asp-317, His-318, Asn-320, Thr-326 and Asp-327 are insensitive to intermediate binding, this may represent a simple conformational change in the Smurf2 WW3. Overall, the majority of the additional chemical shift changes are minor, suggesting that the Smurf2 WW3 domain does not experiencing a significant conformational change upon binding. All perturbed resonances shift together, excluding the possibility that interactions with multiple nonspecific sites are being monitored. At a ratio of 1:6.58, the chemical shifts no longer change and the chemical shift at this ratio is taken to represent the bound state of Smurf2 WW3. Fully binding of U24 peptide gives rise to well-dispersed crosspeaks with uniform line widths. To look into which residues were significantly perturbed, the combined 1HN and 15N chemical shifts of all the residues were calculated (Figure 2.4). The values of combined chemical shifts can be classified as large. Those values can vary greatly between protein complexes[218]. This is attributed to the degree of dynamics within the complex as discussed above.  	   	   59          Figure 2.5 1H-15N chemical shift perturbation (CSP) of Smurf2 WW3 when titrated with U24 peptide. (A) Plot of the combined chemical shift changes in backbone amide imposed on Smurf2 WW3 when titrated with U24 peptide at 5°C versus the residue number of the protein. The combined chemical shift changes were calculated from the chemical shift changes of nitrogen and proton (Figure 2.2(B)) by using equation (2.1). Secondary structure elements are indicated on top. The vertical color strips categorize the changes in combined chemical shifts into very high, high, medium, and low groups for projection onto the three-dimensional structure of Smurf2 WW3. (B) The aforementioned plot mapped onto the ribbon diagram of Smurf2 WW3. Significant chemical shift perturbations were grouped and color-coded into four categories: weak (yellow) if CSP<1σ, medium (light pink) if 1σ<CSP<2σ, strong (pink) if 2σ<CSP<3σ, very strong (red) if 3σ<CSP, where σ is the standard deviation of the mean. The model is derived from solution NMR structure (PDB ID 2DJY) and generated using PyMOL.         (A) (B) 	   	   60                        Figure 2.6 The surface representation of the structure of Smurf2 WW3, colored as described in Figure 2.5. The model is derived from solution NMR structure (PDB ID 2DJY) and generated using PyMOL.  A complete chemical shift map of Smurf2 WW3 for the interaction with U24 peptide was then obtained, based on the known solution NMR structure of Smurf2 WW3 (Figure 2.5(B) and Figure 2.6). In the structure, most of the hydrophobic residues located at or close to binding interface were strongly perturbed by complex formation, while the residues, located on the other side of the Smurf2 WW3 domain, were only weakly perturbed. These residues most likely do not participate in the direct interaction with U24 peptide, simply experiencing a conformational accommodation.  Tyr-314 located on the β2 strand, Thr-323 located at the β3 strand and Phe-325 have previously been shown to form an XP groove to bind the first and second Pro residues in the PPxY motif. In addition, Val-316 located on the β2 strand, His-318 and Arg-321, located in the β2-β3 loop, have also been identified to form a hydrophobic pocket to bind the Tyr residue in the PPxY motif[210]. Tyr-314, Val-316, His-318, Arg-321, Thr-323 and Phe-325 are among the residues that undergo large changes in chemical shift upon interaction with U24 peptide, Rotated by 180° XP groove Hydrophobic Pocket 	   	   61 indicating these residues are most likely in direct contact, as also found for the Smurf2 WW3-smad7 interaction[210]. The residues Gln-324 and Thr-326, positioned on the other side of binding interface, are also strongly perturbed presumably due to a relatively large conformational change that arises from neighboring residues (such as Thr-323 and Phe-325), which are strongly perturbed upon complex formation. In addition, it has been reported that the residues on the β1 strand and in the β1-β2 loop can interact with the residues outside the PPxY region to enhance the affinity[210]. The residues Arg-306 and Thr-308 from the β1 strand, positioned on the side of binding interface, are not perturbed as strongly as the residues involved in the direct interaction, indicating that they may interact with the side chain of residues flanking the PPxY motif. The backbone of Thr-310, Gly-311, Arg-312 and Val-313 in the β1-β2 loop is perturbed very weakly, implying there may be no interactions between U24 peptide and the β1-β2 loop or very weak interaction between U24 peptide and side chains of those residues in the β1-β2 loop.  According to the model, the complex of Smurf2 WW3 domain with U24 peptide should have a stoichiometry of 1:1. On the basis of the homology within the PPxY motifs, the U24 peptide is expected to bind in an extended PPII conformation within the complex of Smurf2 WW3 domain and U24 peptide[210]. To estimate the affinity between Smurf2 WW3 domain and U24 peptide, the dissociation constant (Kd) was calculated. In the case of fast exchange, the observed chemical shifts are a population-weighted average of two forms of the molecule (bound and free state); therefore, fitting curves of apparent chemical shift as a function of added titrant depends on the dissociation constant as a parameter, i.e. equation (2.2) is only applicable in the case of fast exchange. Among the signals that move most during the titration, the resonances of Arg-321, Thr-323, Phe-325 and Gln-324 are obscured because of intermediate exchange. We fitted curves of the 	   	   62 remaining three peaks (Tyr-314, Val-316 and His-318) to a 1:1 binding model (Figure 2.7(A)), with individual amplitudes and offsets for each curve. Eventually, a 1:1 model fits the data well and yields an average Kd value of 123 ± 4 µM at 5°C (Figure 2.7(B))        Figure 2.7 Binding affinity of Smurf2 WW3 and U24 peptide. (A) Binding curves derived from a representative set of residues experiencing high chemical shift perturbations at 5°C, describing combined 1HN and 15N chemical shift perturbation of Smurf2 WW3 residues (in ppm) observed upon addition of increasing amount of U24 peptide. (B) Dissociation constant (Kd) at 5°C was obtained by fitting of titration curve of three well-resolved, significantly perturbed peaks, identified in (A).  Residues Kd (µM) Y314 127.1 H318 120.4 V316 121.2 Average 123 ± 4 (A) (B) H318 Y314 V316 	   	   63 2.3.4 Understanding the Secondary Structure by Circular Dichroism The weak binding of Smurf2 WW3 domain to PPxY motif within U24 may be due in part to the inability of the WW domain to properly fold into a conformation best suited for recognizing cognate ligands. In order to determine whether the WW domain was properly folded, the secondary structure was determined. Specifically, to analyze the secondary structure of Smurf2 WW3 domain and U24 peptide, circular dichroism (CD) spectra in the far-UV region were recorded. At relatively low temperature (from 5°C to 20°C), the spectra of Smurf2 WW3 domain exhibit a positive band at around 230 nm due to aromatic contribution and a negative band around 205 nm due to random-coil contribution (Figure 2.8(A)). These spectral features are characteristic of well-folded WW domains as reported previously, implying that the Smurf2 WW3 domain, in the unbound form, is correctly folded. The NMR titration experiment was performed at 5°C, where Smurf2 WW3 is folded, as supported by the CD data. The spectrum of U24 peptide displays a positive band around 226 nm and a negative band around 205 nm, which is indicative of the PPII helix in the U24 peptide (Figure 2.8(C)).   	   	   64     Figure 2.8 Analysis of the secondary structure by CD spectroscopy. Far-UV CD spectra of (A) Smurf2 WW3 domain, (B) Complex of Smurf2 WW3 and U24 peptide (1:5.4), and (C) U24 peptide were measured in a temperature range of 5°C to 65°C. Spectra were corrected for buffer contributions. Inset: thermodynamics of temperature-induced changes at 230 nm.    (A) (B) (C) 	   	   65   Far-UV CD spectra were recorded with increasing temperature to investigate how the temperature influences the secondary structure of Smurf2 WW3 domain, U24 peptide and the complex of the two. To further test this, changes in the far-UV CD signal at around 230 nm was monitored as a function of temperature. For Smurf2 WW3 alone, the melting curve shows cooperative signal changes with well-defined linear pre- (5°C to 20°C) and post- (55°C to 65°C) transition baselines (Figure 2.8(A)). This is consistent with an apparent temperature-induced two-state unfolding of WW domains[219]. The β1-β2 loop is identified to be the rate-limiting substructure in the WW domains[220]. Hydrophobic interstrand contacts (between Trp-303, Pro-328, Phe-315 and Gln-324 in Smurf2 WW3 domain) gain importance at high temperature[210, 220]. The melting temperature of Smurf2 WW3 is estimated to be around 40°C. It has been identified that Ala-309 and Thr-310 in the β1-β2 loop have poor structural statistics[210], possibly resulting in relatively low melting temperature. The melting curve of Smurf2 WW3 with U24 peptide, compared with that of Smurf2 WW3 domain, suggests that the complex has a higher estimated melting temperature (Figure 2.8(B)), indicating that the WW3 structure is stabilized upon complexation. It should be noted that the melting temperature of WW3 with U24 peptide could only be estimated because there were not enough data points collected at the higher temperatures. These conformational changes in Smurf2 WW3 domain alone and with U24 peptide were completely reversible. The effects of the interaction on the secondary structures were also evaluated by CD spectroscopy; the data show conformational changes induced by the binding of U24 peptide to the Smurf2 WW3 domain. All spectra of Smurf2 WW3 domain and the complex were next fitted using three different programs (CDSSTR[221], CONTINLL[222], SELCON3[223]), using the full data set (points at every 1 nm in the range between 200 and 250 nm) to estimate the percentage 	   	   66 of β-strand. The calculated results (Figure 2.9) indicate that there are the same percentages of β-strands contained in the Smurf2 WW3 domain with U24 peptide than Smurf2 WW3 alone at one specific temperature, within the error associated with the analysis. This suggests that the interaction with U24 peptide only enhances the stability of Smurf2 WW3 minutely.  