Medicine, Faculty of
Biochemistry and Molecular Biology, Department of
DSpace
UBCV
Hui, Lok Tin
Master of Science - MSc
University of British Columbia
The ABC transporter Rv1747, which is important for Mycobacterium tuberculosis (Mtb) growth in infected host cells, has a cytoplasmic regulatory module with two phosphothreonine-binding FHA domains joined by a flexible linker with multiple phospho-acceptor threonines (pThr). In vitro, the isolated regulatory module, Rv1747¹⁻³¹⁰, undergoes phosphorylation-enhanced phase separation upon linker phosphorylation by Mtb serine/threonine kinases. Phosphorylation-based phase separation was proposed to result from multivalent intra/intermolecular FHA-pThr interactions. To further dissect the mechanisms of Rv1747 phase separation, I have investigated the factors that influence droplet formation. Visualized with a temperature-controlled microscope, I found that warming phosphorylated Rv1747¹⁻³¹⁰ droplets from 22 oC to 37 oC modestly reduced their size. Studies with Rv1747 truncation fragments and mutants demonstrated that the FHA-1 domain has a predominant role in phosphorylation-enhanced phase separation. Substitution of a key binding residue, S47, in the FHA-1 domain to alanine impaired droplet formation. In contrast, variants with the analogous mutation, S248A, in the FHA-2 domain, or lacking the FHA-2 domain entirely, still underwent phase separation. However, the resulting droplets were gel-like, indicating both FHA domains are required for “regular” droplet formation. The role of the intrinsically disordered linker was also studied. A polypeptide corresponding to the isolated linker did not phase separate. Also, a comparison of Rv1747¹⁻²¹³ (FHA-1 domain and linker) versus Rv1747¹⁻¹¹⁰ (FHA-1 domain only) by NMR spectroscopy indicated the linker residues do not interact with the FHA-1 domain. Substitution of threonine residues in the linker to alanines revealed that T152 is a key residue for the phosphorylation-enhanced phase separation of the regulatory module. I also found that Rv1747¹⁻³¹⁰ treated with PknF in the absence of ATP still underwent phase separation. Based on studies using inactive PknF variants, I hypothesize that PknF with activating pThr residues directly contributes to droplet formation by bridging the FHA domains of Rv1747¹⁻³¹⁰. Lastly, I observed that protein partnerships with the stress protein Rv2623, and Rv1747 linker phosphopeptides influenced Rv1747¹⁻³¹⁰ droplet formation. Although the underlying mechanisms have not been fully established, my studies support the notion that Rv1747 can be controlled by cellular pathways impacting the phase separation of its regulatory module.
DISSECTING THE BIOPHYSICAL MECHANISMS OF PHASE SEPARATION BY THE MYOBACTERIUM TUBERCULOSIS ABC TRANSPORTER RV1747 by Lok Tin Hui B.Sc., University of Manitoba, 2018 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Biochemistry and Molecular Biology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) September 2021 © Lok Tin Hui, 2021 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the thesis entitled: Dissecting the biophysical mechanisms of phase separation by the myobacterium tuberculosis ABC transporter rv1747 submitted by Lok Tin Hui in partial fulfillment of the requirements for the degree of Master of Science in Biochemistry and Molecular Biology Examining Committee: Lawrence McIntosh, Department of Biochemistry and Molecular Biology, UBC Supervisor Jörg Gsponer, Department of Biochemistry and Molecular Biology, UBC Supervisory Committee Member Calvin Yip, Department of Biochemistry and Molecular Biology, UBC Supervisory Committee Member Yossef Av-Gay, Department of Microbiology and Immunology, UBC Additional Examiner iii Abstract The ABC transporter Rv1747, which is important for Mycobacterium tuberculosis (Mtb) growth in infected host cells, has a cytoplasmic regulatory module with two phosphothreonine-binding FHA domains joined by a flexible linker with multiple phospho-acceptor threonines (pThr). In vitro, the isolated regulatory module, Rv17471-310, undergoes phosphorylation-enhanced phase separation upon linker phosphorylation by Mtb serine/threonine kinases. Phosphorylation-based phase separation was proposed to result from multivalent intra/intermolecular FHA-pThr interactions. To further dissect the mechanisms of Rv1747 phase separation, I have investigated the factors that influence droplet formation. Visualized with a temperature-controlled microscope, I found that warming phosphorylated Rv17471-310 droplets from 22 oC to 37 oC modestly reduced their size. Studies with Rv1747 truncation fragments and mutants demonstrated that the FHA-1 domain has a predominant role in phosphorylation-enhanced phase separation. Substitution of a key binding residue, S47, in the FHA-1 domain to alanine impaired droplet formation. In contrast, variants with the analogous mutation, S248A, in the FHA-2 domain, or lacking the FHA-2 domain entirely, still underwent phase separation. However, the resulting droplets were gel-like, indicating both FHA domains are required for “regular” droplet formation. The role of the intrinsically disordered linker was also studied. A polypeptide corresponding to the isolated linker did not phase separate. Also, a comparison of Rv17471-213 (FHA-1 domain and linker) versus Rv17471-110 (FHA-1 domain only) by NMR spectroscopy indicated the linker residues do not interact with the FHA-1 domain. Substitution of threonine residues in the linker to alanines revealed that T152 is a key residue for the phosphorylation-enhanced phase separation of the regulatory module. I also found that Rv17471-310 treated with PknF in the absence of ATP still underwent phase separation. Based on studies using inactive PknF variants, I hypothesize that PknF with activating pThr residues directly contributes to droplet formation by bridging the FHA domains of Rv17471-310. Lastly, I observed that protein partnerships with the stress protein Rv2623, and Rv1747 linker phosphopeptides influenced Rv17471-310 droplet formation. Although the underlying mechanisms have not been fully established, my studies support the notion that Rv1747 can be controlled by cellular pathways impacting the phase separation of its regulatory module. iv Lay Summary Protein phase separation is a recently discovered phenomenon shown to be crucial in biological processes including in cellular organization. The ABC transporter Rv1747, which is important for Mycobacterium tuberculosis (Mtb) growth in infected host cells, has a cytoplasmic regulatory module that undergoes phosphorylation-enhanced phase separation. The overarching goal of my thesis research was to dissect the mechanisms underlying Rv1747 phase separation and thereby provide a foundation for understanding the roles and regulation of this process in Mtb growth and infectivity. v Preface This thesis is a follow-up to the publication: Florian Heinkel, Libin Abraham, Mary Ko, Joseph Chao, Horacio Bach, Lok Tin Hui, Haoran Li, Mang Zhu, Yeou Mei Ling, Jason C. Rogalski, Joshua Scurll, Jennifer M. Bui, Thibault Mayor, Michael R. Gold, Keng C. Chou, Yossef Av-Gay, Lawrence P. McIntosh, and Jörg Gsponer (2018) “Phase separation and clustering of an ABC transporter in Mycobacterium tuberculosis” Proc. Natl. Acad. Sci. USA 116: 16326-16331. As a co-author, I contributed experimental data to this paper demonstrating that the isolated linker of Rv1747 does not undergo phase separating into liquid droplets. Under the guidance of Dr. Lawrence P. McIntosh, I designed, conducted and analyzed all of the experiments presented in chapters 3 and 4. Dr. Mark Okon assisted with recording and interpreting the 15N-HSQC spectra of the 15N-labeled Rv1747 constructs. Dr. Jason Rogalski recorded the ESI mass spectra presented herein. My laboratory colleagues, Karlton Scheu and Chloe Gerak, helped with designing and cloning Rv1747 variants. I wrote the thesis with comments provided by Dr. McIntosh. vi Table of Contents Abstract ................................................................................................................................................... iii Lay Summary ......................................................................................................................................... iv Preface ....................................................................................................................................................... v Table of Contents ................................................................................................................................... vi List of Tables ......................................................................................................................................... viii List of Figures ......................................................................................................................................... ix List of Abbreviations .............................................................................................................................. xi Acknowledgements ............................................................................................................................... xiii Dedication ............................................................................................................................................. xiv 1. Introduction .......................................................................................................................................... 1 1.1 Protein-protein liquid phase separation ..................................................................................... 1 1.1.1 General background ............................................................................................................... 1 1.1.2 Discovery and history ............................................................................................................ 3 1.1.3 Formation and regulation of droplets in different systems ............................................... 4 1.1.4 Protein-protein interactions within the phase separation scaffold ................................... 5 1.2 Mycobacterium tuberculosis (Mtb) ............................................................................................ 6 1.2.1 Features of Mtb ....................................................................................................................... 6 1.2.2 Virulence mechanism and infectious pathways of Mtb ..................................................... 7 1.2.3 PIM synthesis and macrophage resistance ......................................................................... 8 1.3 Rv1747 ABC Transporter ............................................................................................................. 9 1.3.1 Introducing Rv1747 ................................................................................................................ 9 1.3.2 Rv1747 regulatory module undergoes phosphorylation-dependent phase separation . 12 1.3.3 Rv1747 phosphorylation by the Mtb STPKs .................................................................... 14 1.3.4 Regulation of Rv1747 by Rv2623 ...................................................................................... 16 1.3.5 Rv1747 Clustering in vivo .................................................................................................. 16 1.4 Thesis goals ................................................................................................................................. 18 2. Materials and Methods ..................................................................................................................... 19 2.1 Cloning ..................................................................................................................................... 19 2.2 Protein expression and purification ...................................................................................... 20 vii 2.3 Imaging Rv1747 constructs using bright-field microscopy .............................................. 21 2.4 Image processing .................................................................................................................... 22 2.5 Microscopy studies of Rv17471-310 droplets as a function of temperature ...................... 22 2.6 MALDI mass spectrometry ................................................................................................... 22 2.7 Electrospray ionization mass spectrometry (ESI-MS) ........................................................ 23 2.8 15N-HSQC NMR for Rv17471-110 and Rv17471-213 ............................................................. 23 2.9 Interaction studies of Rv17471-310 and Rv2623 ................................................................... 23 2.10 Interaction between Rv1747 and a phosphopeptide ......................................................... 24 3. Results: Intrinsic factors determining Rv1747 phase separation ................................................. 32 3.1 Increasing temperature reduces phosphorylated Rv17471-310 droplet size ........................... 32 3.2 FHA-1 is required for phase separation by the Rv1747 regulatory module .................................. 34 3.3 Rv17471-213 forms gel-like droplets ............................................................................................... 37 3.4 The role of the linker in phase separation ...................................................................................... 39 3.5 The roles of the linker phosphoacceptors in phase separation ...................................................... 41 3.6 Summary ....................................................................................................................................... 43 4. Results: Intermolecular interaction for Rv1747 regulatory domain ............................................. 44 4.1 Rv1747 phase separation due to PknF docking ....................................................................... 44 4.2 Rv2623 inhibit Rv1747 phase separation ...................................................................................... 47 4.3 Rv1747 linker peptide enhances droplet size ................................................................................ 49 4.4 Summary ....................................................................................................................................... 51 5. Conclusions ......................................................................................................................................... 53 5.1 Dissecting the mechanism of PknF dependent Rv1747 phase separation .......................... 53 5.2 Possible follow-up studies ......................................................................................................... 55 References .............................................................................................................................................. 