The yeast Fus1p SH3 domain recognizes non-PXXP motif targets. This is contrary to most SH3 domains, which recognize PXXP-containing sequences. In this thesis, I characterized atypical binding of the Fus1p SH3 domain and provide insight into atypical SH3 domain interactions.
I demonstrated that the Fus1p SH3 domain binds R(S/T)(S/T)SL-containing peptides derived from yeast proteins, Bnr1p and Ste5p. Through mutagenesis studies and comparisons to other SH3 domains, I showed that the Fus1p SH3 domain utilizes a portion of the same binding surface as typical SH3 domains. However, the PXXP-binding surface is debilitated in the WT domain by the substitution of unusual residues at three key conserved positions. By replacing these residues, I created a version of the Fus1p SH3 domain that binds a PXXP-containing peptide with extremely high affinity. Based on my data and analysis, I clearly delineated two distinct surfaces comprising the typical SH3 domain binding interface, and show that one of these surfaces is the primary mediator of almost every “non-canonical” SH3 domain-mediated interaction.
I demonstrated that the Fus1p SH3 domain also binds Bni1p and Pea2p through yeast two-hybrid experiments, which do not contain either PXXP or R(S/T)(S/T)SL motifs. Through mutagenesis studies and two-hybrid experiments, I showed that the Fus1p SH3 domain utilizes a binding surface comprised of two sub-surfaces to interact with Bni1p and Pea2p. The sub-surfaces include the same portion of the binding surface in typical SH3 domains utilized in R(S/T)(S/T)SL binding of the Fus1p SH3 domain, and a novel binding site. I also showed that the protein binding surface of the Fus1p SH3 domain has a role(s) for efficient mating.
Based on my findings in this thesis, I propose that dramatic alterations in SH3 domain specificity can be simply explained as a modulation of the binding strengths of sub-binding sites within the binding surface.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/24355 |
| Date | 21 April 2010 |
| Creators | Kim, JungMin |
| Contributors | Davidson, Alan R. |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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