Altogether, Smurf2 WW3 is not that stable at ambient temperature and binding to U24 peptide is suggested to slightly enhance the stability of Smurf2 WW3. The relative instability of Smurf2 WW3, compared to other WW domains, may affect binding affinity but more evidence is required to conclusively prove this.     Figure 2.9 Plot of the percentage of beta-strands at different temperatures in Smurf2 WW3 domain with and without U24 peptide. The values with errors are listed in the following: 0.29±0.03 (5°C), 0.29±0.03 (15°C), 0.28±0.02 (25°C), 0.27±0.02 (35°C), 0.23±0.10 (45°C), 0.22±0.08 (55°C), 0.21±0.02 (65°C) for Smurf2 WW3; 0.32±0.01 (5°C), 0.31±0.03 (15°C), 0.29±0.05 (25°C), 0.28±0.09 (35°C), 0.26±0.03 (45°C), 0.23±0.03 (55°C), 0.22±0.05 (65°C) for Smurf2 WW3 with U24 peptide.      	   	   67 2.4 Discussion 2.4.1 Strength of U24-Smurf2 WW3 Interaction Using NMR spectroscopy, the binding affinity for Smurf2 WW3 domain with the U24 peptide was determined at 5°C. To confirm the binding specificity and affinity, isothermal titration calorimetry (ITC) was used to determine the dissociation constant for the interaction (work carried out by Y. Sang in our group). The Kd value obtained by ITC (Table 2.1) agrees with that obtained from fitting of the NMR chemical shift perturbations for the 15N-labeled Smurf2 WW3 domain titrated with U24 peptide (Kd=123 ± 4 µM at 5°C). The ITC data shows in addition that binding of Smurf2 WW3 domain to PPxY motif is largely driven by favorable enthalpic forces (ΔH° < 0) accompanied by unfavorable entropic changes (ΔS° < 0), implying that the intermolecular interactions such as hydrogen bonding and van der Waals contacts likely account for the specificity of this protein-ligand interaction. This is a hallmark of molecular recognition underlying WW-ligand interactions in general[224, 225].   Table 2.1 Thermodynamic parameters obtained from ITC measurements for the binding of the Smurf2 WW3 domain to U24 peptide. T (°C) Kd (µM) ΔH° (kJ/mol) ΔS° (J/molK) 5 132 ± 2 -28.6 ± 0.3 -29 ± 1 15 193 ± 11 -24 ± 3 -11 ± 9 25 329 ± 15 -24 ± 3 -14 ± 9  The strength of the interaction lies within the range of Kd determined for other WW domains (Kd=3-300 µM)[208, 226, 227]. However, the binding affinity is relatively low when compared with other WW-ligand interactions. For example, the analysis of the Smurf2 WW3 	   	   68 domain by intrinsic fluorescence revealed that it can bind to the Smad7 peptide with an affinity of 40.0 ± 0.1 µM at 20°C, significantly better binding than that of the Smurf2 WW3 domain with U24 peptide.    2.4.2 Comparison with the Smurf2 WW3-Smad7 PY Complex Residues within the polyproline-tyrosine (PY) motif and the PY motif biding pockets are well conserved in both the Smurf2 WW3-Smad7 complex and Smurf2 WW3-U24 complex, and are involved in direct ligand binding as well. In the Smurf2 WW3-Smad7 complex[210] (Figure 2.10(B)), the first and second Pro residues in the PY motif bind in a hydrophobic pocket, stacking against Phe-325 and Tyr-314 (XP groove), respectively. A hydrogen bond is formed between the carbonyl group of the second Pro residue and the hydroxyl group of Thr-323. The Tyr of the PY motif lies in a hydrophobic pocket formed by Val-316, an uncharged His-318 and Arg-321. Our results of mapping the binding site are completely consistent with this interaction mechanism. In addition, the majority of resonances observed from 1H-15N HSQC spectra of both [15N]Smurf2 WW3-Smad7 complex and [15N]Smurf2 WW3-U24 complex at 25°C are virtually identical, indicating that two complexes share a common three-dimensional structure of Smurf2 WW3 domain (data not shown). In general, the XP grooves and Tyr binding pockets of group I WW domains are very similar[206, 207, 228].  The Smurf2 WW3 domain has been shown to bind the PY motif region from Smad7. It is the first example of a WW domain that has a Phe in place of the canonical Trp, yet still binds a PY motif[210]. It has been demonstrated that the Phe decreases the affinity of the Smurf2 WW3 domain for the Smad7 PY motif significantly (Kd from wild type Smurf2 WW3 is 40.0 ± 0.1 µM and from F325W WW3 is 0.17 ± 0.01 µM), suggesting that additional interactions modulate 	   	   69 binding affinity. Analysis of the contacts between the Smurf2 WW3 domain and the Smad7 peptide has revealed that it extended beyond the interface with the PY motif proper to include an interaction with the six Smad7 residues C-terminal to the PY motif (PY-tail)[210]. The PY-tail lies parallel to the β-strands of the Smurf2 WW3 domain and binds residues in the first and second β-strands and the intervening β1-β2 loop. There are extensive backbone interactions between the PY-tail and the Smurf2 WW domain, as well as specific side chain contacts involving Arg-213’ with His-318, Pro-215’ with Arg-306, and Asp-217’ with Thr-308 and Ala-309. Additionally, to facilitate these interactions, the Smad7 peptide adopts a loop structure with residues in the PY motif lying anti-parallel to the last four residues of the PY-tail. This loop structure can be stabilized by extensive intrapeptide contacts involving Pro-209’, Pro-210’, Ser-212’, Tyr-214’ and Met-216’. It has recently been suggested that the N-terminal site of Smad7 (Glu-205’) is also involved in the contact with Arg-312[229]. The mutation (Arg312Glu) introduced in Smurf2 WW3 significantly reduces the affinity by approximately 10-fold.                	   	   70                          Figure 2.10 Comparison of the Smurf2 WW3-U24 complex and the Smurf2 WW3-Smad complex. (A) Alignment of U24 and Smad7 PY motif. PPxY motif is highlighted in red. Residues with negative and positive charges are highlighted in cyan and yellow, respectively. (B) The surface representative of the structure of Smurf2 WW3 from Smurf2 WW3-U24 (left) and Smurf2 WW3-Smad7 (right) complexes. In Smurf2 WW3-U24 complex (left), residues of Smurf2 WW3 are colored as described above (Figure 2.6). In Smurf2 WW3-Smad7 complex (right), residues of Smurf2 WW3 involving in binding the PY motif are colored in red and residues interacting with peptide residues outside the PY motif are colored in green based on the NOEs. Smad7 peptide is shown as stick diagram and PPxY motif is highlighted in red. The XP groove and Tyr binding pocket are labeled. Some residues are labeled as well. The model is derived from solution NMR structure (PDB ID 2DJY) and generated using PyMOL.  Although the XP groove and Tyr binding pocket of group I WW domains are very similar, they are not sufficient for discrimination among group I ligands. It has been recognized that Smurf2 WW3 domain binds to PY motif from both U24 peptide and Smad7 by using the same residues forming the XP groove and Tyr binding pocket, but there is significant difference in their binding affinity. This discrimination may primarily lie in regions outside of the core PY motif and its cognate binding pocket. In the Smurf2 WW3-U24 complex (Figure 2.10(B)), Arg-306 and Thr-308, located near the binding interface, are colored in pink based on the Smad7 202’-217’  U24   1-15  SELESPPPPYSRYPMD         MDPPRTPPPSYSEVL  (A) (B) Tyr binding pocket Tyr binding pocket XP groove XP groove Arg-306 Thr-308 Arg-306 Thr-308 Ala-309 	   	   71 chemical shift perturbations, implying that these residues are perturbed moderately. Thus, the two residues are likely involved in interaction with residues C-terminal and/or N-terminal to the PY motif in U24 peptide. However, there are several differences between Smad7 and U24 peptide sequence (Figure 2.10(A)). Arg-213’, which binds to His-318, has a positive charge, but at the equivalent position of U24 peptide, there is a Glu with negative charge. More importantly, U24 peptide has Arg residue with positive charge and in contrast, Smad7 peptide has Glu-205’ with negative charge at the equivalent position. Consequently, residues with the opposite charge may disrupt the interactions. Furthermore, the charge distribution on the binding interface of Smurf2 WW3 (Figure 2.11) shows that the region near the XP groove that likely binds to the residues N-terminal to PY motif has positive electrostatic potential, while the region near the Tyr binding pocket that likely binds to the residues C-terminal to PY motif has negative electrostatic potential. In U24 peptide, however, there is the Arg residue with positive charge N-terminal to the PY motif and the Glu residue with negative charge C-terminal to the PY motif. This analysis that charge distribution in peptide U24 may affect its interaction with Smurf2 WW3 domain, is consistent with the explanation discussed above. Overall, these differences between U24 and Smad7 peptide may explain why a lower binding affinity of U24 peptide with Smurf2 WW3 domain was observed.  