58 viii List of Tables Table 1. Reagents for PCR amplification ....................................................................................19 Table 2. Typical PCR thermal cycling ........................................................................................19 Table 3. Typical restriction enzyme digestion conditions ...........................................................20 Table 4: Expression constructs ....................................................................................................25 Table 5: Protein purification ........................................................................................................30 ix List of Figures Figure 1. Typical membraneless organelles in eukaryotic cells ...................................................2 Figure 2. A variety of biomolecules can phase separate ...............................................................6 Figure 3. Schematic summary of the PIM/LAM biosynthetic pathway ........................................9 Figure 4. Schematic of the assumed homodimeric Rv1747 ABC transporter ...........................10 Figure 5. Schematic of the Rv1747 ABC transporter regulatory module (residues 1-310) ........11 Figure 6. Conserved FHA structure and pThr-binding site ........................................................11 Figure 7. The Rv1747 regulatory module undergoes phosphorylation-enhanced phase separation into liquid-like droplets .............................................................................................13 Figure 8. Rv17471-310 droplets diffuse freely in solution and coalesce upon contact ..................13 Figure 9. A cartoon model of phase separation by the Rv1747 regulatory module resulting from a myriad of multivalent intra- and intermolecular interactions ...................................................14 Figure 10. Fluorescence images of Oregon Green-labeled Rv17471-310 droplets formed upon treatment by 10 STPKs from Mtb ...............................................................................................15 Figure 11. Homology model of the PknF kinase domain from the Swiss-Model repository (P9WI75 PKNF_MYCTU) ........................................................................................................16 Figure 12. A working model for phosphorylation-enhanced clustering and regulation of Rv1747 .....................................................................................................................................................17 Figure 13. Reconstructed ESI-MS of Rv17471-310 with PknF treatment .....................................32 Figure 14. Phosphorylated Rv17471-310 droplets shrink upon warming from 22 oC to 37 oC .....33 Figure 15. Quantitation of Rv17471-310 droplet images using Cellprofiler .................................33 Figure 16. Analyzing the effect of concentration and sample temperature on phosphorylated Rv17471-310 droplet parameters using Cellprofiler ......................................................................34 Figure 17. A functional FHA-1 domain is required for droplet formation by the phosphorylated regulatory module ........................................................................................................................35 Figure 18. A functional FHA-1 domain is also required for droplet formation by the unmodified regulatory module .......................................................................................................................36 Figure 19. Rv1747148-310, lacking the FHA-1 domain, does not phase separate .........................36 Figure 20. Reconstructed ESI-MS of Rv1747148-310 after treatment with PknF ..........................37 Figure 21. Rv17471-213 forms gel-like droplets ............................................................................38 x Figure 22. Rv17471-213 gel droplets cluster in various ways ........................................................38 Figure 23. Rv17471-213 gel droplets cluster instead of fusing ......................................................39 Figure 24. The isolated regulatory module linker, Rv1747105-228, does not phase separate ........40 Figure 25. 15N-HSQC spectra of 15N-labeled Rv17471-110 and Rv17471-213 ...............................41 Figure 26. T152 in the linker is important for phase separation .................................................42 Figure 27. Reconstructed ESI-MS of Rv17471-310 with all linker threonines, except T152, mutated to serines ........................................................................................................................43 Figure 28. PknF induces phosphorylation-independent phase separation of the Rv1747 regulatory module ........................................................................................................................45 Figure 29. Mutation of Rv1747 and PknF impact their binding and droplet formation ............47 Figure 30. Rv2623 impairs droplet formation by the phosphorylated Rv1747 regulatory module .....................................................................................................................................................48 Figure 31. Rv2623 does not perturb the structure of Rv17471-310 ...............................................49 Figure 32. The addition of a phosphopeptide mimic of pT152 did not diminish Rv17471-310 phase separation ..........................................................................................................................50 Figure 33. The presence of the phosphopeptide leads to larger droplets ....................................51 Figure 34. A cartoon figure illustrating the proposed mechanism of phase separation .............55 xi List of Abbreviations ABC ATP binding cassette AF647 Alexa Fluor 647 amu atomic mass unit ATP Adenosine triphosphate BSA Bovine serum albumin Ddx4 DEAD-box helicase 4 DNA Deoxyribonucleic acid EEA1 Early endosomal autoantigen 1 ESI-MS Electrospray ionization mass spectrometry FHA Forkhead-Associated FRAP Fluorescence recovery after photobleaching FUS Fused in Sarcoma GDP Guanosine diphosphate GTP Guanosine triphosphate HIV Human immunodeficiency virus hnRNP Heterogeneous nuclear ribonucleoprotein IAPP Islet amyloid polypeptide ID Intrinsically disordered IDR Intrinsically disordered region IFN-γ Gamma interferon LAM Lipoarabinomannan LM Lipomannan MALDI-ToF Matrix assisted laser desorption/ionization time-of-flight mRNA Messenger RNA Mtb Mycobacterium tuberculosis NBD Nucleotide binding domain Nck Non-catalytic region of tyrosine kinase NMR Nuclear magnetic resonance xii NPM1 Nucleophosmin 1 N-WASP Neural Wiskott-Aldrich syndrome protein OG Oregon Green PI3P Phosphatidylinositol-3-phosphate PIMs Phosphatidyl-myo-inositol mannosides PstP Serine/threonine protein phosphatase pThr Phosphothreonine Rab Rabaptin RGG domain Arginine–glycine–glycine repeat domain RNA Ribonucleic acid rRNA Ribosomal ribonucleic acid scFv Single chain variable fragment STORM Stochastic optical reconstruction microscopy STPK Serine/threonine protein kinase TB Tuberculosis TMD Transmembrane domain UV Ultraviolet WHO World Health Organization WT Wild type xiii Acknowledgements I would also like to extend my deepest gratitude to my supervisor Dr. Lawrence P. McIntosh. I’m extremely grateful to the patience and effort he had offered in the past three years. Studying aboard was not an easy thing, especially being a research student which I had to read, learn, and present my work using a secondary language. Dr. Lawrence P. McIntosh had been providing a calm and relax environment for me to work in, which I had enough time to adept and overcome the difficulties that I had faced. I would also like to thank Dr. Lawrence P. McIntosh for inspiring me and guiding me for the whole time, it feels secure when I know my supervisor is always being here and I could get help anytime. Lawrence had done a lot for me academically, he had contributed in most of my posters and the thesis writing, he would also provide guidance and point me a way out when I am stuck in research. Besides, he is a very caring person, I remember countless time that he was asking and concerning my situation, especially during the Covid-19 pandemic. Thanks for being a truly outstanding teacher, a mentor and hope he would be continuous to enlighten the others. I would also like to give thanks to my dearest group members in the McIntosh Lab, Karlton Scheu, Chloe Gerak, Jacob Brockerman, Mark Okon, Sophia Cho, Maggie Ling and Mine Sher. The joy and laughter was one of my most precious time when I am working with all of you. We work, we talk, and we support each other. Thanks for working along in my research journey and I wish you all the best in the bright future. xiv Dedication TO MY FAMILY 1 1. Introduction 1.1 Protein-protein liquid phase separation 1.1.1 General background Cellular compartments are one of the features differentiating eukaryotic cells from prokaryote cells. The majority of these intracellular compartments are bounded by layer(s) of membranes, composed of lipids with associated proteins and carbohydrates. These membranes play crucial roles in cellular organization, promoting spatiotemporal control of biological processes. Such processes including regulating the localization of metabolites between cellular organelles and enabling signal transduction between different cell compartments (1). A preeminent example of a eukaryotic cellular compartment is the nucleus in which genetic material is stored. The nuclear membrane serves as a physical barrier, separating transcription within the nucleus from translation in the cytoplasm. This is associated with the evolution of post-transcriptional regulation comprising mechanisms such as RNA capping, splicing and polyadenylation that control gene expression at the RNA level (2,3). In addition to membrane-bound organelles, it has long been recognized that some cellular compartments are not surrounding by any membranes (4). These non-membrane bound compartments are composed of a wide range of proteins, DNAs, and RNAs (4,5). How these macromolecules organize into a densely packed confined cellular space was poorly understood until the recent discovery that some proteins and nucleic acids can spontaneously condense and separate into liquid-like phases (2,4). The outer-phase, or the dilute phase, involves macromolecules dissolved in the aqueous environment of the cytosol. The inner-phase, or the dense phase, is composed of these macromolecules highly concentrated into liquid-like “droplets” (e.g. RGG-domain proteins in P-granules) (6,7), gels (e.g. type II diabetes IAPP amyloid precursors (8)) and more solid-like forms (e.g. FUS proteins in aberrant aggregated states (9)). In the context of my studies, the phenomenon of macromolecules transitioning between the outer-phase and the inner-phase is referred as phase separation or droplet formation. Phase separation is exploited by living cells to form membraneless organelles. A 2 summary of some typical eukaryotic membraneless organelles is shown in Figure 1. These organelles are composed proteins and/or nucleic acids. For example, nucleolus is a dynamic structure that serves as the site of ribosomal RNA (rRNA) transcription, pre-rRNA processing and ribosome subunit assembly (10). Similarly, Cajal bodies found in the nucleus harbor essential components for histone, messenger RNA (mRNA) and rRNA processing (11). Nuclear speckles, also called interchromatin granule clusters, contribute as storage sites of splicing factors and serve other roles in RNA metabolism (12). Processing bodies (P-bodies) are primarily composed of translationally repressed mRNA and proteins (13). In the case of proteasome storage granules, a yeast organelle that protects proteasomes from autophagic degradation (14), the phase-separated materials are mainly composed of protein molecules. In addition to membraneless organelles, phase separation also underpins processes such as membrane protein clustering, as exemplified by the association of the adhesion receptor Nephrin with its cytoplasmic partner proteins, Nck and N-WASP (15) and the Par complex proteins in Drosophila neuroblasts (16). Figure 1. Typical membraneless organelles in eukaryotic cells. Unlike the classical cell compartments, such as the nucleus and mitochondria, which have distinct membranes separating the internal content with the surrounding medium, the 3 membraneless organelles have boundaries created by phase separation. Within the phase-separated state, the constituent proteins and/or nucleic acids are highly concentrated, and hence with reduced solvent content. However, the boundary of the membraneless droplet is not impermeable. Instead, reversible diffusive exchange of the macromolecules between dilute and dense phases readily occurs. For example, using photobleaching recovery experiments, the contents of phase-separated droplets can be seen to exchange with the surrounding phase on typical timescales of seconds to minutes (6). Moreover, the membraneless droplets without physical barriers are generally spherical, yet readily deform and coalesce with another droplets. This differs from classical cell compartments for which membrane fusion requires complex mechanisms, for example as seen with the participation of p97 and syntaxin 5 in the assembly of the endoplasmic reticulum (17). 1.1.2 Discovery and history The history of biological phase separation can be traced back to as early as 1830s, when the first membraneless organelle was found inside the nucleus of neuronal cells using bright-field microscopy (6,18). Although discovered, the formation and function of the nucleolus remained to be defined. A century later, researchers had a breakthrough that brought attention to phase separation and membraneless structures, such as the nucleolus, in the cell (19). It is believed that the first report of phase separation was from a study of the cytoplasm of star fish and sea urchin eggs by Wilson et al., in which they described a “...fine emulsion consisting of a continuous substance in which are suspended drops” (20). Similar to the case of nucleolus, it was difficult to further investigate this phenomenon without using high-resolution microscopy imaging. Nowadays, we can adopt various kinds of microscopy to reveal the characteristics of phase-separated droplets, including their liquid-like properties, spherical shape, diffusive exchange, and coalescence with other droplets. Many other membraneless compartment have been studied for a long time, including Cajal bodies, P-bodies, stress granules and signaling centers formed by transmembrane receptors (21). However, the physical mechanisms underlying the formation of these compartments were also poorly understood. In 2009, a crucial piece of evidence came from investigations of P granules in germ cells of Caenorhabditis elegans (22). The seminal studies from Brangwynne et 4 al. showed that these granules are formed by RNA and protein molecules, phase separating into a liquid-like compartment. Soon afterwards, similar observations were reported for the nucleolus by the same group (23). In 2012, another seminal paper by Rosen and co-workers showed that weak reversible multivalent interactions between macromolecules can lead to highly cooperative liquid-liquid “demixing” (phase separation) to generate micrometer-sized droplets in aqueous solutions (24). Since then, scientists have shown that phase separation is associated with the formation of many membraneless organelles. Furthermore, protein phase separation has been well studied in vitro, including its relationships with diverse processes such as tau protein aggregation (25), hemoglobin phase separation at high concentration (26) and the behavior of proteins in crystallization trials (27). 1.1.3 Formation and regulation of droplets in different systems Over the past decade, great progress has been made in understanding the mechanisms and biological functions of phase separation. Although much remains to be learned, in general, proteins or other macromolecules have to be “sticky” to maintain a network of multivalent interactions within a phase-separated state. Furthermore, binding between the components needs to be reversible and relatively weak to enable rapid diffusive exchange of molecules between phases (28). Due to these multivalent interactions, phase separation occurs sharply once molecules cross threshold conditions, such as temperature, sample pH value or ionic strength, and protein concentration, which we will discuss further in chapter 3. This sharp threshold allows a sensitive control of droplet formation, both in vivo and in vitro (6). System undergoing phase separation have specific components with distinct regulatory mechanisms. For instance, in ribonucleoprotein (RNP) granules, the formation is driven by the low-affinity interactions of multivalent proteins that contain intrinsically disordered regions (IDRs) (29,30). In contrast, Balbiani bodies in the giant oocytes of Xenopus laevis frogs involve a protein called Xvelo that undergoes phase separation utilizing its prion-like domain. These domains are disordered it a soluble state and assemble in the oocytes as a tandem beta-sheet amyloid coil. Once the eggs are mature, Balbiani bodies will disperse due to the disassembly of the Xvelo amyloid back to the soluble state (31). Not surprisingly, aberrant phase separation can lead to diseases such as Alzheimer’s disease and Lou Gehrig's disease (32). 