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Probing the interaction between U24 from HHV-6A and Smurf2 WW domains

University of British Columbia

This thesis describes the study of the interaction of the U24 membrane protein, encoded by Human Herpesvirus type-6A (HHV-6A), with WW domains from Smad ubiquitylation regulatory factor 2 (Smurf2) via its PPxY motif. U24 from HHV-6A is of interest because it acts to block endosomal recycling, as mediated by its PPxY motif interacting with WW domain-containing proteins. We have used a multidisciplinary approach to study the interactions between the Smurf2 WW domains and the PPxY motif-containing region of U24. The GST pull-down experiment demonstrated a difference in affinity between WW domains for the full-length U24 protein, prompting binding studies on interactions between the PPxY motif-containing region of U24 and isolated WW3 domain or WW2 and WW3 domains in tandem (WW23). In Chapter 2, studies were focused on interactions between the third WW domain (WW3) of Smurf2, and the 15-mer U24 peptide containing the PPxY motif. NMR studies demonstrated that the PPxY motif of U24 peptide bound to Smurf2 WW3 domain in a similar way as the interaction between Smurf2 WW3 domain and its cognate ligand Smad7. The dissociation constant was determined to be 123 ± 4 μM at 5°C, reflecting weak binding affinity between WW3 domain and U24 peptide, possibly due to the lack of additional interactions between WW3 domain and the region of U24 peptide beyond the PPxY motif. Circular Dichroism (CD) experiments suggested that isolated WW3 domain was not as stable as other WW domains and binding to U24 peptide enhanced its stability slightly. In Chapter 3, interactions between tandem Smurf2 WW23 and U24 peptide were studied. NMR studies demonstrated that the interaction between Smurf2 WW23 and U24 peptide mainly relied on the U24 peptide binding to its WW3 domain and that the WW2 domain played only a minor role in the interaction. CD experiments were also carried out to detect the stability of Smurf2 WW23 with and without U24 peptide.

Text

2015-12-31

Attribution-NonCommercial-NoDerivs 2.5 Canada

http://hdl.handle.net/2429/53953

Chemistry

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/2.5/ca/