Therefore, although the topology of the PY motif in the complex is the same with other group I interactions, the Phe weakens the interaction with the PY motif relative to the Trp and likely restricts the Smurf2 WW3 domain to binding PY motif regions that should have additional complementary binding surfaces.    	   	   72  Figure 2.11 The charge distribution on the surface of the Smurf2 WW3. Blue and red represent positive and negative electrostatic potential, respectively. The XP groove and Tyr binding pocket are labeled. The residue R312 is labeled as well. The model is derived from solution NMR structure (PDB ID 2DJY) and generated using PyMOL.  2.4.3 Comparisons with Other WW Domain-Ligand Complexes Although the PY motif is critical for group I WW domain interactions, residues outside the region that binds to the core PY motif in WW domains and residues outside the core PY motif that bind to WW domains in ligands are a recent focal point for determining the specificity of WW domain-PY motif interaction[210]. Interactions with regions N- and C-terminal to the PY motif may be a general requirement for achieving optimal affinity. Smad7 peptide can bind to hYAP WW1, hYAP WW2, hNedd4L WW2, Smurf1 WW2 and Smurf2 WW3 domains with different affinity (Figure 2.12)[229]. In the Nedd4L WW2-Smad7 complex, Pro-215’ is perpendicular to the β-sheet plane and is bound by the Thr-391, Val-384, and Arg-374 side chains. The electrostatic interactions between Glu-205’, located upstream of the PY motif, with the side chains of Arg-380 and of Asp-217’ with Lys-378 and Arg-380 are observed. Either Arg-380 or Lys-378, or both, are replaced by a negatively charged residue (Glu), Tyr binding pocket XP groove R312 	   	   73 which disrupts the electrostatic interactions. Single changes reduce the affinity for the Smad7 peptide 4- to5-fold when compared to the wild-type and more than 6-fold in the double mutant.  In Smurf1 WW2- and Smurf2 WW3-Smad7 complexes, the Smad7 peptide forms a turn, but it does not form a long hairpin as in the case of the Nedd4L complex. A comparison of these two complexes with that of Nedd4L revealed some additional differences; for instance, Glu-205’ is interacting with Arg-295/Arg-312 in the second strand, but no contact with the peptide are observed for the other residues located in the β1-β2 loop of the WW domains. As a consequence, Glu-205’ and Asp-217’ are less defined in the Smurf1 WW2 and Smurf2 WW3 complexes. In contrast, the previously characterized Smurf2 WW3-Smad7 complex previously indicates there is contact between Asp-217’ and Ala-309, Thr-308[210]. Therefore, whether there is any contact between the peptide and the residues located in the β1-β2 loop of Smurf2 WW3 domain needs to be confirmed. Smurf1 WW2 domain and Smurf2 WW3 domain share a high sequence homology and also have the similar affinity for the Smad7 peptide.  YAP WW1 and WW2 domains adopt the canonical WW fold and bind to the Smad7 PY core in a similar manner. The main differences between both complexes are the contacts observed between Smad7 Tyr-214’ and Tyr-247, Ile-249 and Glu-237 in the YAP WW2 complex and more significantly, the absence of interactions between the residues located in the β1-β2 loop and the residues preceding or following the PY site, which are observed in the complex with YAP WW1, and in the complexes corresponding to the three E3 ubiquitin ligases-Nedd4L, Smurf1 and Smurf2. The absence of these interactions could be interpreted on the basis of the charge distribution of the YAP WW2 domain, which differs from that of the YAP WW1 domain. The presence of negatively charged residues in the β1-β2 loop (Asp-243 and Glu-245) repels both the accommodation of the N-terminal part of Smad7 as well as the 	   	   74 negatively charged residues located in the C-terminal extension of the PY motif (Figure 2.12(A)). Point mutations introduced in Asp-243 and Glu-245 residues to glutamine resulted in an improved affinity by a factor of two. Therefore, the affinity for YAP WW1 domain is 10-fold higher than YAP WW2 domain (Figure 2.12(B)).                 Figure 2.12 Binding affinity of five WW domains and Smad7 peptide. (A) Alignment of group I WW domains that Smad7 can bind to. The second conserved Trp is highlighted in red. Residues binding to the region outside the PY motif are highlighted in purple. Asp-243 and Glu-245 in YAP WW2 domain, which repel the interaction with the region outside the PY motif, are highlighted in green. The values after the sequences indicate the sequence homology with Smurf2 WW3 domain. (B) ITC affinity values for the recombinant fragments of Nedd4L, Smurf1, Smurf2 and YAP and Smad7 peptide at 15°C[229].     WW domain Kd (µM) hYAP WW1 6.9 ± 0.3 hYAP WW2 59.8 ± 3.4 hNedd4L WW2 4.2 ± 0.1 hSmurf1 WW2 4.1 ± 0.1 hSmurf2 WW3 4.5 ± 0.2  VPLPAGWEMAKTSSGQRYFLNHIDQTTTWQDPRKA  (47%)  GPLPDGWEQAMTQDGEIYYINHKNKTTSWLDPRLDP (53%)  PGLPSGWEERKDAKGRTYYVNHNNRTTTWTRPIG   (56%)  GPLPPGWEVRSTVSGRIYFVDHNNRTTQFTDPRLH  (82%)  GPLPPGWEIRNTATGRVYFVDHNNRTTQFTDPRLSAN   ELESPPPPYSRYPMD     hYAP WW1    171-205 hYAP WW2    230-265 hNedd4L WW2 365-398 hSmurf1 WW2 280-314 hSmurf2 WW3 297-333  Smad7      203’-217’     (A) (B) 	   	   75 The results discussed above suggest the importance of the interaction between the region outside the core PY motif and WW domains for binding affinity. WW domains have two conserved Trp residues in most cases: the first Trp residue is on one side of the sheet in a hydrophobic cluster that is important for domain stability; the second Trp residue is on the opposite face of the sheet in a hydrophobic pocket and is integrally involved in binding to polyproline sequences of ligands[205]. It has been identified that introduction of a Phe residue at the position of the first Trp residue produces a natively unfolded protein while substitution of the second Trp residue by a Phe residue does not affect the general structure of the WW domain, but abolishes ligand binding activity[205, 230]. Although Smurf1 WW2 and Smurf2 WW3 have a Phe residue in place of the second conserved Trp, which are expected to decrease the affinity for Sma7 peptide, both of them have high affinity as the same as other WW domains due to additional interactions that are provided by the residues flanking the PY motif. In addition, the WW domains that contain two conserved Trp residues may have relatively low affinity due to the absence of interactions provided by the residues outside the PY motif. Therefore, altogether these data support a model in which the PY motif functions as an anchor with regions N- and C-terminal to the PY motif increasing (or decreasing) the affinity and modulating specificity[210]. Particularly for Smurf2 WW3 domain, which has a Phe residue in place of the second conserved Trp, additional interactions play more significant role in binding affinity and specificity. The results discussed above also reveal an absence of selectivity in the interactions of Smad7 with these proteins, illuminating the versatility of different WW domains as mediators of convergent interactions with a common Smad7 target. Thus, there is possibility that different WW domains may target binding to U24 to modulate its function. 	   	   76  In addition to the WW-Smad7 complex, Figure 2.13 summarizes determinants of binding affinity and specificity, indicating which WW domain residues interact with the core L/PPXY motif or regions N- or C-terminal to it. Three of the six examples show interactions with peptide residues C-terminal to the core PY sequence. Su(dx) WW4 and dNedd4 WW3 are examples where specific interactions occur both N- and C-terminal to the PY core. There are different opinions about Smurf2 WW3. The core PY motif mainly binds to six residues at the equivalent positions, which are conserved in those WW domains. The residues in the β1-β2 loop are critical for binding to the region outside the PY motif. The features of the Su(dx) WW4 interaction is similar with Smurf2 WW3[230]: a weakened interaction of the binding site Trp-Phe substitution with the first Pro of the PY motif and the involvement of β1-β2 loop residues in specificity determining interactions with peptide residues outside the core PY motif. Although the β1-β2 loop can undoubtedly provide a specificity determining binding pocket, how an interaction occurs is still less predictable. There is some evidence that the positively charged residues on the surface of many WW domains are conserved and acidic peptide residues or phosphorylation can enhance the interaction with those WW domains[231, 232]. Therefore, in addition to predominantly hydrophobic interactions present in all known WW domain complex structures, the presence of the electrostatic contacts N- and C-terminal to the core PY motif also plays an important role in binding affinity and specificity.     	   	   