5 Researchers have also found that post-translation modifications can alter phase separation, particularly in the cases of intrinsically disordered proteins (29). Varying key functional groups on the intrinsically disordered proteins will impact their propensity to undergo intermolecular interactions (electrostatic, hydrophobic, hydrogen bonding, etc.). For example, arginine methylation on human heterogeneous nuclear ribonucleoprotein (hnRNP) may disfavor liquid-liquid phase separation of the C-terminal IDR at the RGG domain and thereby control stress granules formation. Another example is lysine acetylation that disrupting the electrostatic interactions involved in tau phase separation. In contrast, as discussed in my thesis, phosphorylation of threonine residues in the Mycobacterium tuberculosis transporter protein Rv1747 favors its phase separation. 1.1.4 Protein-protein interactions within the phase separation scaffold A wide variety of proteins can undergo phase separation. According to one review (30), they can be classified into four groups: (A) associative polymers with multiple folded binding domains; (B) patchy colloids with distinct surface interaction sites; (C) disordered linear multivalent proteins with “stickers” and “spacers”; and (D) branched multivalent proteins (Figure 2). Some phase-separating proteins, such as the adhesion receptor Nephrin and its cytoplasmic partners, Nck and N-WASP, have a composition of multiple binding domains joined by intrinsically disorder polypeptide linkers. The binding domains on both proteins contribute the necessary multivalent interactions to facilitate droplet formation. A second class of droplets is seen when proteins interact via more precisely defined surface patches. This can lead to well-oriented condensates as only specific components that fit on interacting patches can bind (33). Alternatively, fully intrinsically disordered proteins can polymerize via sticker motifs and spacer linkers. One such example is the human DEAD-box RNA helicase 4 (Ddx4) with a disordered N-terminal region that can spontaneously self-associate and thereby phase separate (34). This results from electrostatic interactions between sticker regions that are connected by flexible spacers. Lastly, a phase-separating protein can gain multivalency through branching. This is exemplified by nucleophosmin (NPM1), with a central oligomerization domain and five branched nucleic acid binding domains, connected by disordered linkers (35). 6 Figure 2. A variety of biomolecules can phase separate. (A) Associative polymers with folded binding domains joined by flexible linkers. (B) Patchy colloids with distinct surface patches (specific interaction sites). (C) Fully disordered linear proteins with “stickers” and “spacers.” (D) Branched multivalent proteins with a central oligomerization domain. (Figure from 30.) 1.2 Mycobacterium tuberculosis (Mtb) 1.2.1 Features of Mtb Mtb is the causative agent of the infectious disease tuberculosis (TB), first identified by the German physician Robert Koch in 1882 (36). According to a recent 2020 report, the World Health Organization (WHO) estimates about one-quarter of the world's population has a TB infection (37). However many have latent tuberculosis, which means the infected person has not yet developed any symptoms. The WHO stated that there will be 5-15% lifetime risk for people who get infected to develop active tuberculosis, and if they do not receive proper treatment, 45% may die from the disease. And for those who have compromised immune systems, such as HIV patients, there is a higher risk of dying from TB (37). 1.2.2 Virulence mechanism and infectious pathways of Mtb 7 Mtb is spread through airborne transmission from coughing and sneezing by active tuberculosis patients. Upon inhalation of the bacteria-containing droplets, Mtb can be recognized and engulfed by alveolar macrophages and other innate immune cells (38). In most cases, phagocytosed bacteria would be killed and digested by macrophage lysosome. However, Mtb has developed a series of strategies to manipulate host metabolic pathways and thereby survive and replicate in the host cell. First, Mtb is recognized by macrophages and several innate immune cells via their cell surface receptors. These include toll-like receptors (39), as well as complement receptors (40), and the mannose scavenger receptors (41). In normal cases, upon uptake by the macrophages, the presence of the pathogen triggers a series event of the endocytic pathway of the phagosome, resulting in phagosomal maturation (42). From uptake of the pathogen at the early phagosome stage to degradation of the prey in the phagolysosome stage, the pH will drop inside the phagosome due to the increasing number of vacuolar H+-ATPases (42). The acidic environment and large number of hydrolases at the phagolysosome stage can effectively degrade most engulfed pathogens (42,43). However, Mtb has the ability to arrest the phagosomal maturation and inhibit progress into phagolysosome stage. In the normal phagosomal pathway, the small GTP-binding proteins Rab5 and Rab7 are used for endosomal sorting (44). Rab5 binds to inactivating GDP during the early phagosome stage and is later replaced with Rab7, which binds to activating GTP during the late phagolysosome stage (45). The rapid replacement from Rab5 to Rab7 is termed Rab conversion, and the signal molecules for the regulation of this conversion are not known (46). However, in recent studies, Mtb has been shown to block Rab conversion by interacting with Rab5 effector early endosomal autoantigen 1 (EEA1). This in turn inhibits the production of phosphatidylinositol-3-phosphate (PI3P) on the phagosome membrane (47). Without the present of both Rab7 and PI3P, Mtb can arrest the macrophages into the less acidic and less hydrolytic early phagosome stage, thereby increasing its chances for survival. 1.2.3 PIM synthesis and macrophage resistance 8 Macrophages have adopted other strategies such as gamma interferon (IFN-γ) immunological activation, which may relieve the Rab conversion block and acidify the macrophages (48). However, Mtb exploits a counter-virulence factor utilizing the thick mycobacterial cell wall. The cell wall contains a layer of peptidoglycan with attached arabinogalactan and long-chain mycolic acids (49), along with glycolipids and lipids that are non-covalently linked on the outer membrane (50). Although the glycolipids vary from species to species, the most abundant and common ones are lipoarabinomannan (LAM), phosphatidyl-myo-inositol mannosides (PIMs) and lipomannan (LM) (50,51). PIMs and LM are synthesized by a series of mannosyltransferases biosynthesis reactions, followed by glycosylation with arabinan to form LAM (52,53). These glycolipids and lipids modification on the membrane result in an unusual, waxy cell wall that is effective in acidic resistance and blocks the host phagosome hydrolases. All of the above glycolipids are essential to the Mtb cell wall resistance. However in this thesis, the main focus will be on the PIM synthesis pathways. PIM biosynthesis is the foundation of all the glycolipid modifications, as schematically illustrated in Figure 3 (50). The chemical structures of PIMs are well established, but an understanding of the detail enzymatic events and the transportation of PIMs materials is still lacking (54). One of the unsolved mysteries on PIM biosynthesis is how the PIMs are transported across the Mtb cell membrane. It has been proposed that the transportation of PIMs might involve the transporter Rv1747, discussed in the next section (55). 9 Figure 3. Schematic summary of the PIM/LAM biosynthetic pathway. (Figure from 50) 1.3 Rv1747 ABC Transporter 1.3.1 Introducing Rv1747 The ATP binding cassette (ABC) transporter Rv1747 is a putative exporter of Mtb cell wall biosynthesis intermediates. Experiments on Rv1747 knockout strains revealed altered Mtb sedimentation phenotypes, decreased levels of PIMs in lipid extracts (55), and increased expression of the efflux pump-related iniBAC operon (56). Collectively, these data link Rv1747 with the transport of cell wall biosynthesis intermediates. However, the exact materials that Rv1747 transports remain to be identified as pinpointing a specific intermediate is hard to achieve, especially when PIMs have the same phosphatidylinositol foundation and only differ from one another by one mannose or arabinan. Nonetheless, the ΔRv1747 mutant shows significantly attenuated growth in macrophages and mice (55, 57) and thus it is clear that Rv1747 function is associated with Mtb infectivity. The domain structure of Rv1747 is shown in Figure 4. In addition to nucleotide binding (NBD) and transmembrane (TMD) domains, Rv1747 has an N-terminal regulatory module (residues 1-310; Figure 5). This module contains phosphothreonine (pThr)-binding FHA-1 and 10 FHA-2 domains, joined by an intrinsically disordered (ID) linker with multiple acceptor sites for phosphorylation. Previous research from our group using X-ray crystallography and NMR spectroscopy illustrated that both the FHA-1 and FHA-2 domains have β-sandwich structures diagnostic of a canonical Forkhead-Associated (FHA) domain (Figure 6A, B). However, FHA-2 is circularly permuted and lacking one β-strand. Both FHA-1 and FHA-2 have a conserved pThr-binding pocket (Figure 6C, D) (58). The FHA-2 domain is less stable and more dynamic than the FHA-1 domain and shows a higher μM-range binding affinity for model pThr-containing peptides (58). Importantly, Mtb strains with the phosphor-ablative T152A/T210A mutations in the linker exhibit reduced growth in macrophages and mice, and those with a mutation in the canonical pThr-binding site of FHA-1 have impaired growth in macrophages (59). Thus the function(s) of Rv1747 are phosphorylation dependent, but very little is currently known about the exact roles played by the regulatory module. Figure 4. Schematic of the assumed homodimeric Rv1747 ABC transporter. Highlighted are the cytoplasmic regulatory module composed of the FHA-1 (blue) and FHA-2 (red) domains joined by an ID linker with phospho-acceptor sites (green dots). 11 Figure 5. Schematic of the Rv1747 ABC transporter regulatory module (residues 1-310). The FHA-1 (1-104, blue) and FHA-2 (230-310, red) domains are joined by an ID linker with multiple phospho-acceptor sites (green dots). Both FHAs domains have a conserved serine residue (S47 in FHA-1 and S248 in FHA-2) that is essential for pThr-binding. The linker has many possible phosphor-accepter sites including T152. The cartoon is not to scale. Figure 6. Conserved FHA structure and pThr-binding site. Structure of (A) the FHA-1 domain (Rv17473-116, PDB: 6CCD) determined by X-ray crystallography and (B) the FHA-2 domain (Rv1747206-310, PDB: 6CAH) determined by NMR spectroscopy. Highlighted in red are 12 the N-terminal β-strands 1 and 2 of FHA-1 and the corresponding C-terminal strand 1* of circularly permuted FHA-2. (C and D) Overlaid representations of the X-ray crystallographic structure of pThr peptide-bound Rad53 FHA-1 domain (PDB: 1G6G, gray) with (C) the crystal structure of unbound FHA-1 (green) and (D) the lowest energy member of the NMR-derived structural ensemble of unbound FHA-2 (green). Important residues in the binding interfaces are shown in stick format (oxygen, red; nitrogen, blue). The Rad53-bound peptide has the carbon and phosphorous atoms of pThr in orange and remaining carbons in black. (Figure from 58.) 1.3.2 Rv1747 regulatory module undergoes phosphorylation-dependent phase separation The presence of tandem FHA domains and multiple phospho-acceptor threonines in the Rv1747 regulatory module sets the stage for phase separation. Initially, based on NMR relaxation and chemical shift perturbation measurements, our group demonstrated that, when joined within a polypeptide chain, either FHA domain can bind different linker pThrs to form intra- and intermolecular complexes (58). Subsequently, we found that the full regulatory module (Rv17471-310) with both FHA domains joined by the linker undergoes phase separation in vitro (60). As shown in Figure 7, Rv17471-310 droplets can be visualized using a bright-field microscope. The droplets are transparent to visible light and mostly spherical with a clear boundary that separates phases. Individual droplets vary in size at any time point and freely diffuse throughout solution. However, upon contact, the droplets can also coalesce to form larger droplets, with the time scale for the fusion event being about 10 s (Figure 8). Fragmentation into smaller droplets does not occur. In early studies, Rv17471-310 at relatively low concentrations (~ 50 μM) phase separated after phosphorylation by PknF, a Mtb serine-threonine protein kinase (STPK; Figure 7A). Using mass spectrometry, we found that multiple threonines and serines within the Rv17471-310 linker were phosphorylated by PknF (60). The mutation of two previously reported sites (58), T152 and T210, to alanines impaired, but did not prevent, droplet formation (60). Thus, phosphorylation of additional sites in the linker also contribute to phase separation. Subsequently we observed that upon concentration to ~ 300 μM, unmodified Rv17471-310 also formed droplets (Figure 7B). This phosphorylation-enhanced phase separation points to a synergy between specific FHA domain/pThr interactions along with intrinsic interaction properties of the regulatory module (Figure 9). Dissecting the contributions of these interactions is a central goal of my thesis research. 13 Figure 7. The Rv1747 regulatory module undergoes phosphorylation-enhanced phase separation into liquid-like droplets. Both samples were in 100 mM NaCl, 20 mM sodium phosphate, pH 6.0 at room temperature. (Left) Rv17471-310 at 50 μM was phosphorylated by treatment with a 1/10 molar ratio of protein kinase PknF (plus 5 mM ATP and 5 mM MgCl2) and incubated for 1 hour at room temperature before plating on the microscope slide. (Right) Unmodified Rv17471-310 was slowly concentrated using centrifugal filtration from 50 μM to 300 μM, followed by plating on the side. The images were captured under a bright-field microscope, with each pixel corresponding to 25 nm. The reference bar is 10 μm. Figure 8. Rv17471-310 droplets diffuse freely in solution and coalesce upon contact. The protein was at 50 μM in 100 mM NaCl, 20 mM sodium phosphate, pH 6.0 at room temperature. The protein was treated with PknF (5 μM with 5 mM ATP and 5 mM MgCl2) for 1 hour before imaging. The time course of the fusion events was recorded under a bright-field microscope, with each pixel corresponding to 25 nm. The reference bar is 10 μm. 14 Figure 9. A cartoon model of phase separation by the Rv1747 regulatory module resulting from a myriad of multivalent intra- and intermolecular interactions. These include specific phosphorylation-dependent FHA domain/pThr interactions along with intrinsic interaction properties of the unmodified regulatory module. 