77   Figure 2.13 Comparison of various WW domains and their binding partners. (A) Alignment of other members of Group I WW domains for which structures in complex with a peptide ligand are available. Residues are color highlighted according to the regions of the PY peptide ligand with which interactions are observed: interaction with core PY residues, pink; interaction with specific residues C-terminal to the core PY motif, yellow; interaction with specific residues N-terminal to the core PY motif, blue. Weak interactions are highlighted in a paler color and boxed. Residues in the β1-β2 loop are shown in blue text. (B) Alignment of the PY motif peptides. Residues that interact with WW domain residues are highlighted and colored according to the description of their location in the peptide: core PY residues (pink); C-terminal to the core PY motif (yellow); N-terminal to the core PY motif (blue). (adapted from [230] with modification)  2.5 Conclusion Based on the discussion, it is confirmed again that the interactions between the residues preceding or following the PY motif and the residues located in the β1-β2 loop of WW domains are critical for binding affinity of WW domain-PY motif interaction. Therefore, it is assumed that the presence of positively charged Arg residue instead of Glu residue in U24 peptide and the absence of residues interacting with the residues located in the β1-β2 loop of WW domains strongly affect the tightness of interaction but does not change the binding interface. U24 acts in a general block in the endosomal recycling, dependent on the interaction of its PPxY motif with WW domain-containing proteins. Smurf2 WW3 domain has the relatively low affinity for U24 (A) (B) 	  	   	   78 peptide. However, we are tempted to suggest that Smurf2 is still a potential WW-containing protein that interacts with PPxY motif to modulate its function. The binding experiments are carried out in vitro, which may have some differences with the behavior in vivo. For example, adjoining domains may assist binding affinity and specificity, such as the EF hand in dystrophin[233]. Smurf2 has three WW domains, and perhaps multiple WW domains may act together to increase the affinity as demonstrated with tandem SH2[234] and PDZ[235] domains. In addition, there is possibility that other WW domain containing proteins modulate the functions of U24 by the interaction with PPxY motif with higher affinity and specificity.                    	   	   79 3.1 Introduction The Nedd4 protein family constitutes a major class of HECT-type E3 ubiquitin ligases implicated in a variety of processes including neurodegeneration[236], blood pressure regulation[230, 237], cell-cell communication[238] and differentiation[239], and viral budding[240]. The Nedd4 protein family exerts their effects though the regulated trafficking and degradation (via ubiquitination) of targets, usually located in the plasma membrane or nucleus, that are recognized by the WW domains[165]. Most HECT-type E3 ubiquitin ligases have multiple WW domains, suggesting that multiple WW domains are required to enhance target-binding affinity. In some cases, such as Rsp5p, Nedd4, and FBP11[206, 241, 242], more than one of the WW domains can bind to the same target. The two consecutive WW domains of FBP11 are both able to individually bind to polyproline target motifs with modest affinity; however, in tandem they bind to a ligand containing two target motifs with much higher affinity. NMR data and molecular modelling suggest that the two FBP11 WW domains bind to the two target motifs in the ligand simultaneously. Thus, multiple WW domain modules may function by binding separate targets or act together to increase specificity as demonstrated with tandem SH2[234] and PDZ[235] domains. Alternatively, they may work together in an autoregulatory manner. The solution structures of paired WW domains for two examples, i.e. yeast Prp40[243] and Drosophila Su(dx)[244], reveal a rigid α-helical linker in the former case and a flexible linker in the latter. In both examples, the nature of the linker and resultant relative domain orientation has functional implications; Prp40 mediating a bridging interaction and Su(dx) likely undergoing regulated, Chapter 3 Probing the Binding of Tandem Smurf2 WW Domains to U24 Protein from HHV-6A  	   	   80 transient interactions with a number of ligands. The Su(dx) tandem pair is in actual fact poorly structured in its apo form, in part because of a domain-domain association that the flexible linker does not prohibit.      As mentioned previously, the isolated Smurf2 WW2 domain cannot bind to PY motifs, however, Smurf2 WW2 domain can cooperate with Smurf2 WW3 domain to bind PY motifs, contributing to optimal target recognition (Figure 3.1). For instance, intrinsic tryptophan fluorescence experiments suggested that a protein containing both of WW2 and WW3 domain of Smurf2 in tandem (Smurf2 WW23) has 20-fold higher affinity for Smad7 PY motif (GPLGSELESPPPPYSRYPMD) than the isolated WW3[209]. Furthermore, solution structure[209] of Smurf2 WW23 tandem bound to Smad7 PY motif revealed that Smurf2 WW2 domain contributes to the enhanced affinity of the tandem WW23 for Smad7 PY motif by forming additional contact with the prolines of the PY motif and a novel E/D-S/T-P motif, which is N-terminal to the PY motif. Thus, it was concluded that interdomain coupling of Smurf2 WW domains not only augmented the interaction with PY motif, but also enhanced the selectivity.    	   	   81  Figure 3.1 Ribbon diagram of the Smurf2 WW23-Smad7 complex (adapted from [209]). The WW2, linker, WW3 are shown in green, gray, and blue, respectively. The backbone of Smad7 peptide is shown in red, whereas the side chains are in yellow. Smad7 residues are labeled with the prime symbol.   However, it has recently been reported that the affinity increase due to the presence of the Smurf2 WW domain pair is about 2-fold with respect to the values obtained with the Smurf2 WW3 domain at a given temperature[229], in contrast to previous observations that suggest an improvement of about 20-fold (for details in section 3.4.3). In addition, Smurf2 WW2 domain is suggested to have a minor role in binding to short PY containing sequences (ELESPPPPYSRYPMD), but an important role in protein oligomerization and aggregation, which also differs from the previously reported interpretation that the Smurf2 WW2 domain participates in hydrophobic and electrostatic interactions with the Smad7 peptide.   We previously demonstrated that the isolated WW3 domain of Smurf2 (but not the WW2 domain) has the ability to directly, albeit weakly, bind to the U24 PY motif (Chapter 2). In order to address the issue of whether the affinity and specificity of U24 peptide binding by Smurf2 WW3 domain is influenced by its neighboring WW2 domain, we further examined the 	  	   	   82 interaction between U24 PY motif region and Smurf2 WW23, a construct containing both the WW2 and WW3 domains of Smurf2 in tandem, using NMR and CD spectroscopy.  3.2 Materials and Methods 3.2.1 Protein and Peptide Preparation The plasmid expressing the GST-Smurf2 WW23 protein was a kind gift from Dr. Julie D. Forman-Kay (Hospital for Sick Children, Toronto). The vector pGET 6P1 with DNA encoding the Smurf2 WW2 and WW3 domains in tandem (Smurf2 WW23, amino acids 250-333) domain was cloned into the BamH1 and XhoI restriction sites. Glutathione S-transferase fusions were expressed in Escherichia coli BL21(DE3) cells at 25°C with a 16-h isopropyl 1-thio-β-D-galactopyranoside (IPTG) induction period. Smurf2 WW23 was labeled with 15N by expressing the protein in M9 minimal medium containing 15NH4Cl as the sole nitrogen sources. Cells were lysed by sonication in 10 mL binding buffer (PBS with 1% Triton X-100, pH 7.3), supplemented with lysozyme, EDTA-free protease inhibitor cocktail and DNase I. The resulting cell lysate was centrifuged for 1 hour and the supernatant was syringe-filtered through a 0.45 µM membrane filter. Approximately 1 mL of Glutathione-Sepharose 4B beads were placed in a 15 mL falcon tube and equilibrated by adding 5 mL binding buffer. The beads were pelleted by centrifugation at 500 ×g for 5 minutes, the buffer removed and the procedure was repeated twice more. The cell lysate was added into the equilibrated beads, supplemented with 6 mM dithiothreitol (DTT) and incubated for at least 2 hours with end-over-end rotation at ambient temperature. The beads were sedimented by centrifugation, and the supernatant was carefully decanted. The beads were washed by addition of 5 mL binding buffer. The washing process was then repeated twice for a total of three washes. Afterwards, Triton X-100 was removed by three 	   	   83 more successive washing steps using Human Rhinovirus 3C (HRV-3C) protease reaction buffer. After the addition of 2 mM DTT, WW23 was cleaved form glutathione S-transferase (GST) by using Human Rhinovirus 3C protease with end-over-end rotation at 4°C for 16 hours. The suspension was centrifuged to pellet the beads and the supernatant was carefully collected. After the HRV 3C protease reaction buffer was added, the beads were suspended and immediately centrifuged. The supernatant was collected and the process was repeated twice. A total volume of 6 mL supernatant containing purified WW23 was finally collected. The still-bound GST protein was removed form the beads with elution buffer, which consisted of 50 mM Tris, 10 mM reduced glutathione, 1% Triton X-100, pH 8.0. The purified WW3 domain was dialyzed against a solution of 40 mM HEPES, 10 mM NaCl, pH 7.2, using a dialysis membrane with MWCO of 2000. A 15-mer peptide representing the N-terminus of U24 form HHV-6A (MDPPRTPPPSYSEVL) were synthesized using the same method as previously described (in section 2.2.3). The synthetic peptide was purified by preparative gradient RP-HPLC on a Waters 600 system with a Waters 2996 photodiode array detector with 229 nm and 278 nm UV detection using a Phenomenex C18 column (10 µm, 2.12 cm × 25 cm) with an acetonitrile gradient. The solution containing purified peptide was lyophilized, and its final mass was confirmed by MALDI-TOF mass spectrometry.  3.2.2 1H-15N HSQC NMR Titrations and Kd Calculations for the Smurf2 WW23 Domain and U24 Peptide Interaction All spectra were measured on Bruker NMR spectrometers operating at proton resonance frequencies of either 600 MHz or 850 MHz and equipped with cryogenic probes. Experiments 	   	   84 were performed in buffer containing 40 mM HEPES, pH 7.2, 10 mM NaCl, 0.05% NaN3, 1% Glycerol, 1 mM TCEP, 10% D2O, unless otherwise stated. The final concentration of [15N]Smurf2 WW23 tandem was calculated from the method of Edelhoch [214] with the extinction coefficients (ε280) of Trp and Tyr determined by the protparam algorithm (http://www.expasy.ch). Binding during the titration series was monitored with 1H-15N HSQC experiments, with a spectral width of 16 ppm in the 1H dimension and 29 ppm in the 15N dimension. Titrations were carried out in sixteen steps, each one adding the U24 peptide from HHV-6A in the following protein/peptide molar ratio: 1:0.25, 1:0.51, 1:0.85, 1:1.28, 1:1.71, 1:2.51, 1:3.45, 1:4.46, 1:5.55, 1:6.78, 1:8.01, 1:9.46, 1:11.05, 1:13.08 and 1:15.25. Combined chemical shift perturbations (Δδ [HN,N]) were calculated using the equation:                Δδ [HN,N] = ([ΔδHNWHN]2 + [ΔδNWN]2)1/2                    (3.1) where WHN and WN are weighting factors for the HN and N shifts, respectively (WHN=1; WN=0.154[215]), and ΔδHN/N = δbound – δfree. Subsequently, the effective dissociation constant Kd, was determined independently for each residue by fitting the titration data, and the concentrations of WW3 domain and U24 peptide using the equation[216]:     Δδobs = Δδmax{([P]t + [L]t + Kd) – [([P]t + [L]t + Kd)2 - 4[P]t[L]t]1/2}/2[P]t       (3.2) where Δδobs is the change in the combined chemical shift from the free state of Smurf2 WW23, Δδmax is the maximum combined chemical shift change on saturation, [P]t is the concentration of Smurf2 WW23 and [L]t is the concentration of U24 peptide.  Tandem Smurf2 WW23 in complex with U24 peptide was in part assigned using three-dimensional (3D) gradient-enhanced [1H-15N]-NOESY-HSQC (τm=150 ms) spectra and [1H-15N]-TOCSY-HSQC (τm=60 ms) spectra.    	   	   85 3.2.3 Circular Dichroism Far-UV CD measurements were conducted on a Jasco J-815 spectropolarimeter equipped with a circulating temperature-controlled water bath. Experiments were conducted on a 200-µL sample of Smurf2 WW23 alone, U24 peptide alone or in the context of the complex with WW23/U24 peptide ratio (1:5.4) in a solution of 10 mM HEPES, 2.5 mM NaCl, pH 7.2. Data were collected using a quartz cuvette with a 1-mm pathlength in the 200-250 nm wavelength range. Starting at 5°C, the temperature was incremented to 65°C in the interval of 5°C. Data were recorded at a rate of 100 nm/min in 0.1-nm intervals. Each data set represents an average of four scans. The raw data were corrected by removing the contribution from the buffer and were converted to mean residue ellipticity [θ] as a function of wavelength of electromagnetic radiation using the equation:                  [θ] = [(106 Δε)/ncl] deg · cm2 /dmol                       (3.3) where Δε is the observed ellipticity in millidegrees, c is the concentration in micromolar, n is the number of residues and l is the cuvette pathlength in millimeters.  3.3 Results 3.3.1 Studying the Secondary Structure by Circular Dichroism To analyze the stability and the secondary structure of tandem Smurf2 WW23, and its complex with U24 peptide, circular dichroism (CD) spectra in the far-UV region (200-250 nm) were recorded. At relatively low temperature (such as 25°C), the spectra of Smurf2 WW23 exhibit a positive band at around 230 nm due to aromatic contribution and a negative band between 205 nm and 210 nm due to random-coil contribution (Figure 3.2(A) and Figure 3.2(B)). These spectral features, which are characteristic of well-folded WW domains, as reported 	   	   86 previously, are identical with Smurf2 WW3 domain, implying that the tandem Smurf2 WW23, in the unbound form, is correctly folded. As a result of the CD findings, the NMR experiments of Smurf2 WW23 were carried out at 25°C, where Smurf2 WW23 is folded.       Figure 3.2 Analysis of the secondary structure by CD spectroscopy. Far-UV CD spectra of (A) Tandem Smurf2 WW23 protein, and (B) Complex of Smurf2 WW23 and U24 peptide (1:5.4) were measured in a temperature range of 5°C to 65°C. Spectra were corrected for buffer contributions. Inset: thermodynamics of temperature-induced changes at 231 or 229 nm.  (A) (B) 	   	   87 In addition, to further test how the temperature influences the stability and the secondary structure of tandem Smurf2 WW23 and its complex with U24 peptide, we monitored changes in the far-UV CD signal at 229 or 331 nm as a function of temperature. For Smurf2 WW23 alone, the melting curve shows cooperative signal changes with well-defined linear pre- (5°C to 25°C) and post- (55°C to 65°C) transition baselines (Figure 3.2(A)). This is consistent with an apparent temperature-induced two-state unfolding of WW domains. Furthermore, the Smurf2 WW23 undergoes a single cooperative transition during thermal melting[209]. This could represent cooperative unfolding of the coupled WW23 or an overlay of WW2 and WW3 unfolding transitions. Compared with Smurf2 WW3 domain described in section 2.4, tandem Smurf2 WW23 seems to have the similar melting temperature. The behavior of Smurf2 WW23 with U24 peptide is almost the same with Smurf2 WW23 alone, except for estimated higher melting temperature (Figure 3.2(B)). It should be noted that the melting temperature of WW23 with U24 peptide could only be estimated because there are not enough data points at the higher temperatures. Furthermore, the melting temperature of Smurf2 WW23 with U24 is suggested to be slightly higher than that of Smurf2 WW3 with U24. These conformational changes in Smurf2 WW23 alone and with U24 peptide were completely reversible.   The interaction and the effects on the components of secondary structure were also evaluated by CD spectroscopy; the data show only a minute conformational stability induced by the binding of U24 peptide to the Smurf2 WW23. All spectra of Smurf2 WW23 and its complex were fitted using three different programs (CDSSTR[221], CONTINLL[222], SELCON3[223]), and the full data set (points at every 1 nm in the range between 200 and 250 nm) to estimate the percentage of β-strand. The calculated results (Figure 3.3) indicate that at low temperatures (from 5°C to 35°C), there is no significant difference between Smurf2 WW23 with and without 	   	   88 U24 peptide. This suggests that interaction with U24 peptide may not significantly change the secondary structure and enhance the stability of tandem Smurf2 WW23 when compared with Smurf2 WW3 domain at low temperature. Overall, interaction with U24 peptide does not enhance the stability of Smurf2 WW23 in a significant manner, based on the associated errors.    Figure 3.3 Plot of the percentage of beta-strands at different temperatures in tandem Smurf2 WW23 with and without U24 peptide. The values with errors are listed in the following: 0.27±0.06 (5°C), 0.27±0.06 (15°C), 0.27±0.08 (25°C), 0.26±0.06 (35°C), 0.25±0.04 (45°C), 0.25±0.05 (55°C), 0.24±0.07 (65°C) for Smurf2 WW23; 0.28±0.04 (5°C), 0.28±0.04 (15°C), 0.28±0.03 (25°C), 0.27±0.03 (35°C), 0.27±0.04 (45°C), 0.27±0.04 (55°C), 0.27±0.06 (65°C) for Smurf2 WW23 with U24 peptide.   3.3.2 Characterization of the Tandem Smurf2 WW23-U24 Interactions by NMR To define the interaction of the WW2 and WW3 domains of Smurf2 in tandem with U24 peptide, NMR was used to obtain a structural explanation. The analysis of the 1H-15N HSQC spectrum (Figure 3.4(B)) recorded from apo Smurf2 WW23 at 25°C indicates that some amide signals are broadened beyond detection and that there is a region (around 8.5 ppm in the 1H dimension) with poor resolution. Previous NMR data showed that Smurf2 WW2 and WW3 coupling with its associated restriction in linker flexibility is not constitutive, suggesting that in the unbound state Smurf2 WW23 may exchange between coupled and uncoupled WW domain 	   	   89 conformations due to its flexible linker. Thus, this clustering of the peaks is likely due to exchange between multiple conformations and/or aggregation[209].         