1.3.3 Rv1747 phosphorylation by the Mtb STPKs The regulation of Rv1747 is closely connected with the STPKs of Mtb. The Mtb genome encodes 11 eukaryotic-like STPKs. Although the physiological substrates of these STPKs have not been fully elucidated, it is well established that these kinases, and the sole serine/threonine phosphatase PstP, have important roles in Mtb signaling transduction pathways (61, 62). In the context of this thesis, Rv1747 can be phosphorylated by several STPKs including PknF. The latter is often called the cognate kinase for Rv1747 since both are encoded in the same operon (61). However, our group found that Rv17471-310 forms droplets with different morphologies when treated with different STPKs (Figure 10) (60). It was suggested that the differences in shapes could be caused by the differences in stoichiometry and sites of phosphorylation by each particular STPK. 15 Figure 10. Fluorescence images of Oregon Green-labeled Rv17471-310 droplets formed upon treatment by 10 STPKs from Mtb. In each case, 0.5 - 2 μM of the purified kinase domain (with 100 μM ATP) was used to phosphorylate 10 μM Rv17471-310. At this concentration, unmodified Rv17471-310 does not phase separate. The reference scale bar is 20 μm. (Images from 60.) A homology-based model structure of the PknF kinase domain is shown in Figure 11 (63). Lysine 41 forms part of the ATP binding site, where PknF captures and holds ATP for the phosphorylation reaction. Multiple research groups have shown that mutation of K41 completely abolishes the interaction of PknF with Rv1747 (57, 64). Aspartate 137 is part of the substate binding site, where PknF interacts with its phosphorylation target (65). The mutation D137N also deactivates the kinase (64, 65). Threonines 173 and 175 are in activation loop and are typically auto-phosphorylated by PknF as part of its activation cascade (62). This auto-phosphorylation occurs “in trans”, and can also be installed by other kinases, suggesting a possible STPK interaction network. For example, PknJ may obtain signal from the frontier master regulator kinase PknH and transduce the phosphorylation signal to the downstream PknF. The mutation of these threonines also decreases the interaction with Rv1747 (57, 66). Furthermore, the recruitment and phosphorylation of Rv1747 may depend on a possible docking interaction between its FHA domains and phosphothreonine residues in auto-phosphorylated PknF. Deciphering the molecular mechanisms underlying interactions between PknF and Rv1747 was also part of my thesis. 16 Figure 11. Homology model of the PknF kinase domain from the Swiss-Model repository (P9WI75 PKNF_MYCTU). Key active site residues are indicated. (Image from 63.) This model closely aligns with the recently published X-ray crystal structure of PknF (PDB: 7NNA). However, in the latter several important regions, including the activation loop with T173 and T175, were not observed presumably due to conformational disorder (83). Thus, for illustrative purposes, the homology model is shown in this figure. 1.3.4 Regulation of Rv1747 by Rv2623 Rv2623 is a universal stress protein reported to interact with Rv1747 and down regulate its transporter activity as related to PIM biosynthesis (55). Furthermore, Rv2623 harbors a phosphothreonine (pT237) recognized by the FHA-1 domain Rv1747. A mutant of Rv2623 with T237 replaced by a non-phosphorylatable alanine showed decreased interactions with the FHA-1 domain, as well as an increase in expression of PIMs. Another goal of my thesis was to clarify the role of Rv2623 in regulating Rv1747 function. 1.3.5 Rv1747 clustering in vivo Purified Rv17471-310 has the intrinsic ability to phase separate in vitro. Furthermore, the regulatory module also phase separates under more native-like conditions. That is, it forms droplets when tethered in “2-dimensions” to supported lipid bilayers, as well as dynamic foci that have characteristics consistent with phase separation (coalescence and recovery of 17 fluorescence after photobleaching) when expressed in both yeast and Mycobacterium smegmatis (60). The latter is a non-pathogenic model of Mtb. Accordingly, we proposed a working model for Rv1747 function and regulation based on the hypothesis that it phase separates in vivo to form higher order complexes within the Mtb cellular membrane (Figure 12). In support of this hypothesis, ultra-high resolution microscopy experiments revealed that endogenous Rv1747 forms nanoclusters in wild-type Mtb (60). Succinctly summarizing these experiments, Mtb H37Rv was grown in liquid culture, fixed, permeabilized, and immunolabeled with a single chain variable fragment (scFv) “intrabody” generated against the FHA-1 domain of Rv1747 via phage display. The scFv was labeled with Alex Fluor 647 (AF647) to enable super-resolution (i.e., 50 nm scale) stochastic optical reconstruction microscopy (STORM). Single molecule localization reconstructions of the imaged Mtb cells revealed that the fluorescent anti-Rv1747 svFc was distributed in clusters distributed non-uniformly along the bacterial surface. Many these clusters were substantially larger in area than expected from a liberal size estimation of a single Rv1747 monomer. Such nanoclustering has emerged as an organization principle for membrane proteins (60, 67). On-going studies in the Gsponer and Av-Gay laboratories are aimed at elucidating the potential role of phase separation, mediated by the regulatory module, in this process and in the regulation of Rv1747 function. Figure 12. A working model for phosphorylation-enhanced clustering and regulation of Rv1747. The transporter (core ABC structure, brown) clusters due to the concentration-dependent phase separation of the regulatory module (FHA domains, blue). Phosphorylation (red 18 circles) by Mtb STPKs reduces the saturation concentration for clustering (upper triangle), and dephosphorylation by PstP reverses this process. Both phosphorylation- and concentration-dependent phase separation may increase the transporter efficiency of Rv1747 (green arrow) for cell wall biosynthesis intermediates via a yet undefined mechanism. Additional interactions with regulatory proteins, such as Rv2623, or transporter substrates could modulate the saturation concentration for clustering. (Figure adapted from 60.) 1.4 Thesis goals Protein phase separation is now at the forefront of molecular and cellular biology research with recognized roles in processes spanning compartmental organization and the regulation of gene expression to diseases including amyotrophic lateral sclerosis (ALS) and cancer (68, 69). However, many aspects of the mechanisms and regulation of phase separation remain to be uncovered. Previous studies led by a former laboratory member, Florian Heinkel, characterized the structures, stabilities, and phosphothreonine-binding properties of the FHA domains of the Rv1747 regulatory module (58), and demonstrated that it undergoes phase separation in vitro (60). This process occurs at a saturation concentration that is reduced upon multisite phosphorylation by several Mtb STPKs. The importance of two threonines in the linker between the FHA domains for phase separation was demonstrated. The regulatory module was also found to form dynamic foci when heterologously expressed in live yeast and M. smegmatis cells. Preliminary evidence for clustering of endogenous Rv1747 in the Mtb membrane led to a model in which phase separation of the regulatory module in response to intracellular signaling controls the association and function of this transporter of cell wall precursors. Although the general parameters governing the phase separation of the Rv1747 regulatory module were established, the goal of my thesis was to address several questions regarding the detailed mechanisms of this process. For example, what is the basis for the phase separation of the unmodified protein? What are the roles played by each FHA domain and by the connecting ID linker? Also, which residues in the linker are key for phosphorylation-enhanced phase separation? Furthermore, do the STPKs such as PknF act only by phosphorylating the regulatory module or do they also have a direct scaffolding role within a phase-separated complex? Similarly, how do partner proteins such as Rv2623 modulate Rv1747 phase separation? In the following chapters, I will present the results of my experiments directed towards answering these questions and conclude with a refined model for the regulation of Rv1747 phase separation. 19 2. Materials and methods 2.1 Cloning A summary of the expression constructs used these studies is provided in Table 4 at the end of this chapter. In the cases of Rv1747 variants with multiple mutations, expression vectors were purchased from Biomatik. For simpler truncations and point mutations, standard PCR methods were used to genetically modify the DNA (70). The reagents was mixed in a sterilized, nuclease free microcentrifuge tube, as summarized in Table 1. The PCR thermal cycling followed the general protocol of Table 2 with adjustments for different plasmid constructs according to the Thermofisher online tool (71). After amplification, the DNA was collected and purified with the QIAGEN QIAquick PCR Purification Kit (72), using 30-50 µL of autoclaved nuclease-free water for elution. DNA concentrations were determined by UV absorbance using predicted ε260 values. Samples of DNA were sent to Genewiz for sequence verification. Table 1. Reagents for PCR mutagenesis Reagents Volume added Final concentration Pfu DNA polymerase 10X buffer with MgSO4 (Thermo Scientific) 5 µL 1X dNTP mix 10 mM each (Thermo Scientific) 1 µL 0.2 mM Forward primer (10 µM, working stock) 2.5 µL 0.5 µM Reverse primer (10 µM, working stock) 2.5 µL 0.5 µM Template DNA (50-100 ng/µL) Variable ~2 ng/µL Pfu DNA Polymerase (Invitrogen) 1 µL 1 unit Autoclaved nuclease-free water to 50 µL total vol. Table 2. Typical PCR thermal cycling Step Temperature (°C) Time Number of cycles Initial denaturation 95 3 min 1 Denaturation 95 30 s 30 Annealing Calculated by Thermofisher online application 30 s Extension 72 2 min Final extension 4 1 20 In most cases, the DNA plasmid purchased, or the modified DNA could be directly expressed after transformation into E. coli BL21. However, some of the DNA had to be cloned into a different base vector (labeled with an asterisk (*) in Table 4). A general summary of conditions used for restriction enzyme digestions is given in Table 3. After digestion at 37 °C overnight, the reaction mix was resolved by electrophoresis on a 0.8% Agarose gel. The desired DNA fragment was cut from the gel after visualization under a UV imager, and purified using the QIAquick Gel Extraction Kit, followed by the QIAGEN QIAquick PCR Purification Kit, as above (72,73). Table 3. Typical restriction enzyme digestion conditions Reagents Volume added Final concentration 10X FastDigestion buffer (Thermo Scientific) 2 µL 1X Forward restriction enzyme (Thermo Scientific) 2.5 µL 2.5X Reverse primer (Thermo Scientific) 2.5 µL 2.5X Template DNA (50 -100 ng/µL) Variable 200 ng/µL Autoclaved nuclease free water to 20 µL final vol. 2.2 Protein expression and purification The general procedures for protein expression are outlined below, with additional details in Table 5. Template DNA was transformed into E. coli BL21 cells by the heat shock method. DNA (100 ng) was added to the 200 µL pre-chilled competent cells and incubated on ice for 30 min. The cells were heat shocked in a 42 oC water bath for 45 sec, incubated in 1 mL of SOC media at 37 oC for 1 hr, plated on LB agar with suitable antibiotic (Table 5) and incubated overnight at 37 oC. Desired colonies were picked and inoculated into 50 mL of LB broth with suitable antibiotic, followed by growth at 37 oC overnight. The overnight culture was transferred the next day into 1 L of LB broth with suitable antibiotic and grown with rotary shaking at 37 oC to an OD600 in the range from 0.6 - 0.8. The OD600 was measured in 1 cm pathlength quartz cuvettes using a Biochrom Ultrospec 3000 UV/Vis spectrophotometer. To induce protein expression, 1 mM final concentration of isopropyl β-D-1-thiogalactopyranoside (IPTG) was added. The culture was incubated for overnight at 20 oC. Cells were then harvested by centrifugation in a CLA 3000 rotor under 6000xg for 15 min. After taking a small sample for SDS-PAGE characterization, the cell pellet was stored at -70 oC. 21 The frozen pellet was thawed for 2 hours at room temperature, resuspended in 50 mL lysis buffer (50 mM Tris-HCl, 1 M NaCl, 10 mM imidazole, pH 7.5), and sonicated with a Branson Sonifier 250 sonicator for 3 cycles of 10 min (output 4, duty cycle 60). The lysate was kept on ice during the sonication and inverted several times between cycles. The lysate was then centrifuged in a SS34 rotor for 60 min at 30000xg at 4 oC. The supernatant was collected and filtered through a 0.45 µm pore membrane. Protein purification was performed using GE AKTA Prime Plus FPLC System with the appropriate affinity column (Tables 4 and 5). The column was initially washed with 10 column volumes of binding buffer (Table 5). The supernatant was passing through the column twice and the flow-through was collected and stored as a backup. The column was then washed with buffer A until the UV absorbance at 280 nm (A280) of the flow-through dropped to baseline level. The bound protein was eluted using buffer B over a linear gradient of 50 mL. All fractions with A280 higher than the baseline were collected. The following samples (50 µL) were analyzed with SDS-PAGE during the purification process: lysate, flow-through from the column, and 5 - 8 eluted fractions. All the fractions with the desired protein were pooled and dialyzed with the corresponding tag protease in cleavage buffer (Table 5) overnight at 4 oC. The sample was then passed through the same affinity column as the first purification step to remove the protease, cleaved affinity tag and any uncleaved protein. The desired protein in the flow-through, as confirmed with SDS-PAGE, was dialyzed against 2 L of storage buffer (20 mM sodium phosphate, 100 mM NaCl, pH 6.0) for 2 hours at 4o C. After concentrating using an Amicon Ultra-15 centrifugal filter unit with a 10 kDa molecular weight cutoff, the protein was finally flash frozen in liquid nitrogen and stored at -70 oC. Proteins were characterized by SDS-PAGE and, as needed, ESI-MS, and protein stock concentrations were determined by UV absorbance using predicted ε280 values. 2.3 Imaging Rv1747 constructs using bright-field microscopy Most images were recorded using a bright-field microscope (Nikon Eclipse TW2000-U, courtesy of the Gaynor lab, UBC). After thawing, Rv1747 constructs, kinases and supplemental materials were mixed and incubated on the lab bench at room temperature for 1 hr before imaging. A typical sample included 50 µM Rv17471-310 and 5 µM PknF1-292 in 100 mM NaCl and 20 mM sodium phosphate pH 6.0, with 5 mM ATP and 5 mM MgCl2. The PknF1-292 was 22 expressed and purified as explained above, with conditions listed at Table 5. The PknF kinase was stored in storage buffer (20 mM sodium phosphate, 100 mM NaCl, pH 6.0) at -70 oC. Thawed on lab bench for 20 minutes before adding into the reaction mix. Exact conditions are provided in the Results section. The CoverWell™ imaging chamber gaskets were coated with 30 mg/mL BSA for at least an hour before transferring 300 µL of sample onto the imaging chamber. A microscope slides from Fisherbrand Superfrost was used for capping the imaging chamber. Images were recorded using magnification set to 400x and each pixel at 25 nm. 2.4 Image processing Two imaging programs were used in this research, namely ImageJ (74) and Cellprofiler (75). Both can scan the images recorded on a bright-field or fluorescent microscope and distinguish droplets from the slide background. ImageJ can also be used to determine the fractional intensity, which is the overall area of the droplets on the image compared to the area of whole image. Cellprofiler can be used to quantify details of the droplets such as size, radius and the number count. 2.5 Microscopy studies of Rv17471-310 droplets as a function of temperature The reaction mixture consisted of 10, 30 or 50 µM Rv17471-310 and 5 µM PknF1-292 in 100 mM NaCl and 20 mM sodium phosphate, pH 6.0, with 5 mM ATP and 5 mM MgCl2. After incubation at room temperature for an hour, the mixture was plated on the imaging chamber. The samples were studied using a custom-built bright-field microscope with a temperature-controlled stage slide warmer (Gold lab, UBC) (76). Unfortunately, the temperature of the slide platform could only be increased from room temperature to 37 oC. Thus, initial images of the droplets were recorded at 22 oC. After warming to 37 oC, a second set of images were captured. The images were analyzed with ImageJ and Cellprofiler, as introduced above. 2.6 MALDI mass spectrometry The MALDI mass spectrometry was performed at a Voyager-DE STR Biospectrometry Workstation (courtesy of the Overall Lab, UBC). Rv1747 constructs (5 µL in 20 mM sodium phosphate, 100 mM NaCl, pH 6.0) were coated on the Biosystems Perspective Stainless Voyager V700666 Welled Sample Plate. The sample plate was be transferred into the Voyager workstation 23 and analyzed using Visual Basic software (77). 2.7 Electrospray ionization mass spectrometry (ESI-MS) The ESI-mass spectrometry was performed in Michael Smith Laboratories. Samples, such as the Rv1747 constructs (50 µM of 50 µL in 20 mM sodium phosphate, 100 mM NaCl, pH 6.0), were submitted to Dr. Jason Rogalski. He recorded and analyzed the ESI-MS data, returning the results in pdf format. 