Figure 3.4 NMR titration studies of Smurf2 WW23 and U24 peptide. (A) Alignment of the U24 peptide from HHV-6A and WW domains and the linker from Smurf2. The PPxY motif is highlighted in grey. (B) Overlay of HSQC spectra of a 15N-labeled tandem Smurf2 WW23 in the free (green) and U24 peptide bound state (purple) at 25°C. The chemical shift perturbations caused by U24 peptide binding are highlighted by blue arrows and crosspeaks are labeled on the spectra. 	  	       MDPPRTPPPSYSEV          252  DLPEGYEQRTTQQGQVYFLHTQTGVSTWHDPRV 294  PRDLSNINCEELG 298  PLPPGWEIRNTATGRVYFVDHNNRTTQFTDPRLSAN  U24 (HHV-6A) hSmurf2 WW2 hSmurf2 WW2-3 linker hSmurf2 WW3 (B) (A) G302 F315 G311 T322 N320  R321  R312 Q324 F325 T308 	   	   90                                	          Figure 3.5 Binding affinity of Smurf2 WW3 and U24 peptide. (A) Several portions of Smurf2 WW23 1H-15N HSQC spectrum (green), following addition of U24 peptide at molar ratio of 1:1.71 (orange), 1:4.46 (blue), 1:15.25 (purple) at 25°C. (B) Binding curves derived from a representative set of residues experiencing high chemical shift perturbations at 25°C, describing combined 1HN and 15N chemical shift perturbation of Smurf2 WW23 residues (in ppm) observed upon addition of increasing amount of U24 peptide. (C) Dissociation constant (Kd) at 25°C was obtained by fitting of titration curve of four well-resolved, significantly perturbed peaks, identified in (B). 	    Residues Kd (µM) T326 274.6 R312 322.1 R321 357.4 N320 354.7 Average 327 ± 38 (A)  T322  N320 R312   Q324 (B)  T322  R312  R321  N320 (C) 	   	   91 An exploration of the interactions was carried out by adding unlabeled U24 peptide into 15N-labeled Smurf2 WW23 while monitoring the chemical shift perturbations for backbone residues in HSQC NMR spectra (Figure 3.5(A)). Titration of U24 peptide causes significant chemical shift changes and line broadening of resonances, which indicate binding to Smurf2 WW23, because large effect is not due to nonspecific hydrophobic interactions. Interestingly, there are two peaks corresponding to the side chain of Trp residue from WW3 domain during the whole process of titration, suggesting that the side chain of Trp-303 lies in the slow exchange between two states. The residues Arg-321, Thr-323 and Phe-325 are strongly perturbed, presumably due to interaction with the PPxY motif, as described previously in Smurf2 WW3-U24 complex. Other residues, such as Gln-324, Gly-302, Thr-322, Asn-320, Trp-303 and Arg-312 from WW3, are also found to be perturbed and the movement of those peaks during the titration with U24 peptide into Smurf2 WW23 is identical with that in the titration with U24 peptide into Smurf2 WW3. This suggests that most strongly perturbed peaks correspond to residues of WW3 and they are similarly affected by addition of U24 peptide. Thus, we propose that the residues participating in the binding for U24 peptide are mainly from WW3. In addition, the region in the center of the spectrum, mainly composed of the residues from WW2, is still poorly dispersed even after the saturation point is reached. This suggests that the residues of WW2 are not affected significantly upon binding to U24 peptide. Overall, the residues of WW3 play a significant role in Smurf2 WW23 binding to U24 peptide. It is not possible to fully exclude that WW2 is involved in this interaction, but the effect of WW2 involvement seems subtle. Because the resonances of Arg-321, Arg-312, Asn-320 and Thr-322 show that they are in the fast exchange regime, we fitted the chemical shift perturbations curves (Figure 3.5(B)) of 	   	   92 these four peaks to a 1:1 binding model, with individual amplitudes and offsets for each curve. An average Kd value of 327 ± 38 µM at 25°C is obtained.        3.3.3 HSQC Spectrum of Smurf2 WW23 in the U24 Peptide Bound State in Phosphate Buffer The HSQC spectra of Smurf2 WW23 in the absence and presence of saturating amounts of U24 peptide in different buffer were recorded. The HSQC spectrum of apo Smurf2 WW23 in the phosphate buffer, with the narrow amide proton dispersion, the broadness of the peaks and the limited number of peaks (data not shown), was worse than the one shown in Figure 3.4(B). However, in the phosphate buffer (40 mM phosphate, 20 mM NaCl, 0.05% NaN3, 10% D2O)) at pH 7.8, a well-dispersed HSQC spectrum of Smurf2 WW23-U24 complex can be obtained (Figure 3.6).    Figure 3.6 1H-15N HSQC spectrum of 15N-labeled Smurf2 WW23 in the presence of U24 peptide at 25°C in phosphate buffer (40 mM phosphate, 20 mM NaCl, 0.05% NaN3, 10% D2O) at pH 7.8.  	   	   93 3.4 Discussion 3.4.1 Strength of U24-Smurf2 WW23 Interaction  By carrying out the titration experiment, the binding affinity of tandem Smurf2 WW23 with U24 peptide was determined at 25°C. To confirm the binding affinity, isothermal titration calorimetry (ITC) was used (work carried out by Y. Sang in our group). The dissociation constant (Kd) value (Table 3.1) obtained by ITC agrees with that obtained from fitting of the NMR chemical shift perturbations for the 15N-labeled Smurf2 WW23 titrated with U24 peptide (Kd=327 ± 38 µM at 25°C). In addition, the ITC data shows that binding of Smurf2 WW23 domain to PPxY motif is still largely driven by favorable enthalpic forces (ΔH° < 0) accompanied by unfavorable entropic changes (ΔS° < 0) at 15°C and 25°C, implying that the intermolecular interactions such as hydrogen bonding and van der Waals contacts likely account for the specificity of this key protein-ligand interaction, which is the same as the interaction between Smurf2 WW3 domain and U24 peptide discussed above.   Table 3.1 Thermodynamic parameters obtained from ITC measurements for the binding of the Smurf2 WW23 domain to U24 peptide.  T (°C) Kd (µM) ΔH° (kJ/mol) ΔS° (J/molK) 5 128 ± 4 -19.5 ± 0.2 4.4 ± 0.6 15 225 ± 6 -27.4 ± 0.6 -25 ± 2 25 302 ± 5 -30.6 ± 0.9 -35 ± 3   This binding affinity at 25°C is almost the same with that of Smurf2 WW3 and U24 peptide, which also reflects that the WW2 domain does not likely enhance the binding affinity for U24 	   	   94 peptide. Moreover, the strength of the Smurf2 WW23 and U24 peptide interaction is much weaker than that of Smurf2 WW23 and Smad7 peptide interaction, of which Kd was determined to be 1.7 ± 0.4 µM at 20°C by intrinsic fluorescence[209].  3.4.2 Comparison with Smurf2 WW3-U24 Interaction   This binding affinity of Smurf2 WW23 and U24 peptide is almost the same with that of Smurf2 WW3 and U24 peptide, as described above. This suggests that the WW2 domain does not enhance the binding affinity for U24 peptide. To compare the Smurf2 WW23-U24 interaction with Smurf2 WW3-U24 interaction from a structural view, the HSQC spectrum of Smurf2 WW23 in the presence of saturating amounts of U24 peptide is compared to the spectrum of Smurf2 WW3 bound to U24 (Figure 3.7 and Figure 3.8). Chemical shift changes are observed upon addition of U24 peptide to either the isolated WW3 or the WW23 tandem. Figure 3.7 shows that the residues from the β2 strand, the β3 strand and the β2-β3 loop of WW3 domain (such as Tyr-314, Phe-315, Arg-321, and Thr-323) are strongly perturbed when Smurf2 WW3 and Smurf2 WW23 bind to U24 peptide, indicating that both Smurf2 WW3 and Smurf2 WW23 possibly employ similar residues participating in the interactions with U24 peptide. Thus, it can be proposed that Smurf2 WW23 binds to PY motif of U24 peptide in a similar way with isolated Smurf2 WW3. However, there are possibly some differences between the Smurf2 WW23-U24 interaction and the Smurf2 WW3-U24 interaction.   Furthermore, whether there are additional interactions from WW2 exiting in the Smurf2 WW23-U24 complex when compared with the Smurf2 WW3-U24 complex needs to be discussed. It has been reported that by comparison of 1H-15N HSQC spectrum of WW3 to that of WW23 in the presence of saturating Smad7 peptide, the chemical shifts of many WW3 residues 	   	   95 differed significantly, implying that the WW3 did not bind independently[209]. In our case (Figure 3.8), the chemical shifts of one-third of WW3 resonances (such as Thr-308, Phe-315 and Arg-312) differ. Those resonances are mainly from the β1 strand and the β1-β2 loop. The chemical shifts of the other WW3 resonances, mainly from the β2 strand, the β3 strand and the β2-β3 loop, do not change. This indicates that the residues on the β1 strand and in the β1-β2 loop of WW3 are likely affected due to the existing WW2 domain. In addition, peaks corresponding to the WW2 in the Smurf2 WW23/U24 spectra do not shift extensively as reported in the WW23/Smad7 spectra. Therefore, it is proposed that the WW2 domain may interact with the WW3 domain and/or U24 peptide but this interaction does not significantly affect the Smurf2 WW23 binding to U24 peptide. Overall, it can be concluded that Smurf2 WW23 binds to U24 peptide mainly relying on the residues from WW3, and in a similar way with isolated Smurf2 WW3. WW2 may be also involved in interacting with the WW3 domain and/or U24 peptide, although such interactions have no significant effect on the binding affinity.           	   	   