2.8 15N-HSQC NMR for Rv17471-110 and Rv17471-213 NMR experiments were performed at 25 °C using a cryoprobe-equipped Bruker Avance III 600 MHz spectrometer. 15N-labeled Rv1747 constructs were prepared using M9 minimal media with 1 g/L of 15NH4Cl as the sole nitrogen source. The samples were up to 100 μM in 20 mM sodium phosphate, 100 mM NaCl, at pH 6.0 with 10% lock D2O. The 15N-HSQC spectra were collected overnight and processed using Bruker TopSpin 3.0 software (78). 2.9 Interaction studies of Rv17471-310 and Rv2623 The expression vector for Rv2623 was provided by Dr. John Chan (Albert Einstein College of Medicine & Montefiore Medical Center). The purification conditions are summarized in Table 5. Rv17471-310 (50 μM) was treated with 50 μM Rv2623 in the reaction mix. The reaction mixture consisted with 5 µM PknF1-292 in 100 mM NaCl and 20 mM sodium phosphate, pH 6.0, with 5 mM ATP and 5 mM MgCl2. After incubation at room temperature for an hour, the mixture was plated on the imaging chamber as described. The image was captured and analyzed with ImageJ. NMR experiments were performed at 25 °C using a cryoprobe-equipped Bruker Avance III 600 MHz spectrometer. 15N-labeled Rv17471-310 was prepared using M9 minimal media with 1 g/L of 15NH4Cl as the sole nitrogen source. The samples contained 75 μM of labeled Rv17471-310 without and with 75 μM Rv2623 in phosphate buffer (100 mM NaCl, 20 mM sodium phosphate, pH 7) and HEPES buffer (50 mM NaCl, 2 mM MgCl2, 10% glycerol and 13 mM β-mercaptoethanol pH 8.0). The 15N-HSQC spectra were collected overnight and processed using Bruker TopSpin 3.0 software (78). 24 2.10 Interaction between Rv1747 and a phosphopeptide The phosphopeptide 5-TAMRA-KKYAGQQLPPA(pT)TRIPAA-NH2 corresponding to the reported Rv1747 phosphorylation site pT152, was purchased from Biomatik. Although not used herein, the 5-TAMRA (5-carboxytetramethylrhodamine) fluorophore was included for potential binding studies using fluorescence spectroscopy. The peptide was dissolved in 100 mM NaCl and 20 mM sodium phosphate, pH 6.0, flash frozen in liquid nitrogen and stored at -70 oC. Rv17471-310 (50 μM) was phosphorylated in a similar manner as the above, with 5 µM PknF1-292 in 100 mM NaCl and 20 mM sodium phosphate, pH 6.0, with 5 mM ATP and 5 mM MgCl2. The phosphopeptide was added to the reaction mix in a 20 or 50-fold molar excess before imaging. The samples with or without the phosphopeptide were placed on the lab bench at room temperature for another 16 hrs before the second set of images was taken. 25 Table 4. Expression constructs Construct DNA Sequence Plasmid vector Purification tag Cleaving enzyme Stock DNA Conc. DNA source Rv17471-310 atgcatcatcatcatcatcacagcagcggcagagaaaacttgtatttccagggccatatgccgatgagccaaccagccgccccgcccgtgttgaccgtgcggtatgagggatcggagcgcacgttcgccgcaggacacgatgtcgtcgtcgggcgtgacctgcgcgcggatgtccgcgtcgcacaccccctgatctcccgggcacacctgctgctgcgattcgaccagggtcgctgggtcgccattgacaatggcagcctcaatgggctctacctcaataaccgtcgggtgccagtcgtggacatctacgatgcccagcgagtccatatcggaaaccccgacggtccggcgctggacttcgaagtgggccgccaccggggttcggccgggcgaccaccccagacgacgtcgatacgcctgcccaacctgtccgcgggagcgtggcccaccgacggcccgccgcagaccggcacgctcggctccggccagctacaacagcttccaccggccaccacccggatacccgccgctccgccatcgggaccacagccgcgataccccaccggtgggcaacagttgtggccacccagcggaccgcaacgggcgccgcagatttaccggccacccacggccgcaccgccgccggcgggtgcccgcggcggaactgaggcgggaaacctcgcgacatcgatgatgaagatcctgcggccaggcaggttgacgggggagttgccgcccggtgccgtcaggatcggccgggcgaacgacaacgacatcgtcattcccgaggtgttggcctcacgtcaccacgccaccctggtcccgacgcctggcggcacggagattcgggacaaccgcagcatcaatggcaccttcgtcaacggcgcccgggtcgacgcggcgctgctgcacgacggcgacgtcgtgaccatcggcaacatcgacctcgtcttcgccgacggcaccctggcgcgccgtgaagagaactaa pET28ML PolyHis TEV 135 ng/µL McIntosh Lab Rv17471-310 _Cys atgcatcatcatcatcatcacagcagcggcagagaaaacttgtatttccagggccatatgtgcccgatgagccaaccagccgccccgcccgtgttgaccgtgcggtatgagggatcggagcgcacgttcgccgcaggacacgatgtcgtcgtcgggcgtgacctgcgcgcggatgtccgcgtcgcacaccccctgatctcccgggcacacctgctgctgcgattcgaccagggtcgctgggtcgccattgacaatggcagcctcaatgggctctacctcaataaccgtcgggtgccagtcgtggacatctacgatgcccagcgagtccatatcggaaaccccgacggtccggcgctggacttcgaagtgggccgccaccggggttcggccgggcgaccaccccagacgacgtcgatacgcctgcccaacctgtccgcgggagcgtggcccaccgacggcccgccgcagaccggcacgctcggctccggccagctacaacagcttccaccggccaccacccggatacccgccgctccgccatcgggaccacagccgcgataccccaccggtgggcaacagttgtggccacccagcggaccgcaacgggcgccgcagatttaccggccacccacggccgcaccgccgccggcgggtgcccgcggcggaactgaggcgggaaacctcgcgacatcgatgatgaagatcctgcggccaggcaggttgacgggggagttgccgcccggtgccgtcaggatcggccgggcgaacgacaacgacatcgtcattcccgaggtgttggcctcacgtcaccacgccaccctggtcccgacgcctggcggcacggagattcgggacaaccgcagcatcaatggcaccttcgtcaacggcgcccgggtcgacgcggcgctgctgcacgacggcgacgtcgtgaccatcggcaacatcgacctcgtcttcgccgacggcaccctggcgcgccgtgaagagaactaa pET28a PolyHis TEV 210 ng/µL Biomatik Rv17471-310 _Cys_S47A atgcatcatcatcatcatcacagcagcggcagagaaaacttgtatttccagggccatatgtgcccgatgagccaaccagccgccccgcccgtgttgaccgtgcggtatgagggatcggagcgcacgttcgccgcaggacacgatgtcgtcgtcgggcgtgacctgcgcgcggatgtccgcgtcgcacaccccctgatcgcgcgggcacacctgctgctgcgattcgaccagggtcgctgggtcgccattgacaatggcagcctcaatgggctctacctcaataaccgtcgggtgccagtcgtggacatctacgatgcccagcgagtccatatcggaaaccccgacggtccggcgctggacttcgaagtgggccgccaccggggttcggccgggcgaccaccccagacgacgtcgatacgcctgcccaacctgtccgcgggagcgtggcccaccgacggcccgccgcagaccggcacgctcggctccggccagctacaacagcttccaccggccaccacccggatacccgccgctccgccatcgggaccacagccgcgataccccaccggtgggcaacagttgtggccacccagcggaccgcaacgggcgccgcagatttaccggccacccacggccgcaccgccgccggcgggtgcccgcggcggaactgaggcgggaaacctcgcgacatcgatgatgaagatcctgcggccaggcaggttgacgggggagttgccgcccggtgccgtcaggatcggccgggcgaacgacaacgacatcgtcattcccgaggtgttggcctcacgtcaccacgccaccctggtcccgacgcctggcggcacggagattcgggacaaccgcagcatcaatggcaccttcgtcaacggcgcccgggtcgacgcggcgctgctgcacgacggcgacgtcgtgaccatcggcaacatcgacctcgtcttcgccgacggcaccctggcgcgccgtgaagagaactaa pET28a PolyHis TEV 96 ng/µL Biomatik Rv17471-310 ––_Cys_S248A [binary] TEV 103 ng/µL Biomatik 26 ctgctgcgattcgaccagggtcgctgggtcgccattgacaatggcagcctcaatgggctctacctcaataaccgtcgggtgccagtcgtggacatctacgatgcccagcgagtccatatcggaaaccccgacggtccggcgctggacttcgaagtgggccgccaccggggttcggccgggcgaccaccccagacgacgtcgatacgcctgcccaacctgtccgcgggagcgtggcccaccgacggcccgccgcagaccggcacgctcggctccggccagctacaacagcttccaccggccaccacccggatacccgccgctccgccatcgggaccacagccgcgataccccaccggtgggcaacagttgtggccacccagcggaccgcaacgggcgccgcagatttaccggccacccacggccgcaccgccgccggcgggtgcccgcggcggaactgaggcgggaaacctcgcgacatcgatgatgaagatcctgcggccaggcaggttgacgggggagttgccgcccggtgccgtcaggatcggccgggcgaacgacaacgacatcgtcattcccgaggtgttggccgcgcgtcaccacgccaccctggtcccgacgcctggcggcacggagattcgggacaaccgcagcatcaatggcaccttcgtcaacggcgcccgggtcgacgcggcgctgctgcacgacggcgacgtcgtgaccatcggcaacatcgacctcgtcttcgccgacggcaccctggcgcgccgtgaagagaactaa Rv17471-310 -_Cys_S47A_ S248A atgcatcatcatcatcatcacagcagcggcagagaaaacttgtatttccagggccatatgtgcccgatgagccaaccagccgccccgcccgtgttgaccgtgcggtatgagggatcggagcgcacgttcgccgcaggacacgatgtcgtcgtcgggcgtgacctgcgcgcggatgtccgcgtcgcacaccccctgatcgcgcgggcacacctgctgctgcgattcgaccagggtcgctgggtcgccattgacaatggcagcctcaatgggctctacctcaataaccgtcgggtgccagtcgtggacatctacgatgcccagcgagtccatatcggaaaccccgacggtccggcgctggacttcgaagtgggccgccaccggggttcggccgggcgaccaccccagacgacgtcgatacgcctgcccaacctgtccgcgggagcgtggcccaccgacggcccgccgcagaccggcacgctcggctccggccagctacaacagcttccaccggccaccacccggatacccgccgctccgccatcgggaccacagccgcgataccccaccggtgggcaacagttgtggccacccagcggaccgcaacgggcgccgcagatttaccggccacccacggccgcaccgccgccggcgggtgcccgcggcggaactgaggcgggaaacctcgcgacatcgatgatgaagatcctgcggccaggcaggttgacgggggagttgccgcccggtgccgtcaggatcggccgggcgaacgacaacgacatcgtcattcccgaggtgttggccgcgcgtcaccacgccaccctggtcccgacgcctggcggcacggagattcgggacaaccgcagcatcaatggcaccttcgtcaacggcgcccgggtcgacgcggcgctgctgcacgacggcgacgtcgtgaccatcggcaacatcgacctcgtcttcgccgacggcaccctggcgcgccgtgaagagaactaa pET28a PolyHis TEV 133 ng/µL Biomatik Rv17471-310 _Cys_only_T152 atgcatcatcatcatcatcacagcagcggcagagaaaacttgtatttccagggccatatgtgcccgatgagccaaccagccgccccgcccgtgttgaccgtgcggtatgagggatcggagcgcacgttcgccgcaggacacgatgtcgtcgtcgggcgtgacctgcgcgcggatgtccgcgtcgcacaccccctgatctcccgggcacacctgctgctgcgattcgaccagggtcgctgggtcgccattgacaatggcagcctcaatgggctctacctcaataaccgtcgggtgccagtcgtggacatctacgatgcccagcgagtccatatcggaaaccccgacggtccggcgctggacttcgaagtgggccgccaccggggttcggccgggcgaccaccccagtcttcttcgatacgcctgcccaacctgtccgcgggagcgtggcccagcgacggcccgccgcagtctggctctctcggctccggccagctacaacagcttccaccggccacctctcggatacccgccgctccgccatcgggaccacagccgcgatacccctctggtgggcaacagttgtggccacccagcggaccgcaacgggcgccgcagatttaccggccaccctctgccgcaccgccgccggcgggtgcccgcggcggaagcgaggcgggaaacctcgcgtcttcgatgatgaagatcctgcggccaggcaggttgtctggggagttgccgcccggtgccgtcaggatcggccgggcgaacgacaacgacatcgtcattcccgaggtgttggcctcacgtcaccacgccaccctggtcccgacgcctggcggcacggagattcgggacaaccgcagcatcaatggcaccttcgtcaacggcgcccgggtcgacgcggcgctgctgcacgacggcgacgtcgtgaccatcggcaacatcgacctcgtcttcgccgacggcaccctggcgcgccgtgaagagaactaa pET28a PolyHis TEV 126 ng/µL Biomatik Rv17471-310 _Cys_no_linker_Thr [binary] TEV 96 ng/µL Biomatik 27 tcattcccgaggtgttggcctcacgtcaccacgccaccctggtcccgacgcctggcggcacggagattcgggacaaccgcagcatcaatggcaccttcgtcaacggcgcccgggtcgacgcggcgctgctgcacgacggcgacgtcgtgaccatcggcaacatcgacctcgtcttcgccgacggcaccctggcgcgccgtgaagagaactaa Rv17471-110 atgccgatgagccaaccagccgccccgcccgtgttgaccgtgcggtatgagggatcggagcgcacgttcgccgcaggacacgatgtcgtcgtcgggcgtgacctgcgcgcggatgtccgcgtcgcacaccccctgatcgcgcgggcacacctgctgctgcgattcgaccagggtcgctgggtcgccattgacaatggcagcctcaatgggctctacctcaataaccgtcgggtgccagtcgtggacatctacgatgcccagcgagtccatatcggaaaccccgacggtccggcgctggacttcgaagtgggccgccaccggggttcggcctaa pET28MHL PolyHis TEV 106 ng/µL McIntosh Lab R1747105-228 _Cys* tgccgccaccggggttcggccgggcgaccaccccagacgacgtcgatacgcctgcccaacctgtccgcgggagcgtggcccaccgacggcccgccgcagaccggcacgctcggctccggccagctacaacagcttccaccggccaccacccggatacccgccgctccgccatcgggaccacagccgcgataccccaccggtgggcaacagttgtggccacccagcggaccgcaacgggcgccgcagatttaccggccacccacggccgcaccgccgccggcgggtgcccgcggcggaactgaggcgggaaacctcgcgacatcgatgatgaagatcctgcggccaggcaggttgacgggggagttgccgcccggttaa pGEX4T1 GST Thrombin 127 ng/µL McIntosh Lab Rv17471-213* atgcatcatcatcatcatcacagcagcggcagagaaaacttgtatttccagggccatatgccgatgagccaaccagccgccccgcccgtgttgaccgtgcggtatgagggatcggagcgcacgttcgccgcaggacacgatgtcgtcgtcgggcgtgacctgcgcgcggatgtccgcgtcgcacaccccctgatctcccgggcacacctgctgctgcgattcgaccagggtcgctgggtcgccattgacaatggcagcctcaatgggctctacctcaataaccgtcgggtgccagtcgtggacatctacgatgcccagcgagtccatatcggaaaccccgacggtccggcgctggacttcgaagtgggccgccaccggggttcggccgggcgaccaccccagacgacgtcgatacgcctgcccaacctgtccgcgggagcgtggcccaccgacggcccgccgcagaccggcacgctcggctccggccagctacaacagcttccaccggccaccacccggatacccgccgctccgccatcgggaccacagccgcgataccccaccggtgggcaacagttgtggccacccagcggaccgcaacgggcgccgcagatttaccggccacccacggccgcaccgccgccggcgggtgcccgcggcggaactgaggcgggaaacctcgcgacatcgatgatgtaa pET28MHL PolyHis TEV 109 ng/µL McIntosh Lab Rv1747148-310 cttccaccggccaccacccggatacccgccgctccgccatcgggaccacagccgcgataccccaccggtgggcaacagttgtggccacccagcggaccgcaacgggcgccgcagatttaccggccacccacggccgcaccgccgccggcgggtgcccgcggcggaactgaggcgggaaacctcgcgacatcgatgatgaagatcctgcggccaggcaggttgacgggggagttgccgcccggtgccgtcaggatcggccgggcgaacgacaacgacatcgtcattcccgaggtgttggcctcacgtcaccacgccaccctggtcccgacgcctggcggcacggagattcgggacaaccgcagcatcaatggcaccttcgtcaacggcgcccgggtcgacgcggcgctgctgcacgacggcgacgtcgtgaccatcggcaacatcgacctcgtcttcgccgacggcaccctggcgcgccgtgaagagaactaa pET28MHL PolyHis TEV 114 ng/µL McIntosh Lab PknF1-292 _Cys atgtgcccgctcgcggaaggttcgacgttcgccggcttcaccatcgtccggcagttgggatccggcgggatgggcgaggtgtacctggcccggcatcccagactgccccgccaggacgcgctcaaggtactgcgggccgatgtgtcagccgacggcgaataccgggcacggttcaaccgcgaagccgatgccgcggcgtcgctgtggcatccacacatcgtcgccgtccacgaccgcggcgagttcgacggccagctctggatcgacatggacttcgtcgacggcaccgacaccgtatcccttctcagggatcgttatccgaacgggatgcccggccccgaggtcaccgagatcatcactgcggtggccgaagcgctcgactatgcccacgaacgtcggctgttgcaccgcgacgtcaaacccgccaacatcctgatcgccaatcctgattcacctgatcgtcgaatcatgttggccgacttcgggatcgccggctgggtcgatgatccaagcggattgaccgccacaaacatgactgtgggcaccgtgtcatacgcggctccggaacagcttatgggcaacgagctcgatggacgggccgaccaatacgcactagccgcgacggcgtttcacttgctgaccggctccccgccctttcagcacgccaaccccgccgtggtgatcagccagcatctcagcgcgtcaccccgggcgatcggcgatcgggttcccgagctgacaccgctggacccggtcttcgccaaagcgctggccaagcaacccaaggaccgttaccagcggtgtgtcgacttcgcgcgcgcactcggccatcgtctgggcggcgcgggtgatcctgacgacacgcgggtgtcgcaaccgtgagcggccgcatcgtga pGEX4T3 GST Thrombin 176 ng/µL McIntosh Lab PknF1-280 [binary] TEV 96 ng/µL Biomatik 28 acaccgtatcccttctcagggatcgttatccgaacgggatgcccggccccgaggtcaccgagatcatcactgcggtggccgaagcgctcgactatgcccacgaacgtcggctgttgcaccgcgacgtcaaacccgccaacatcctgatcgccaatcctgattcacctgatcgtcgaatcatgttggccgacttcgggatcgccggctgggtcgatgatccaagcggattgaccgccacaaacatgactgtgggcaccgtgtcatacgcggctccggaacagcttatgggcaacgagctcgatggacgggccgaccaatacgcactagccgcgacggcgtttcacttgctgaccggctccccgccctttcagcacgccaaccccgccgtggtgatcagccagcatctcagcgcgtcacccccggcgatcggcgatcgggttcccgagctgacaccgctggacccggtcttcgccaaagcgctggccaagcaacccaaggaccgttaccagcggtgtgtcgacttcgcgcgcgcactcggccatcgtctgggcggctaa PknF1-280 _K41M atgggcagcagccatcatcatcatcatcacagcagcggcagagaaaacttgtatttccagggccatatgtgcccgctcgcggaaggttcgacgttcgccggcttcaccatcgtccggcagttgggatccggcgggatgggcgaggtgtacctggcccggcatcccagactgccccgccaggacgcgctcatggtactgcgggccgatgtgtcagccgacggcgaataccgggcacggttcaaccgcgaagccgatgccgcggcgtcgctgtggcatccacacatcgtcgccgtccacgaccgcggcgagttcgacggccagctctggatcgacatggacttcgtcgacggcaccgacaccgtatcccttctcagggatcgttatccgaacgggatgcccggccccgaggtcaccgagatcatcactgcggtggccgaagcgctcgactatgcccacgaacgtcggctgttgcaccgcgacgtcaaacccgccaacatcctgatcgccaatcctgattcacctgatcgtcgaatcatgttggccgacttcgggatcgccggctgggtcgatgatccaagcggattgaccgccacaaacatgactgtgggcaccgtgtcatacgcggctccggaacagcttatgggcaacgagctcgatggacgggccgaccaatacgcactagccgcgacggcgtttcacttgctgaccggctccccgccctttcagcacgccaaccccgccgtggtgatcagccagcatctcagcgcgtcacccccggcgatcggcgatcgggttcccgagctgacaccgctggacccggtcttcgccaaagcgctggccaagcaacccaaggaccgttaccagcggtgtgtcgacttcgcgcgcgcactcggccatcgtctgggcggctaa pET28a PolyHis TEV 88 ng/µL Biomatik PknF1-280 _D137N atgggcagcagccatcatcatcatcatcacagcagcggcagagaaaacttgtatttccagggccatatgtgcccgctcgcggaaggttcgacgttcgccggcttcaccatcgtccggcagttgggatccggcgggatgggcgaggtgtacctggcccggcatcccagactgccccgccaggacgcgctcaaggtactgcgggccgatgtgtcagccgacggcgaataccgggcacggttcaaccgcgaagccgatgccgcggcgtcgctgtggcatccacacatcgtcgccgtccacgaccgcggcgagttcgacggccagctctggatcgacatggacttcgtcgacggcaccgacaccgtatcccttctcagggatcgttatccgaacgggatgcccggccccgaggtcaccgagatcatcactgcggtggccgaagcgctcgactatgcccacgaacgtcggctgttgcaccgcaacgtcaaacccgccaacatcctgatcgccaatcctgattcacctgatcgtcgaatcatgttggccgacttcgggatcgccggctgggtcgatgatccaagcggattgaccgccacaaacatgactgtgggcaccgtgtcatacgcggctccggaacagcttatgggcaacgagctcgatggacgggccgaccaatacgcactagccgcgacggcgtttcacttgctgaccggctccccgccctttcagcacgccaaccccgccgtggtgatcagccagcatctcagcgcgtcacccccggcgatcggcgatcgggttcccgagctgacaccgctggacccggtcttcgccaaagcgctggccaagcaacccaaggaccgttaccagcggtgtgtcgacttcgcgcgcgcactcggccatcgtctgggcggctaa pET28a PolyHis TEV 110 ng/µL Biomatik PknF1-280 _D137N-T173A-T175A atgggcagcagccatcatcatcatcatcacagcagcggcagagaaaacttgtatttccagggccatatgtgcccgctcgcggaaggttcgacgttcgccggcttcaccatcgtccggcagttgggatccggcgggatgggcgaggtgtacctggcccggcatcccagactgccccgccaggacgcgctcaaggtactgcgggccgatgtgtcagccgacggcgaataccgggcacggttcaaccgcgaagccgatgccgcggcgtcgctgtggcatccacacatcgtcgccgtccacgaccgcggcgagttcgacggccagctctggatcgacatggacttcgtcgacggcaccgacaccgtatcccttctcagggatcgttatccgaacgggatgcccggccccgaggtcaccgagatcatcactgcggtggccgaagcgctcgactatgcccacgaacgtcggctgttgcaccgcaacgtcaaacccgccaacatcctgatcgccaatcctgattcacctgatcgtcgaatcatgttggccgacttcgggatcgccggctgggtcgatgatccaagcggattggccgccgcaaacatgactgtgggcaccgtgtcatacgcggctccggaacagcttatgggcaacgagctcgatggacgggccgaccaatacgcactagccgcgacggcgtttcacttgctgaccggctccccgccctttcagcacgccaaccccgccgtggtgatcagccagcatctcagcgcgtcacccccggcgatcggcgatcgggttcccgagctgacaccgctggacccggtcttcgccaaagcgctggccaagcaacccaaggaccgttaccagcggtgtgtcgacttcgcgcgcgcactcggccatcgtctgggcggctaa pET28a PolyHis TEV 145 ng/µL Biomatik Rv26231-297 atgtcatcgggcaattcatctctgggaattatcgtcgggatcgacgattcaccggccgcacaggttgcggt pQE PolyHis / 102 ng/µL Albert 29 gcggtgggcagctcgggatgcggagttgcgaaaaatccctctgacgctcgtgcacgcggtgtcgccggaagtagccacctggctggaggtgccactgccgccgggcgtgctgcgatggcagcaggatcacgggcgccacctgatcgacgacgcactcaaggtggttgaacaggcttcgctgcgcgctggtccccccacggtccacagtgaaatcgttccggcggcagccgttcccacattggtcgacatgtccaaagacgcagtgctgatggtcgtgggttgtctcggaagtgggcggtggccgggccggctgctcggttcggtcagttccggcctgctccgccacgcgcactgtccggtcgtgatcatccacgacgaagattcggtgatgccgcatccccagcaagcgccggtgctagttggcgttgacggctcgtcggcctccgagctggcgaccgcaatcgcattcgacgaagcgtcgcggcgaaacgtggacctggtggcgctgcacgcatggagcgacgtcgatgtgtcggagtggcccggaatcgattggccggcaactcagtcgatggccgagcaggtgctggccgagcggttggcgggttggcaggagcggtatcccaacgtagccataacccgcgtggtggtgcgcgatcagccggcccgccagctcgtccaacgctccgaggaagcccagctggtcgtggtcggcagccggggccgcggcggctacgccggaatgctggtggggtcggtaggcgaaaccgttgctcagctggcgcggacgccggtcatcgtggcacgcgagtcgctgacttag Einstein College of Medicine & Montefiore Medical Center Mutations are highlighted in yellow. The codon for a non-native cysteine introduced for fluorophore labeling is in red. 30 Table 5: Protein purification Protein Name Antibiotic (conc.) Tag protease (unit) Affinity column Binding Buffer Buffer A Buffer B Cleavage buffer Stock conc. (µM) Rv17471-310 Kanamycin (50 µg/mL) TEV (150 µg/ fractions) Ni2+-NTA HisTrap HP 20 mM sodium phosphate, 500 mM NaCl, 5 mM imidazole, pH 7.5 20 mM sodium phosphate, 500 mM NaCl, 30 mM imidazole, pH 7.5 20 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, pH 7.5 20 mM sodium phosphate, 500 mM NaCl, 1 mM DTT, 0.5 mM EDTA, pH 7.0 75 Rv17471-310_Cys Kanamycin (50 µg/mL) TEV (150 µg/ fractions) Ni2+-NTA HisTrap HP 20 mM sodium phosphate, 500 mM NaCl, 5 mM imidazole, pH 7.5 20 mM sodium phosphate, 500 mM NaCl, 30 mM imidazole, pH 7.5 20 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, pH 7.5 20 mM sodium phosphate, 500 mM NaCl, 1 mM DTT, 0.5 mM EDTA, pH 7.0 63 Rv17471-310_Cys _S47A Kanamycin (50 µg/mL) TEV (150 µg/ fractions) Ni2+-NTA HisTrap HP 25 mM Tris-HCl, 500 mM NaCl, 5 mM imidazole, pH 7.5 50 mM Tris-HCl, 500 mM NaCl, 30 mM imidazole, pH 7.5 50 mM Tris-HCl, 500 mM NaCl, 500 mM imidazole, pH 7.5 20 mM sodium phosphate, 500 mM NaCl, 1 mM DTT, 0.5 mM EDTA, pH 7.0 43 Rv17471-310_Cys _S248A Kanamycin (50 µg/mL) TEV (150 µg/ fractions) Ni2+-NTA HisTrap HP 25 mM Tris-HCl, 500 mM NaCl, 5 mM imidazole, pH 7.5 50 mM Tris-HCl, 500 mM NaCl, 30 mM imidazole, pH 7.5 50 mM Tris-HCl, 500 mM NaCl, 500 mM imidazole, pH 7.5 20 mM sodium phosphate, 500 mM NaCl, 1 mM DTT, 0.5 mM EDTA, pH 7.0 56 Rv17471-310_Cys _S47A_S248A Kanamycin (50 µg/mL) TEV (150 µg/ fractions) Ni2+-NTA HisTrap HP 25 mM Tris-HCl, 500 mM NaCl, 5 mM imidazole, pH 7.5 50 mM Tris-HCl, 500 mM NaCl, 30 mM imidazole, pH 7.5 50 mM Tris-HCl, 500 mM NaCl, 500 mM imidazole, pH 7.5 20 mM sodium phosphate, 500 mM NaCl, 1 mM DTT, 0.5 mM EDTA, pH 7.0 67 Rv17471-310_Cys _only_T152 Kanamycin (50 µg/mL) TEV (150 µg/ fractions) Ni2+-NTA HisTrap HP 20 mM sodium phosphate, 500 mM NaCl, 5 mM imidazole, pH 7.5 20 mM sodium phosphate, 500 mM NaCl, 30 mM imidazole, pH 7.5 50 mM Tris-HCl, 500 mM NaCl, 500 mM imidazole, pH 7.5 20 mM sodium phosphate, 500 mM NaCl, 1 mM DTT, 0.5 mM EDTA, pH 7.0 63 Rv17471-310_Cys _no_linker_Thr Kanamycin (50 µg/mL) TEV (150 µg/ fractions) Ni2+-NTA HisTrap HP 20 mM sodium phosphate, 500 mM NaCl, 5 mM imidazole, pH 7.5 20 mM sodium phosphate, 500 mM NaCl, 30 mM imidazole, pH 7.5 20 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, pH 7.5 20 mM sodium phosphate, 500 mM NaCl, 1 mM DTT, 0.5 mM EDTA, pH 7.0 72 Rv17471-110 Kanamycin (50 µg/mL) TEV (150 µg/ fractions) Ni2+-NTA HisTrap HP 25 mM Tris-HCl, 500 mM NaCl, 5 mM imidazole, pH 7.5 50 mM Tris-HCl, 500 mM NaCl, 30 mM imidazole, pH 7.5 50 mM Tris-HCl, 500 mM NaCl, 500 mM imidazole, pH 7.5 20 mM sodium phosphate, 500 mM NaCl, 1 mM DTT, 0.5 mM EDTA, pH 7.0 58 R1747105-228_Cys Ampicillin (100 µg/mL) Thrombin (2 Unit) tandem GSTrap™ HP GSTrap™ FF PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4) PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4) 1 0mM reduced glutathione, 50 mM Tris-HCl, pH 7.4) 200 mM Tris-HCl pH 8.4, 1.5 M NaCl, 25 mM CaCl2 54 Rv17471-213 Kanamycin (50 µg/mL) TEV (150 µg/ fractions) Ni2+-NTA HisTrap HP 20 mM sodium phosphate, 500 mM NaCl, 5 mM imidazole, pH 7.5 20 mM sodium phosphate, 500 mM NaCl, 30 mM imidazole, pH 7.5 20 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, pH 7.5 20 mM sodium phosphate, 500 mM NaCl, 1 mM DTT, 0.5 mM EDTA, pH 7.0 48 Rv1747148-310 Kanamycin (50 µg/mL) TEV (150 µg/ fractions) Ni2+-NTA HisTrap HP 20 mM sodium phosphate, 500 mM NaCl, 5 mM imidazole, pH 7.5 20 mM sodium phosphate, 500 mM NaCl, 30 mM imidazole, pH 7.5 20 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, pH 7.5 20 mM sodium phosphate, 500 mM NaCl, 1 mM DTT, 0.5 mM EDTA, pH 7.0 64 PknF1-292_Cys Ampicillin (100 µg/mL) Thrombin (2 Unit) tandem GSTrap™ HP GSTrap™ PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4) PBS (140 mM NaCl, 2.7mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4) 10mM reduced glutathione, 50mM Tris-HCl, pH 7.4) 200 mM Tris-HCl pH 8.4, 1.5 M NaCl, 25 mM CaCl2 51 31 The TEV protease was prepared in-house. Thrombin protease was purchased from Sigma Aldrich. The affinity columns (5 mL) were manufactured by GE Healthcare Life Sciences. FF PknF1-280 Kanamycin (50 µg/mL) TEV (150 µg/ fractions) Ni2+-NTA HisTrap HP 25 mM Tris-HCl, 500 mM NaCl, 5 mM imidazole, pH 7.5 50 mM Tris-HCl, 500 mM NaCl, 30 mM imidazole, pH 7.5 50 mM Tris-HCl, 500 mM NaCl, 500 mM imidazole, pH 7.5 20 mM sodium phosphate, 500 mM NaCl, 1 mM DTT, 0.5 mM EDTA, pH 7.0 50 PknF1-280_K41M Kanamycin (50 µg/mL) TEV (150 µg/ fractions) Ni2+-NTA HisTrap HP 25 mM Tris-HCl, 500 mM NaCl, 5 mM imidazole, pH 7.5 50 mM Tris-HCl, 500 mM NaCl, 30 mM imidazole, pH 7.5 50 mM Tris-HCl, 500 mM NaCl, 500 mM imidazole, pH 7.5 20 mM sodium phosphate, 500 mM NaCl, 1 mM DTT, 0.5 mM EDTA, pH 7.0 63 PknF1-280_D137N Kanamycin (50 µg/mL) TEV (150 µg/ fractions) Ni2+-NTA HisTrap HP 25 mM Tris-HCl, 500 mM NaCl, 5 mM imidazole, pH 7.5 50 mM Tris-HCl, 500 mM NaCl, 30 mM imidazole, pH 7.5 50 mM Tris-HCl, 500 mM NaCl, 500 mM imidazole, pH 7.5 20 mM sodium phosphate, 500 mM NaCl, 1 mM DTT, 0.5 mM EDTA, pH 7.0 64 PknF1-280_D137N-T173A-T175A Kanamycin (50 µg/mL) TEV (150 µg/ fractions) Ni2+-NTA HisTrap HP 25 mM Tris-HCl, 500 mM NaCl, 5 mM imidazole, pH 7.5 50 mM Tris-HCl, 500 mM NaCl, 30 mM imidazole, pH 7.5 50 mM Tris-HCl, 500 mM NaCl, 500 mM imidazole, pH 7.5 20 mM sodium phosphate, 500 mM NaCl, 1 mM DTT, 0.5 mM EDTA, pH 7.0 70 Rv26231-297 Kanamycin (50 µg/mL) TEV (150 µg/ fractions) Ni2+-NTA HisTrap HP 25 mM Tris-HCl, 500 mM NaCl, 5 mM imidazole, pH 7.5 50 mM Tris-HCl, 500 mM NaCl, 30 mM imidazole, pH 7.5 50 mM Tris-HCl, 500 mM NaCl, 500 mM imidazole, pH 7.5 20 mM sodium phosphate, 500 mM NaCl, 1 mM DTT, 0.5 mM EDTA, pH 7.0 62 32 3. Results: Intrinsic factors determining Rv1747 phase separation 3.1 Increasing temperature reduces phosphorylated Rv17471-310 droplet size As summarized in Chapter 1, protein liquid-liquid phase separation is dependent upon several factors. These include, but are not limited to, protein sequence, structure and concentration, sample pH value and ionic strength, temperature, and post-translational modifications such as phosphorylation. To further understand the mechanisms of Rv1747 phase separation, in this chapter I investigated how temperature, protein concentration, and phosphorylation influence droplet formation by its isolated regulatory module, and I dissected the roles of the FHA domains and ID linker in this process. The effect of temperature on the phase separation of phosphorylated Rv17471-310 was coarsely investigated using a temperature-controlled microscope. Purified Rv17471-310 was phosphorylated at multiple sites by treatment with the active catalytic domain of PknF (Figure 13). As illustrated in Figure 14, droplets formed at 22 oC decreased in size upon heating to 37 oC. To quantitate this phenomenon, I captured 10 frames from random slide positions at each temperature (around 150 droplets each in total) for quantitation with the image analysis software Cellprofiler (Figure 15). The program automatically detects droplets in an image based on contrast differences in order to provide information such as their total number and average size. Figure 13. Reconstructed ESI-MS of Rv17471-310 with PknF treatment. Rv17471-310 (50 µM) 33 was treated before (left) and after (right) with PknF (0.1 molar ratio plus 5 mM ATP and 5 mM MgCl2) at room temperature for 1 hour. The major peaks in the right spectrum correspond to Rv17471-310 with 3, 4, and 5 covalently attached phosphates (each increasing the mass by 80 Da). Figure 14. Phosphorylated Rv17471-310 droplets shrink upon warming from 22 oC to 37 oC. Rv17471-310 (50 μM) was treated with PknF (5 μM with 5 mM ATP and 5 mM MgCl2) at room temperature for 1 hour. The images were recorded with a temperature-controlled microscope. The size of each pixel is 160 nm and the reference bar is 8 μm. Figure 15. Quantitation of Rv17471-310 droplet images using Cellprofiler. Rv17471-310 (50 μM) was treated with PknF (5 μM with 5 mM ATP and 5 mM MgCl2) at room temperature for 1 hour before imaging. On the right, the boundaries of identified droplets (cyan) are overlaid on the initial image (left). The upper and lower limits for droplet detection were 48 μm and 480 nm, respectively. The lower bounds on the threshold was set to default (0.1) in Cellprofiler. The size of each pixel is 160 nm and the reference bar is 8 μm. 34 At 22 oC, both the droplet count and average cross-sectional area increased with increasing Rv17471-310 concentration (Figure 16). However, upon warming, the droplet count remained approximately constant while the average droplet area decreased for all three protein concentrations tested. These results suggest that phase separation of the Rv1747 regulatory module is an exothermic process that is less favored with increasing temperature. Unfortunately, due to limitations of the microscope, a greater temperature range with both heating and cooling could not be explored as required to definitely measure the reversible temperature-dependent parameters governing this process. Previous studies by Ceballos at el. (81), and Luo at el. (82) have shown that protein phase separation can also be monitored using an absorbance spectrophotometer to measure sample turbidity. The availability of a spectrophotometer with heating and cooling capabilities would offer a possible future route for exploring the temperature dependence of Rv17471-310 phase separation. Figure 16. Analyzing the effect of concentration and sample temperature on phosphorylated Rv17471-310 droplet parameters using Cellprofiler. 3.2 FHA-1 is required for phase separation by the Rv1747 regulatory module Based on earlier studies, a working model for phosphorylation-enhanced Rv17471-310 phase separation was proposed. In this model, both FHA domains interact intra- and intermolecularly with linker pThr residues to generate a multivalent cross-linked protein network that leads to droplet formation. Furthermore, the FHA-2 domain was found to bind short synthetic phosphopeptides with higher affinity than did the FHA-1 domain (KD ~ 50 μM versus ~ 500 μM) (58). This suggested that, if unequal, the FHA-2 domain might have a predominant role in phase separation. To refine this 35 model, I undertook further studies using a series of Rv1747 truncation fragments and mutants. The binding of phosphothreonines by FHA domains is strongly dependent upon a small number of conserved residues, including Ser47 in FHA-1 and Ser248 in FHA-2 (Figures 5 & 6). Accordingly, I tested the phase separation behaviour of Rv17471-310-S47A, Rv17471-310-S248A and Rv17471-310-S47A/S248A with impaired FHA domains. Upon phosphorylation by PknF, Rv17471-310-S248A still formed droplets visible by light microscopy whereas Rv17471-310-S47A and Rv17471-310-S37A/S248A did not (Figure 17). Unexpectedly, these experiments showed that FHA-1, but not FHA-2, is required for the phosphorylation-enhanced phase separation of the Rv1747 regulatory module. Figure 17. A functional FHA-1 domain is required for droplet formation by the phosphorylated regulatory module. Samples of Rv17471-310 (50 μM) with mutations in the FHA-1 (S47A) and/or FHA-2 (S248A) binding sites were treated with PknF (5 μM with 5 mM ATP and 5 mM MgCl2) at room temperature for 1 hour before imaging with light microscopy (400x). The reference bar is 10 μm. I also tested how mutations in the FHA domains impacted phase separation by the unmodified regulatory module. As illustrated in Figure 18, at 250 μM, Rv17471-310-S248A formed droplets visible by light microscopy whereas Rv17471-130-S47A and Rv17471-310-S47A/S248A did not. Increasing sample concentration to 250 μM did not rescue the crippling effect of the mutated FHA-1 domain in the latter two proteins (not shown). Note that the droplets were significantly smaller in concentration-enhanced phase separation (Figure 18) than the phosphorylated-enhanced phase separation (Figure 19). Also note that the folding of FHA domains mutants (Rv17471-310-S47A, (Rv17471-130-S248A and Rv17471-310-S47A/S248A) was not confirmed by NMR or circular dichroism experiments. 36 Figure 18. A functional FHA-1 domain is also required for droplet formation by the unmodified regulatory module. Samples of Rv17471-310 (250 μM) with mutations in the FHA-1 (S47A) and/or FHA-2 (S248A) binding sites were incubated in 100 mM NaCl, 20 mM sodium phosphate at pH 6.0 for 15 min before light microscopy imaging (400x). The reference bar is 10 μm. These experiments showed that FHA-1, but not FHA-2, is important for both phosphorylated-enhanced and concentration-dependent phase separation of the Rv1747 regulatory module. A similar lack of phase separation was found for Rv1747148-310 with the FHA-1 domain removed (Figure 19). That is, visible droplets were not observed for unmodified Rv1747148-310 at 566 μM or phosphorylated Rv1747148-310 at 50 μM. Modification of the truncated regulatory module by PknF was confirmed using mass spectrometry (Figure 20). Given these somewhat unexpected results, the role of the FHA-1 domain in phase separation was further examined. Figure 19. Rv1747148-310, lacking the FHA-1 domain, does not phase separate. (Left) Rv1747148-310 (50 μM) was treated with PknF (5 μM with 5 mM ATP and 5 mM MgCl2) for 1 hour before imaging. (Right) Rv1747148-310 (566 μM) was incubated within 100 mM NaCl, 20 mM sodium phosphate at pH 6.0 for 15 min before imaging. All the images were captured using light microscopy (400x). The reference bar is 10 μm. 37 Figure 20. Reconstructed ESI-MS of Rv1747148-310 after treatment with PknF. Rv1747148-310 (50 μM) was treated with PknF (0.1 molar ratio plus 5 mM ATP and 5 mM MgCl2) at room temperature for 1 hour. The expected mass of unmodified Rv1747148-310 is 17212 Da. The major peaks at 17503 Da and 17583 Da correspond to protein with 4 and 5 covalently attached phosphates, respectively. 