96   Figure 3.7 Comparison of chemical shift perturbations from Smurf2 WW3 and Smurf2 WW23 upon binding to U24 peptide. (A) HSQC spectra of Smurf2 WW3 in apo (black) and bound (red) form at 25°C (on top). Plot of the normalized chemical shift changes versus the residue number of Smurf2 WW3 (on the bottom). The chemical shift perturbations caused by U24 peptide binding are highlighted by blue arrows and crosspeaks are labeled. The normalization factor was 3.218 for the nitrogen shifts (blue bars) and 0.343 for the proton shifts (red bars). (B) HSQC spectra of tandem Smurf2 WW23 in apo (black) and bound (red) form at 25°C (on top). Plot of the normalized chemical shift changes versus the residue number of Smurf2 WW23 (on the bottom). The chemical shift perturbations caused by U24 peptide binding are highlighted by blue arrows and crosspeaks are labeled. In the plot, residue E304 is labeled with the star because upon the saturation with U24 peptide, there are two peaks corresponding to E304, indicating the slow exchange. The normalization factor was 3.190 for the nitrogen shifts (blue bars) and 0.205 for the proton shifts (red bars). 	   	   97  Figure 3.8 Superposition of Smurf2 WW3 (blue) and ww23 (red) 1H-15N HSQC spectra recorded in the presence of U24 peptide at 25°C. Peaks are labeled for several residues. 	  3.4.3 Comparison with the Smurf2 WW23-Smad7 Complex Studies has shown that the Smurf2 WW2 domain augments the interaction of Smurf2 WW3 and Smad7 PY motif by binding to the WW3 and making auxiliary contacts with the PY motif and a novel E/D-S/T-motif, which is N-terminal to all Smad PY motifs[209]. In the Smurf2 WW23-Smad7 complex[209] (Figure 3.9(B)), the second and third β-strand of the WW2 pack against the third β-strand of the WW3, with the strands of the two domains oriented approximately perpendicular to each other. The coupled WW domains form an interaction with the Smad7 peptide that restricts the motion of 10 Smad7 residues (from Glu-205’ to Tyr-214’). The WW3 forms the primary contacts with the Smad7 peptide. The PY motif and PY tail, formed by the six residues C-terminal to the PY motif, bind in a conformation that is similar to their interaction with the isolated WW3, as observed in Figure 3.9(B). The PY tail loops around and binds residues on the first and second β-strands of WW3 and is stabilized by intrapeptide interactions. The WW2 forms additional contacts with the prolines of the PY motif T308 	  	   R312 F315 	  Q324 T308 T323 G311 T322 N307 N319 R321 H318 D317 N333 R306 	   	   98 (Pro-208’-Pro-210’) and the proline preceding the PY motif (Pro-207’)[209]. Glu-205’ and Ser-206’, which are N-terminal to the PY motif, interact with Trp-279. The WW domain coupling places Trp-279 of WW2 directly opposite the position normally occupied by the binding pocket tryptophan of WW3, where it functions to compensate for the lack of this binding pocket tryptophan. The slightly reduced affinity of Smurf2 WW23 for mutant Glu205’Ala Smad7 peptide relative to wild type, suggests that the glutamic acid residue in position 205’ makes a small energetic contribution to the interaction. In Figure 3.9(B), the region that is N-terminal to the PY motif has a different conformation between Smurf2 WW23-Smad7 and Smurf2 WW3-Smad7 complex. Thus the Smurf2 WW2 domain makes auxiliary contacts with the PY motif and binds to an E/D-S/T-P motif N-terminal to the PY motif enhancing the affinity of Smurf2 for Smad7 over 20-fold. The Smurf2 WW23 also binds to Smad1 and Smad2 peptides, suggesting that the interaction of the E/D-S/T-P sequence with WW2 serves as a specific marker for Smad recognition by Smurf2[209]. Phosphorylation of the threonine in the E/D-S/T-P motifs of Smad2 and Smad3 can modulate their interaction with Smurf2 WW2[245].          	   	   99     Figure 3.9 Comparison of the Smurf2 WW23-Smad7 complex and the Smurf2 WW3-Smad7 complex. (A) Alignment of U24 and Smad7 PY motif. PPxY motif is highlighted in red. Residues with negative and positive charges are highlighted in cyan and yellow, respectively. (B) Ribbon diagram of the Smurf2 WW23-Smad7 complex (left) and the Smurf2 WW3-Smad7 complex (right). The backbone of Smad7 is shown in green. The first and second proline, and tyrosine in PY motif are shown in yellow. The WW2, linker, WW3 are shown in blue, grey and red, respectively. The models are derived from solution NMR structures (PDB ID 2DJY and 2KXQ) and generated using PyMOL. 	  However, a different principle that governs the interactions of Smurf2 ubiquitin ligases with Smad7 has been proposed. Neither the contacts between the two WW domains nor the contacts between the WW2 domain and Smad7, which was described above, are observed[229]. Instead, it is suggested that Smurf2 WW-WW pairs have a high tendency to form homo-dimers via its WW2 domain. The presence of Smad7 peptide does not prevent this dimerization since the WW-WW domain pairs bind the Smad7 mainly through contacts with the WW3 domain. Thus in this case, Smurf2 WW2 domain has a minor role in binding to short PY containing sequences, but an important role in protein oligomerization and aggregation[229]. The affinity increase due to Smad7 202’-217’  U24   1-15  SELESPPPPYSRYPMD         MDPPRTPPPSYSEVL  (A) (B) β1 β2 β3  β1 β2  β3  β1  β2  β3 	   	   100 the presence of the WW2-WW3 domain pair is about 2-fold with respect to the values obtained with the Smurf2 WW3 domain at a given temperature[229], in contrast to previous observations[209]. In our case, the data possibly supports that the WW2 domain plays a minor role in the Smurf2 WW23 interaction with U24 peptide. The binding affinity of tandem Smurf2 WW23 and U24 peptide is almost the same with that of Smurf2 WW3 and U24 peptide, indicating that the WW2 domain does not play a significant role in enhancing the binding affinity. In addition, the titration experiments suggest that significant perturbations of resonances mainly occur in the WW3 domain upon binding to U24 peptide. And in the HSQC spectrum of Smurf2 WW23-U24 complex, the peaks are less dispersed when compared with the HSQC spectrum of Smurf2 WW23-Smad7 complex previously reported[209] (data not shown), implying that Smurf2 WW2 domain has more significant effects on the Smurf2 WW23-Smad7 interaction than on the Smurf2 WW23-U24 interaction. As a result, the data suggests that Smurf2 WW2 domain likely plays only a very minor role in binding to U24 peptide.   3.4.4 Implication for the WW Domains and PY Motifs Interaction The rules governing target recognition by HECT type E3 ubiquitin ligases are open questions, and the work to date reflects a more complex scenario than originally expected[229]. Although they all have multiple WW domains, some family members recognize PY motifs using a single WW domain, as is the case of Itch binding to the Epstein Barr virus protein LMP2A[246] and Nedd4 binding to the voltage gated sodium channel[206] and to Commissureless[228]. In other cases, such as the binding of Smurf1 and Nedd4L to R-Smads, the proteins use a pair of WW domains to expand the binding interface with a composite binding site that includes 	   	   101 pSer/pThr-Pro elements in addition to a canonical PY motif, a combination that allows regulation of the interaction by input-driven protein kinases[245, 247, 248]. WW domain clustering results in a variety of tertiary interactions, which refine their function[209]. For example, FBP11’s two consecutive WW domains likely enable simultaneous binding to two polyproline target motifs in forming homology 1 regions, enhancing the affinity of this interaction[242]. The two WW domains of Prp40 are joined by a stable α-helical linker that fixes the relative orientation of the WW domains, such that their binding pockets face in opposite directions[243]. This may enable the Prp40 WW domains to interact simultaneously with two proline-rich target sites and function as an adapter. The WW3 of suppressor of deltex [Su(dx)] interacts with the Su(dx) WW4 domain inhibiting the WW4 from binding to a Notch-PY motif. Binding of a ligand to the Su(dx) WW3 stabilizes it and reduces its inhibitory interactions with the WW4[230]. Thus, the Su(dx) WW3 enables context-dependent regulation of WW4 target binding. In contrast, the WW domains of Smurf2 are proposed to have a joint binding surface that enhances target selectivity[209]. Therefore, interactions between adjacent WW domains introduce added complexity to their functions. The length and structure of the interdomain linkers also appear to be crucial. For example, the increased linker length in the longer Smurf1 isoform reduces the affinity of the WW domains for each other and for Smad7 and alters the binding mechanism[209]. Short conserved linkers indicate a requirement for interaction between the WW domains. One interesting example is hWWP2 and hItch, which have no linker between the first two WW domains, suggesting that they are fused into a super-WW domain structure[209]. In addition, the presented data up to date suggest a versatility of WW domains depending on the target protein in that a given HECT E3 ubiquitin ligase can use WW domains singly or in a combinatorial manner[229, 245], depending on the target. For example, in case of Nedd4L, the 	   	   102 interaction with Smad7 peptide involves a single WW domain (WW2 preferentially)[229]. The affinity of the Nedd4L WW2 domain for the Smad7 PY region is based on aggregate contacts with a canonical PY motif, a C-terminal extension of this motif that aligns on the first strand of the WW2 domain, and an electrostatic balance between a glutamic and an aspartic acids (Glu-205 and Asp-217) in Smad7 and two positively charged residues positioned in loop 1 of the Nedd4L WW2 domain, as described in section 4.3 of Chapter 2. Furthermore, Nedd4L binding to Smad2/3 involves a second WW domain, WW3, for contacts with a separate, dephosphorylated region down stream of the PY motif. In Smad2/3 a phosphothreonine N-terminal to the PY motif (pThr-179) makes a critical contribution in the binding to a Nedd4L WW domain[245]. In the case of Smad7 a corresponding serine residue (Ser-206) does not need to be phosphorylated for high affinity binding to Nedd4L; instead, an acidic residue, Glu-205, plays the part of pThr-179 in Smad3. Therefore, Nedd4L binds Smad7 using a single WW domain interaction, while it binds Smad2/3 using two WW domains that require multiple phosphorylation of the target region by different protein kinases.   