3.3 Rv17471-213 forms gel-like droplets Initially our expectation was that the FHA-1 and FHA-2 domains, along with the intervening linker, are required for Rv1747 regulatory module phase separation. However, mutation of the FHA-2 domain did not disrupt this process. Accordingly, I investigated a truncation fragment of the regulatory module, Rv17471-213. Despite lacking the FHA-2 domain, the protein still underwent phase separation in both its unmodified and phosphorylated forms (Figure 21). However, in contrast to Rv17471-310 which formed spherical droplets that fused upon diffusive collision into larger spherical droplets (Figure 8), the phosphorylated Rv17471-213 droplets concatenated into distinctive clusters. Furthermore, the Rv17471-213 droplets clustered in various ways, forming long chains, branched chains, and large clumps (Figure 22). Also, rather than fusing over time, the Rv17471-213 clusters stuck together to form larger clusters (Figure 23). This is reminiscent of gel-like droplets described by Wang et al (79). Such gels have a slow or negligible fusing rate and may form irregular shaped clusters over time. It is interesting to note that Rv17471-310-S248A, with an inactive FHA-2 domain, 38 formed “regular” droplets, whereas Rv17471-213, lacking this domain, formed such gels. Also, species with an inactive (Rv17471-310-S47A) or deleted (Rv1747148-310) FHA-1 domain did not phase separate under the conditions examined. Thus, the propensity of the regulatory module to phase separate and the viscoelastic properties of the resulting condensates appears to be a delicate balance of possible intermolecular interactions involving the FHA domains and the linker. Figure 21. Rv17471-213 forms gel-like droplets. (Left) Rv17471-213 (50 μM) was treated with PknF (5 μM with 5 mM ATP and 5 mM MgCl2) for 1 hour before imaging. (Middle) More concentrated Rv17471-213 (400 μM) was incubated within 100 mM NaCl, 20 mM sodium phosphate at pH 6.0 for 15 min before imaging. Although phosphorylation reduced the concentration required for phase separation, the unmodified protein also exhibited this behaviour of forming gel-like droplets. The images were captured using light microscopy (400x). The reference bar is 10 μm. (Right). Reconstructed ESI-MS of Rv17471-213 after treatment with PknF (0.1 molar ratio plus 5 mM ATP and 5 mM MgCl2) at room temperature for 1 hour. The expected mass of unmodified Rv17471-213 is 22729 Da. The major peaks at 23025 Da and 23106 Da correspond to protein with 4 and 5 covalently attached phosphates, respectively. Figure 22. Rv17471-213 gel droplets cluster in various ways. Rv17471-213 (50 μM) was treated with PknF (5 μM with 5 mM ATP and 5 mM MgCl2) for 3 hours before imaging with light microscopy (400x). The two pictures are from the same sample. The reference bar is 10 μm. 39 Figure 23. Rv17471-213 gel droplets cluster instead of fusing. Rv17471-213 (50 μM) was treated with PknF (5 μM with 5 mM ATP and 5 mM MgCl2) for 3 hours before imaging with light microscopy (400x). The images were recorded sequentially with a delay of 30 s. The smaller clusters diffuse and collide to form larger clusters, but the individual spherical entities do not fuse. The reference bar is 10 μm. 3.4 The role of the linker in phase separation We have published a model for the phase separation of phosphorylated Rv17471-310 in which phosphothreonines in the intrinsically disordered linker bind to the FHA domains in both intra- and inter-molecular manners to generate multivalent crosslinked complexes (60). However, the mechanisms by which unmodified Rv17471-310 undergoes phosphorylation-independent phase separation remained uncertain. One possibility is that, like some intrinsically disordered polypeptides (30), the Rv1747 linker can weakly self-associate to drive phase separation. To test this, I heterologously expressed in E. coli and purified the isolated linker, Rv1747105-228. Droplets were not observed with PknF-treated Rv1747105-228 at 50 μM or the unmodified linker at 250 μM (Figure 24). Upon further concentrating, Rv1747105-228 irreversibly precipitated. Thus, under the conditions examined, the isolated linker does not phase separate into droplets. 40 Figure 24. The isolated regulatory module linker, Rv1747105-228, does not phase separate. (Left) Rv1747105-228 (50 μM) was treated with PknF (5 μM with 5 mM ATP and 5 mM MgCl2) at room temperature for 1 hour before imaging. (Right) Rv1747105-228 at 250 μM formed precipitates when incubated with 100 mM NaCl and 20 mM sodium phosphate at pH 6.0 for 15 min before imaging. All the samples were captured using light microscopy (400x), and the reference bar is 10 μm. Alternatively, it is possible that the linker binds one or both FHA domains in a non-canonical manner to create multivalency underpinning phase separation. To test this, I compared the 15N-HSQC spectra of Rv17471-213 (FHA-1 domain and linker) versus that of Rv17471-110 (FHA-1 domain only) (Figure 25). Even though covalently linked, the presence of the linker did not perturb the amide chemical shifts of the FHA-1 domain. Also, the linker residues had amide 1HN chemical shifts diagnostic of an unstructured polypeptide. This indicates that the linker residues are indeed intrinsically disordered and do not measurably interact with the FHA-1 domain. 41 Figure 25. 15N-HSQC spectra of 15N-labeled Rv17471-110 and Rv17471-213. The Rv17471-110 (left) and Rv17471-213 (right) have spectra with dispersed 1HN-15N peaks that arise from amides in the structured FHA-1 domain. The additional peaks with 1H shifts ~ 8.0 - 8.5 ppm seen for Rv17471-213 arise from amides in the disordered linker. The presence of the linker does not perturb the dispersed peaks, indicating that it does bind to the FHA-1 domain. Both proteins were 100 μM in 100 mM NaCl, 20 mM sodium phosphate at pH 6.0, and the spectra were recorded overnight at room temperature. Previously our group reported that the 15N-HSQC spectra of the FHA-1 (Rv17471-156) and FHA-2 (Rv1747206-310) domains with partial linkers overlapped closely with that of the full regulatory module (Rv17471-310) (58). This also indicated that the unmodified linker does not bind the FHA-2 domain, nor do the FHA-1 and FHA-2 domains interact measurably. Thus, the mechanism for phase separation of unmodified Rv17471-310 remains unclear and likely involves the compounded effect of many interactions that are individually too weak to detect even with techniques such as NMR spectroscopy. 3.5 The roles of the linker phosphoacceptors in phase separation The observations that phosphorylation of the regulatory module reduces its saturation concentration for phase separation, and that mutation of S47 in the FHA-1 domain alter droplet 42 formation, clearly shows that pThr-FHA domain interactions contribute to this process. However, the linker has 12 threonines and treatment of Rv17471-310 with PknF leads to the phosphorylation of multiple residues. In an earlier study (60), we found that phase separation was impaired, but not fully abrogated, by alanine substitutions of the previously reported (59) phospho-acceptors T152 and T210. Residual levels of condensation likely resulted from phosphorylation of alternative linker residues. Indeed, mass spectrometry phospho-acceptor mapping revealed that besides T152, T116, T117, S161, and T169 were also phosphorylated by PknF, whereas T210 was not confirmed. To complement these studies, we generated variants of Rv17471-310 with all 12 linker threonines mutated to serines, or with all threonines except T152 substituted. As shown in Figure 26, Rv17471-310, with T152 as the only linker threonine, still underwent phosphorylation-enhanced phase separation. This species was phosphorylated by PknF at 3 to 4 serines and/or threonines. (Figure 27) Presumably this occurred across a distribution of acceptor sites, with T152 phosphorylated at least in a fraction of the sample. In contrast, the Rv17471-310 variant lacking any linker threonines only formed small droplets upon phosphorylation. Thus, pT152 is a key residue for the phase separation of the regulatory module. However, phosphoserines may also weakly contribute, perhaps through non-canonical interactions with the FHA domains or through modification of the electrostatic properties of the linker. Figure 26. T152 in the linker is important for phase separation. Samples at 50 μM of (left) wild-type Rv17471-310, (middle) Rv17471-310 with all the linker threonines, except T152, mutated to serine, and (right) Rv17471-310 with all the linker threonines mutated to serines were treated with PknF (5 μM with 5 mM ATP and 5 mM MgCl2) at room temperature for 1 hour before imaging by light microscopy (400x). The reference bar is 10 μm. 43 Figure 27. Reconstructed ESI-MS of Rv17471-310 with all linker threonines, except T152, mutated to serines. Both samples were at 50 μM and were treated with 5 μM PknF at room temperature for 1 hour before sample submission. (Left) No ATP was supplied and only a single species as observed with a mass of 33045 Da. (Right) ATP and MgCl2 at 5 mM each were included with the sample. The species with masses 33286 Da and 33364 Da have 3 and 4 covalently attached phosphates, respectively. 3.6 Summary In conclusion, in this chapter I have investigated how properties intrinsic to Rv1747 impact its phase separation. I discovered that droplets of phosphorylated Rv17471-310 shrink upon heating. This result suggested that the phase separation is an exothermic process as it is disfavored with increasing temperature. Using a series of mutation and truncation studies, I unexpectedly discovered that FHA-1, but not FHA-2, is important for phosphorylated-enhanced and concentration-dependent phase separation of the Rv1747 regulatory module. However, both FHA-1 and FHA-2 are required for “regular” droplet formation. The truncated sample (Rv17471-213), lacking FHA-2, formed gel-like droplets. Rather than coalescing over time, the gel droplets formed increasingly larger irregular shaped clusters, and thus have viscoelastic properties distinct from those of the full regulatory module. When expressed as an isolated polypeptide, the linker itself did not phase separate. Also, even when covalently linked, the linker did not interact with the FHA-1 domain. Thus, the basis for phase separation of the unmodified regulatory module remains to be determined. Finally, T152 was confirmed to be the key residue for Rv17471-310 phase separation, and substitution of all threonines in the linker to serines severely impaired, but did not fully eliminate, phosphorylation-enhanced phase separation of the regulatory module. In the next chapter, I will present my investigations of the intermolecular interaction with additional Mtb proteins influence Rv17471-310 phase separation. 44 4. Results: Intermolecular interactions impacting Rv1747 phase separation 4.1 Rv1747 phase separation due to PknF docking There is abundant evidence that the Mtb STPKs can modulate Rv1747 activity. In the context of my thesis, one mechanism is that the kinases phosphorylate threonines, including T152, within the regulatory module linker. These in turn are bound by the FHA domains to create a network of multivalent interactions leading to phase separation of regulatory module and clustering of the membrane-bound transporter. However, we previously observed that Rv17471-310 treated with PknF in the absence of ATP still formed droplets, albeit at a dramatically reduced level compared to samples with ATP (60). This was attributed to low level phosphorylation due to residual ATP that co-purified with the bacterially expressed kinase. Such an argument seemed rather speculative, and thus I further investigated the role of PknF in inducing Rv1747 phase separation. As expected, in the presence of ATP, Rv17471-310 is phosphorylated by PknF at multiple sites and readily forms regular phase-separated droplets (Figure 28). In the absence of ATP, Rv17471-310 is not phosphorylated. Nevertheless, irregular droplets were observed that were non-spherical and fused slowly. This is reminiscent of the gel-like droplets discussed in section 3.3. Note that at this concentration (50 μM), Rv17471-310 alone does not form droplets. Thus, even without phosphorylation, PknF appears to interact with the regulatory module to induce phase separation. The irregular properties of the resulting droplets reflect their different composition and mechanism of formation. 45 Figure 28. PknF induces phosphorylation-independent phase separation of the Rv1747 regulatory module. Rv17471-310 (50 μM) was treated with 5 μM PknF (with 5 mM MgCl2) in the presence (top) or absence (bottom) of 1 mM ATP at room temperature for 1 hour before imaging. In the presence of ATP, Rv17471-310 is phosphorylated at 3 to 5 sites as shown by the accompanying ESI-MS and forms spherical droplets. In the presence of PknF, but absence of ATP, Rv17471-310 is unmodified and forms irregular droplets. The reference bar is 10 μm. A possible explanation for this phenomenon lies with the fact that PknF is activated by phosphorylation of T173 and T175 within the activation loop adjacent to its catalytic site (57). These phosphothreonines could be bound by the Rv1747 FHA domains, thereby positioning the Rv1747 phospho-acceptors near the active site of the kinase to facilitate their modification. This “docking” alone might provide multivalent interactions between Rv1747 and PknF, thereby causing phase separation of the protein-kinase complex. In very recent paper, Cabarca et al. used small angle X-ray scattering and isothermal titration calorimetry to show a direct interaction between the FHA domains of Rv1747 with the catalytic domain of PknF (83). Parenthetically, in the crystal structure of PknF reported by these authors, the activation loop appeared to be disordered (83). This may provide conformational plasticity to enable the observed FHA domain binding. Indeed, others have previously reported that Mtb serine/threonine protein kinases (STPKs) such as PknF could bind to the FHA-1 domain and promote phosphorylation of the Rv1747 linker (59). There are many STPK in Mtb that act on Rv1747 (65). However, I only focused on the 46 interaction between PknF and Rv1747 since the two are expressed from the same operon (59). The interaction of PknF with the Rv1747 FHA-1 domain has been studied by Curry at el. (57), showing that it is crucial for Mtb virulence (57). Using a yeast two-hybrid assay, they also identified key amino acids in the kinase (K41 and T173/T175) and Rv1747 (S47A) that are saturation for binding (Figure 29). To test the possibility of PknF “docking” and the effect on phase separation, I mutated K41 or D137 in the active site, as well as T173/T175 in the activation loop of the kinase. As summarized in Figure 29, the K41M, D137N and T173A/T175A substitutions of PknF all completely abolished Rv17471-310 droplet formation. A similar result occurred when the S47A mutation was introduced into the FHA-1 domain of Rv17471-310. In contrast, the S248A mutation of the FHA-2 domain only modestly reduced droplet formation. These results correlate well with the two-hybrid assay studies of Curry et al. (57), suggesting that the interaction of PknF with the FHA-1 domain of Rv17471-310 leads to kinase-induced phosphorylation-independent phase separation. The docking could involve the binding of the Rv17471-310 phospho-acceptors to active site of the kinase, forming a Michaelis enzyme-substrate complex, and thus could be directly disrupted by the K41M or D137N mutations. However, a more likely scenario is that docking involves FHA domain binding to the pThr residues in the activation loop of the kinase, and this is disrupted directly by the T173A/T175A mutations (i.e., removing the pThr residues) or indirectly by the active site mutations (i.e., preventing auto-phosphorylation of the activation loop threonines). These possibilities could be distinguished by treating the inactive PknF variants with active PknF, allowing trans-phosphorylation of the activation loop. The active kinase could be removed using an alternative affinity tag before droplet formation studies. Fluorescence microscopy studies using PknF and Rv17471-310 variants with different fluorophore tags could also be pursued to test if, as predicted, they co-localize within the resulting droplets. 47 Figure 29. Mutation of Rv1747 and PknF impact their binding and droplet formation. (Left) A summary of two-hybrid yeast assay conducted by Curry et al. (57). Mutation of S47 in the Rv1747 FHA-1 domain, or mutation of K41 or T173/T175 in the catalytic domain of PknF disrupted their interaction as monitored by β-galactosidase activity. Mutation of S248 in the FHA-2 domain had an intermediate effect. (Right) A summary of droplet formation captured using bright-field microscopy and analyzed for area with Cellprofiler. The samples of Rv17471-310, Rv17471-310-S47A, and Rv17471-310-S248A (50 μM), were treated with WT PknF (5 μM with 5 mM ATP and 5 mM MgCl2) at room temperature for 1 hour before imaging. Conversely, Rv17471-310 (50 μM) was treated with PknF-K41M, PknF-D137N or PknF-T173A-T175A (5 μM with 5 mM ATP and 5 mM MgCl2) at room temperature for 1 hour before imaging. Similar to the two-hybrid yeast assay, mutation of mutation of S47 in the Rv1747 FHA-1 domain, or mutation of K41, D137 or T173/T175 in the catalytic domain of PknF eliminated droplet formation, whereas mutation of S248 in the FHA-2 domain had an intermediate effect. 