3.5 Conclusion The binding affinity of tandem Smurf2 WW23 and U24 peptide is determined to be almost the same with that of Smurf2 WW3 domain and U24 peptide. In addition, the NMR results suggest that the contacts between Smurf2 WW2 domain and U24 peptide were almost not observed. Overall, Smurf2 WW2 domain is suggested to play a very minor role in increasing the binding affinity of Smurf2 and U24. We have proposed that Smurf2 may potentially interact with PY motif of U24 protein through its WW domains to modulate the function of U24, a general block in endosomal recycling. In terms of weak interaction between Smurf2 WW23 and U24 	   	   103 peptide, Smurf2 may not be an ideal binding partner of U24 PY motif. However, the interaction between Smurf2 WW domains and phosphorylated U24 peptide should be investigated because phosphorylated U24 peptide may have a much better binding affinity for Smurf2 WW domains. Other WW domain containing proteins, such as Nedd4, also need to be further investigated in order to find an optimal binding partner for U24 PY motif, which is also able to modulate the function of U24.                         	   	   104     4.1 Summary WW domain – ligand interactions are essential for many biological processes. The associations between WW domains and PY motif-containing region at the N-terminus of U24 protein from HHV-6 are critical for regulation of U24 function, which U24 acts to block the early endosomal recycling[141]. A general block of endosomal recycling by U24 may be related to the latency of HHV-6 and associated with many diseases, such as multiple sclerosis[145] (Chapter 1). In order to recognize the specific WW domain-containing protein and expand our understanding into the mechanisms of specific recognition involved in WW domain – U24 complexes, we have used a multidisciplinary approach to study Smurf2 WW domain – U24 PY motif (from HHV-6A) interactions. Using GST pull-down assay, we have determined that full-length U24 binds to the Smurf2 WW3 domain and Smurf2 WW23 (Smurf2 WW2 and WW3 in tandem) via its PPxY motif, while there is no interaction between Smurf2 WW2 and U24 protein. Initial structural studies focused on the complex between the U24 peptide and Smurf2 WW3 domain by using NMR. The PPxY motif binds to the XP groove and Tyr binding pockets of Smurf2 WW3, which are identical with other Smurf2 WW3 – ligand interactions. However, the Smurf2 WW3 binding affinity for U24 peptide was determined to be low by titration experiments, when compared with other Smurf2 WW3 – ligand interactions. The difference of binding affinity may be result from the lack of extra interactions that are provided by the regions outside the PPxY motif. The Smurf2 WW3 domain has a Phe in place of the canonical binding pocket Trp and the Phe Chapter 4 Conclusions and Future Work 	   	   105 decreases the affinity of the WW3 domain for the PPxY motif, restricting the Smurf2 WW3 to binding PPxY motif regions that have additional complementary surfaces with which to bind (Chapter 2). Subsequent structural studies focused on the complex between the U24 peptide and tandem Smurf2 WW23 by using NMR. The PPxY motif should bind to the residues of the WW3 domain, which are identical with other Smurf2 WW23 – ligand interactions. The Smurf2 WW23 binding affinity for U24 peptide was determined to be low by titration experiments, which is almost the same with the Smurf2 WW3 binding affinity for U24 peptide. This suggests that Smurf2 WW2 domain may paly a minor role in the interaction between Smurf2 WW23 and U24 peptide (Chapter 3). Taken together, both Smurf2 WW3 and Smurf2 WW23 have relatively low binding affinity for U24 peptide.  4.2 Future Work  Binding Analysis of Phosphorylated U24 Peptide In some cases, phosphorylated PY motif-containing peptides are necessary for high affinity binding to WW domains. For example, Nedd4L binds Smad2/3 using two WW domains that require multiple phosphorylation of the target region by different protein kinases. In Smad2/3 a phosphothreonine N-terminal to the PY motif (pT197) makes a critical contribution in the binding to a Nedd4L WW domain[245]. As discussed above, the residue Arg with the positive charge in U24 peptide may disrupt some interactions that are found between Arg residue from Smurf2 WW3 and Glu residue from Smad7 peptide. Phosphothreonine in U24 peptide may play the part of Glu to interact with Smurf2 WW3. Thus, it can be predicted that phosphorylation of 	   	   106 U24 (U24: PRTPPPSY; phosphorylation site underlined) may result in enhanced WW domain binding. It has been demonstrated that U24 protein from HHV-6A can be efficiently phosphorylated by MAPK in vitro[249].  Binding Analysis of Other Possible WW Domains Neither the WW2 nor the WW3 of Smurf2 is canonical. Smurf2 WW2 domain has a tyrosine at the site normally occupied by the first conserved tryptophan and Smurf2 WW3 domain has a phenylalanine at the site normally occupied by the second conserved tryptophan. This feature is discriminative with other classic WW domains and restricts the Smurf2 WW3 domain to binding PY motif regions that have additional complementary binding surfaces. Except for Smurf2, there are many other members in the Nedd4 family of Ubiquitin ligases, such as Nedd4 protein and WWP protein. They are involved in the endosomal pathway and all contain multiple WW domains, which have two conserved tryptophan residues. Thus, there is possibility that they can have high affinity for binding U24 peptide when compared with Smurf2 WW domains and this interaction is likely critical for regulation of U24 function. In addition, the structural analysis of other WW domain – U24 peptide complexes may shed light into understanding results form thhe binding analysis described above. Mutagenesis experiments of WW domains would complement these structural studies. Overall, structural studies on those interactions, which also can be compared to those already solved, will further advance our understanding into specific recognition of WW domain – ligand complexes.   Binding Analysis Using Cell Lines The cell studies of WW domain – U24 interactions, followed by the in vitro studies, are 	   	   107 necessary, because they can demonstrate whether in cell lines U24 protein from HHV-6A can bind those WW domains, which have been identified in vitro, and whether this interaction can affect the function of U24. Performing in vivo expression of U24 followed by immunoprecipitation and probing for WW domain-containing protein is a possible next step. In addition, monitoring of certain enzyme activity and protein levels participating in the endosomal recycling in the presence of unbound U24 protein and U24 – WW domain interaction may show which WW domain can bind to U24 protein for regulating its function in vivo.    	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	   	   108 References 	  [1] Berneman, Z.N., Ablashi, D.V., Li, G., Eger-Fletcher, M., Reitz, M.S., Hung, C.-L., Brus, I., Komaroff, A.L., and Gallo, R.C., Human herpesvirus 7 is a T-lymphotropic virus and is related to, but significantly different from, human herpesvirus 6 and human cytomegalovirus. Proceedings of the National Academy of Sciences, 1992. 89(21): p. 10552-10556. [2] Mori, Y., Recent topics related to human herpesvirus 6 cell tropism. Cell Microbiol, 2009. 11(7): p. 1001-6. 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FEBS letters, 2008. 582(18): p. 2685-2688.    	   	   125 Appendices 	  Appendix	  A	   	   	  [1H-15N]-NOESY-HSQC and [1H-15N]-TOCSY-HSQC data for Smurf2 WW3-U24 complex A.1 Strips from the [1H-15N]-NOESY-HSQC and [1H-15N]-TOCSY-HSQC experiments were taken at the 15N and 1HN chemical shifts of residues L299 to R312 of Smurf2 WW3 domain.    	  	  	  	  	  	  	  	  L299 G302 W303 E304 I305 R306 N307 T308 T310 G311 R312 	   	   126 	  	  	  	  	  A.2 Strips from the [1H-15N]-NOESY-HSQC and [1H-15N]-TOCSY-HSQC experiments were taken at the 15N and 1HN chemical shifts of residues V313 to T323 of Smurf2 WW3 domain. 	  	  	  	  	  	  	  	  	  	  	  	  V313 Y314 F315 V316 D317 H318 N319 N320 R321 T322 T323 	   	   127 	  	  	  	  	  A.3 Strips from the [1H-15N]-NOESY-HSQC and [1H-15N]-TOCSY-HSQC experiments were taken at the 15N and 1HN chemical shifts of residues Q324 to L330 of Smurf2 WW3 domain. 	  	  	   	   	   	   	   	   	   	   	   Q324 F325 T326 D327 R329 L330 

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