4.2 Rv2623 inhibits Rv1747 phase separation Rv2623 is a universal stress protein that has been proposed to interact with Rv1747 and attenuate the transport of PIM-related cell wall precursors (55). Furthermore, has been reported that Rv2623, phosphorylated at T237 by PknG, binds to the FHA-1 domain of Rv1747 (55). If so, Rv2623 could counteract Rv1747 phase separation and transporter clustering. As a preliminary step towards 48 testing this hypothesis, I examined the effect of Rv2623 on the phase separation of Rv17471-310 (Figure 30). When added in a 1:1 molar ratio, Rv2623 diminished droplet formation by the phosphorylated regulatory module. The physical appearance of the resulting droplets also differed from that of a Rv17471-310 -only control. Thus, the presence of Rv2623 does alter Rv1747 phase separation, suggesting an interaction between the two proteins. Figure 30. Rv2623 impairs droplet formation by the phosphorylated Rv1747 regulatory module. Rv17471-310 (50 μM) was treated with PknF (5 μM with 5 mM ATP and 5 mM MgCl2) at room temperature for 1 hour before imaging. The presence of 50 μM Rv2623 (right) reduced the number and sizes of droplets relative to the control without Rv2623 (left). The reference bar is 10 μm To further explore the possible interaction between these two proteins, I used NMR spectroscopy to monitor the effect of Rv2623 on Rv17471-310. The 15N-HSQC spectrum of 15N-labeled Rv17471-310 was not perturbed upon addition of unlabeled Rv2623 at a 1:1 molar ratio (Figure 31). Thus, Rv2623 did not measurably change the structure of Rv17471-310, and the two proteins did not bind one another under these conditions. However, unlike the microscopy experiments of Figure 30, the Rv17471-310 was not phosphorylated as its phase separation would have precluded analysis by NMR spectroscopy. 49 Figure 31. Rv2623 does not perturb the structure of Rv17471-310. The 15N-HSQC spectra of unmodified 15N-lanled Rv17471-310 (75 μM) in the absence (left) or presence of unlabeled Rv26231-292 (75 μM; right) are very similar, indicating the lack of a detectable interaction. Both samples were in a 1:1 mix of phosphate buffer (100 mM NaCl, 20 mM sodium phosphate, pH 7) and HEPES buffer (50 mM NaCl, 2 mM MgCl2, 10% glycerol and 13 mM β-mercaptoethanol. pH 8.0). It is also note-worthy that, based on the crystal structure of Rv2623 (55), T237 is within a β-strand whereas FHA domains typically bind phosphothreonines within intrinsically disordered polypeptides. An initial attempt was made to phosphorylate Rv2623 with PknG. However, a check with ESI-MS indicated that the Rv2623 was not modified (data not shown). Thus, at this stage, it appears that Rv2623 can inhibit Rv1747 phase separation, although the underlying mechanisms need to be further investigated. 4.3 Rv1747 linker peptide enhances droplet size Phosphorylation-dependent phase separation of Rv17471-310 involves binding of pThr residues in the linker by the FHA domains. Accordingly, we would expect that phosphopeptide models of the linker would compete for FHA domain binding and thereby disrupt phase separation. To test this, I added 20:1 molar ratio of a phosphopeptide (KKYAGQQLPPA(pT)TRIPAA, corresponding to 50 pThr152) to phosphorylated Rv17471-310 droplets. Although slightly smaller in size, the preformed droplets did not vanish (Figure 32). Figure 32. The addition of a phosphopeptide mimic of pT152 did not diminish Rv17471-310 phase separation. All the samples are 50 μM Rv17471-310 treated with 5 μM PknF (5 mM ATP and 5 mM MgCl2) at room temperature for 1 hour before imaging. (Left) Phosphopeptide absent. (Right) The phosphopeptide was added at a 20-fold molar ratio into the reaction mix before imaging. The reference bar is 10 μm. Any effects of the phosphopeptide on the droplets might need time. To further investigate this, samples of phosphorylated Rv17471-310 without and with 20- and 50-fold molar ratios of the phosphopeptide were placed on the lab bench for 16 hrs, and the slides then imaged on the bright-field microscope. The expected results would be for the droplet to diminish, particularly with higher concentrations of added peptide. Unexpectedly, the control Rv17471-310 droplets remained the same over time, whereas the presence of the phosphopeptide actually induced “gigantic” droplets (Figure 33). It is plausible that the binding of the phosphopeptide to the FHA domains changes the balance of intra- and intermolecular FHA-linker interactions and thereby alters the viscoelastic properties of the phase separated protein that establish their droplet size. 51 Figure 33. The presence of the phosphopeptide leads to larger droplets. Samples of phosphorylated Rv17471-310 (50 μM with 5 μM PknF, 5 mM ATP and 5 mM MgCl2) were incubated without (left) and with phosphopeptide 20-fold (middle) and 50-fold (right) molar ratios at room temperature for 16 hours before imaging. The reference bar is 10 μm. 4.4 Summary To sum up this chapter, I have examined several intermolecular interactions impacting Rv1747 phase separation. I discovered that, in the absence of ATP, PknF could induce phosphorylation-independent phase separation of Rv17471-310 to form gel-like droplets. This effect was dependent upon the presence of K41 and D137 in the active site, as well as T173/T175 in the activation loop of the kinase. Furthermore, mutation of S47 in the FHA-1 domain Rv17471-310 eliminated PknF-mediated phase separation, whereas mutation of S248 in the FHA-2 domain only weakened it. This is both in agreement with the yeast two-hybrid studies on the Rv1747-PknF interaction reported by Curry et al. (57), as well as the results of Chapter 3 showing the dominant role of the FHA-1 domain in Rv1747 phase separation. A provisional explanation for this phenomenon is that Rv1747 docks via its FHA-1 domain, and to a lesser extent, its FHA-2 domain on auto-phosphorylated PknF, thereby creating an intermolecular network that favors phase separation. I also investigated the effect of the universal stress protein Rv2623 on Rv1747 droplet formation. When combined in an equimolar ratio, Rv2623 diminished phosphorylated-enhanced Rv17471-310 droplet formation as observed by microscopy. However, based on NMR spectroscopy, Rv2623 did not bind to non-phosphorylate Rv17471-310. Thus, Rv2623 can inhibit the phase separation of Rv1747, although the detailed mechanisms of this phosphorylation-dependent process remains to be elucidated. Finally, I also discovered that the addition of a phosphopeptide model of 52 pThr152 did not competitively disrupt phase separation by phosphorylated Rv17471-310, but rather led to the slow growth of “gigantic” droplets. The implications of these three examples with PknF, Rv2623, and a phosphopeptide is that intermolecular interactions can modulate Rv1747 phase separation in vitro. This in turn provides possible routes to the regulated phase separation of Rv1747 in vivo and the control of its function in the export of cell wall precursors. 53 5. Conclusion 5.1 Dissecting the mechanism of PknF dependent Rv1747 phase separation In the previous two chapters, I divided the parameters that influenced Rv1747 phase separation into two main categories: the intrinsic properties of Rv1747 regulatory module and its intermolecular interactions with additional agents. In this concluding chapter, I will integrate my findings with previously published research into a more refined model of the mechanisms and regulation of Rv1747 phase separation. Firstly, although containing tandem FHA domains, several lines of evidence demonstrate that FHA-1 has the predominant role in governing both phosphorylated-enhanced or concentration-dependent phase separation. Mutation (Rv17471-310-S47A) or deletion (Rv1747148-310) of FHA-1 completely crippled droplet formation. Similarly, mutation of S47 in the FHA-1 domain of Rv17471-310 eliminated PknF-mediated phase separation, whereas mutation of S248 in the FHA-2 domain only weakened it. This effect was also found in a cellular context. As part of his MSc thesis, my colleague Hao Ran (Charles) Li in the Gsponer Lab studied a series of fluorescently-tag Rv1747 variants expressed heterologously in Mycobacterium smegmatis, surrogate model of Mtb (80). The wild-type protein formed fluorescent foci, suggestive of phase separation in the living bacterial cell. In contrast, the Rv17471-310-S47A mutant exhibited no foci formation, where Rv17471-310-S248A mutant showed a mixed phenotype. Collectively, these studies consistently demonstrated that FHA-1 has a key role in phase separation. However, gel-like droplets formed when the FHA-2 domain was removed by truncation (Rv17471-213). Thus, both FHA-1 and FHA-2 (even with the S248A substitution) are required for “regular” droplet formation. In contrast to the “regular” droplet characteristic of the full regulatory model, these distinctly different droplets did not coalesce but rather assembled into irregular clusters. This surprising result underscores the delicate balance of interactions that can define the viscoelastic properties of a phase separated protein. I also investigated that the isolated linker (Rv1747105-228), showing that it alone did not phase separate when concentrated or PknF treated. Furthermore, in the context of Rv17471-213, the unmodified linker did not interact with the FHA-1 domain as demonstrated by NMR spectroscopy. 54 Thus, the mechanism for phase separation of the unmodified regulatory module remains unclear, and likely reflects the cooperative action of many interactions that are individually too weak to detect. Continuing to focus on the linker, substitution of all threonines to serines weakened but did not eliminate phosphorylation-enhanced phase separation. In contrast, a variant with all threonines in the linker, except T152, changed to serines formed “regular” droplets. Thus, T152 is key residue for the phosphorylation-dependent Rv1747 phase separation. Turning to intermolecular interactions, I discovered that PknF could induce phosphorylation-independent phase separation and form gel-like droplets in the absence of ATP. Mutation of K41 or D137 in the active site of the kinase, or Thr173/T175 in its activation loop, eliminated this process. Furthermore, substitution of S47 in the FHA-1 domain of Rv17471-310 also precluded PknF-induced phase separation, whereas substitution of S248 in the FHA-2 domain had an intermediate effect. This agrees well with the yeast two-hybrid studies on the Rv1747-PknF interaction reported by Curry et al. (57), in which the mutation of PknF (K41A or T173A/T175A) or Rv1747 (S47A) completely (or with S248A partially) disrupted the interaction between the two proteins. Accordingly, I hypothesize that phosphothreonines in auto-phosphorylated PknF could bridge Rv1747 via its FHA-1 domain, or to a lesser extent it FHA-2 domain. This bridging would create an intermolecular network, leading to phase separation in the absence of Rv1747 phosphorylation (Figure 34). The gel-like nature of the resulting droplets attests to their different composition as compared the “regular” droplets formed by Rv1747 alone. Previously, we proposed that phosphorylation of the Rv1747 regulatory module by PknF leads to intra- and intermolecular FHA domain-pThr interactions and “regular” droplet formation (Figures 9 & 12) (60). We can now expand this model to include a role of PknF docking to favor phase separation, even in the absence of Rv1747 phosphorylation. Furthermore, my observations that Rv2623 appeared to inhibit Rv17471-310 phase separation, whereas a phosphopeptide led to “gigantic” droplets, illustrates that additional intermolecular interactions can modulate its behavior. This provides numerous possible routes to regulate functions of the transport Rv1747 linked to its phase-dependent properties such as clustering at the bacterial membrane. 55 Figure 34. A cartoon figure illustrating the proposed mechanism of phase separation. Rv17471-310 has FHA-1 (blue) and FHA-2 (red) domains joined by a disordered linker with multiple phosphoacceptor sites (green dots). In the absence of ATP, PknF (cyan) docks on FHA-1 domain via pThr residues in its activation loop and may also bind phosphoacceptor sites in the linker via its active site. This provides multivalency to drive phase separation. In the presence of ATP, phosphorylation of the Rv1747 regulatory module by PknF leads to intra- and intermolecular FHA domain-pThr interactions and droplet formation. 5.2 Possible follow-up studies One of major discoveries of my thesis research is that new droplet states were observed for Rv1747. That is, the droplets formed by Rv17471-213 with FHA-2 deleted or by non-phosphorylated Rv17471-310 in the presence of PknF without ATP had gel-like features. Similar to those described by Wang et al (79), the gel-like droplets appeared as irregular shaped clusters that did not coalesce. Rather they slowly concatenated into long chains, branched chains, and large clumps. Also, in terms of size and shape, the exact features of the gel-like droplets differed for the truncated versus PknF-treated protein. One way to further investigate the properties of these irregular droplets would involve the use of confocal fluorescence microscopy and Fluorescence Recovery after Photobleaching (FRAP) experiments. As done previously (60), the Rv1747 and PknF variants could be chemically labeled 56 with a fluorescent compound such as Oregon Green 488 (green) or Alexa Fluor 488 (red), or genetically modified to include a fluorescent protein tag. With different fluorescent labels, co-localization of Rv1747 and PknF into the gel-like droplets could be confirmed. Also, through FRAP measurements, the diffusive and coalescing properties of droplets formed by Rv17471-213 and kinase-bound Rv17471-310 could be monitored and compared to those of the “regular” droplets formed by unmodified and phosphorylated wild-type Rv17471-310. I hypothesize that the gel droplets could be less mobile, and it would take longer for the materials to transfer within or between the dilute and concentrated phases. If so, then detailed studies using mutations and sample conditions could be pursued to further dissect the mechanisms of phase separation. The regulation of Rv1747 is certainly an area warranting more investigation. It was reported that phosphorylated Rv2623 can bind FHA-1 domain of Rv1747 and thereby counteract its phase separation and transporter clustering (55). Broadly consistent with this proposal, I did obverse via light microscopy that Rv2623 impaired droplet formation by the phosphorylated Rv1747 regulatory module. However, binding of Rv2623 to unmodified Rv17471-310 was not detected using NMR spectroscopy. An initial attempt to phosphorylate Rv2623 with PknG was not successful. More detailed attempts to control the phosphorylation states of both Rv2623 and Rv17471-310 are needed, followed by approaches such as fluorescent microscopy and FRAP measurements to test their binding (co-localization) and establish the properties of any resulting droplets. Similar strategies could be used to investigate the interaction of the phosphopeptide with Rv17471-310. The phosphopeptide did not diminish the Rv17471-310 droplets as expected, but rather induced the formation of “gigantic" droplets over time. Fluorescent microscopy and FRAP measurements would give insight regarding the interaction between the FHAs and the phosphopeptide or linker. Another important set of studies would also focus on the role of the phosphatase, PstP in controlling the phosphorylation state and phase separation of Rv1747. Collectively, such investigation would provide a foundation to understand the role of phase separation in the regulated function of Rv1747 within Mtb. Lastly, the fact that my studies of the Rv1747-PknF system via microscopy agreed well with 2-hybrid yeast assay by Curry at el. (57), shows that droplet formation intensity can be used as a projection of how well two proteins interact. For example, in both approaches, mutation of S47 in the FHA-1 domain and S248 in the FHA-2 domain had strong or intermediate, respectively, on droplet 57 formation and 2-hybrid interactions. Thus, quantitative monitoring of phase separation could be used to screen for additional factors, including interacting partners as well as potential transporter substrates, that modulate the behavior of Rv1747. 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Thesis/Dissertation
eng
Biochemistry and Molecular Biology
Vancouver : University of British Columbia Library
Attribution-NoDerivatives 4.0 International
Graduate
McIntosh, Lawrence
Dissecting the biophysical mechanisms of phase separation by the myobacterium tuberculosis ABC transporter rv1747
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