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.

Protein-protein interactions are an integral part of virtually all aspects of cellular function. Many of these interactions are mediated by small modular units called protein interaction domains (PIDs). We do not yet understand, however, how much functional information is encoded in these modules. It has previously been shown that Nbp2SH3 and Bem1SH3b domains in S. cerevisiae bind several target peptides with the same consensus sequence, yet display finely tuned affinities for each. In this study, I have shown that there exists an evolutionarily conserved ability of orthologous fungal Nbp2SH3, Bem1SH3b, and Abp1SH3 domains to discriminate between target peptides within the same species. In addition, I have developed a method to quantitatively probe SH3 domain specificity using purified SH3 domains and naturally occurring proline-rich constructs (PRRs) in the context of cell lysate from S. cerevisiae. Expansion of this work may yield valuable insights into intrinsic SH3 domain specificity.

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.

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Protein interactions are often mediated through binding of protein interaction domains to short peptide motifs. However, how the specificity of interactions is regulated within domain families is not well understood. In this thesis, I investigated this issue through the study of two cross-reactive SH3 domains from the yeast adaptor proteins Nbp2p and Bem1p. Despite low sequence similarity and different cellular roles, the Nbp2p SH3 and Bem1p SH3b domains recognize targets containing the same consensus sequences, raising the question of how the specificity of these domains is controlled. The Bem1p SH3b domain is also unusual in containing a 40 amino acid long C-terminal extension required for folding and interaction with the Cdc42p GTPase, which I also investigated in this thesis. I demonstrated that although Nbp2p SH3 and Bem1p SH3b domains recognize the same consensus sequence, each domain possesses its own unique specificity within the context of eight yeast peptides. Through structural and mutagenesis studies, I demonstrated that the Nbp2p SH3 and Bem1p SH3b domains employ different mechanism for binding, explaining how each domain can still possess its own unique specificity, while recognizing the same consensus sequence. I found that the specificity of Bem1 SH3b domain is largely regulated through inhibitory interactions, where a conserved residue in the binding interface serves to decrease binding to non-specific targets. Most significantly I observed that reducing the specificity of Bem1 SH3b domain, without reducing its binding affinity decreases its ability to function in vivo. My structural and mutagenesis studies with the Bem1p SH3b domain also demonstrated that its C-terminal extension is involved in peptide binding surface and creates a separate surface required for interaction with Cdc42p. I also showed that the Bem1p SH3b domain is capable of simultaneously interacting with peptide targets and Cdc42p, suggesting a role for this domain in Cdc42p mediated activation. Overall, my findings demonstrated the ability of SH3 domains to perform complex functions and bind their targets with finely tuned specificity. Most importantly, I demonstrated the significance of negative interactions for specificity determination and the importance of precise specificity for in vivo function.

Protein interactions are often mediated through binding of protein interaction domains to short peptide motifs. However, how the specificity of interactions is regulated within domain families is not well understood. In this thesis, I investigated this issue through the study of two cross-reactive SH3 domains from the yeast adaptor proteins Nbp2p and Bem1p. Despite low sequence similarity and different cellular roles, the Nbp2p SH3 and Bem1p SH3b domains recognize targets containing the same consensus sequences, raising the question of how the specificity of these domains is controlled. The Bem1p SH3b domain is also unusual in containing a 40 amino acid long C-terminal extension required for folding and interaction with the Cdc42p GTPase, which I also investigated in this thesis. I demonstrated that although Nbp2p SH3 and Bem1p SH3b domains recognize the same consensus sequence, each domain possesses its own unique specificity within the context of eight yeast peptides. Through structural and mutagenesis studies, I demonstrated that the Nbp2p SH3 and Bem1p SH3b domains employ different mechanism for binding, explaining how each domain can still possess its own unique specificity, while recognizing the same consensus sequence. I found that the specificity of Bem1 SH3b domain is largely regulated through inhibitory interactions, where a conserved residue in the binding interface serves to decrease binding to non-specific targets. Most significantly I observed that reducing the specificity of Bem1 SH3b domain, without reducing its binding affinity decreases its ability to function in vivo. My structural and mutagenesis studies with the Bem1p SH3b domain also demonstrated that its C-terminal extension is involved in peptide binding surface and creates a separate surface required for interaction with Cdc42p. I also showed that the Bem1p SH3b domain is capable of simultaneously interacting with peptide targets and Cdc42p, suggesting a role for this domain in Cdc42p mediated activation. Overall, my findings demonstrated the ability of SH3 domains to perform complex functions and bind their targets with finely tuned specificity. Most importantly, I demonstrated the significance of negative interactions for specificity determination and the importance of precise specificity for in vivo function.

SH3 domains are excellent models for probing folding and protein interactions. This thesis describes NMR studies of several SH3 domains, including the N-terminal SH3 domain of the Drosophila adaptor protein Drk (drkN SH3 domain), the SH3 domain of the proto-oncogene tyrosine-kinase Fyn, and the SH3 domains of the human adaptor protein CIN85, involved in Cbl-mediated downregulation of epidermal growth factor receptor (EGFR) and other receptor tyrosine kinases (RTKs). The drkN SH3 domain is an ideal system for studying disordered states. The unique quality of this isolated domain is that it exists in an approximately 50/50 equilibrium between its folded and unfolded states under non-denaturating buffer conditions. Interestingly, the single T22G mutation dramatically stabilizes the domain. Here the NMR structures of the drkN SH3 domain and its T22G mutant are determined and compared in order to illuminate the causes of the marginal stability of the domain. Solvent exposure of the folded and the unfolded drkN SH3 domains are probed and compared with a novel NMR technique using molecular oxygen dissolved in solution as a paramagnetic probe. The changes in partial molar volume along the folding trajectories of the drkN SH3 and Fyn SH3 domains are also studied and analyzed here in terms of changes in protein hydration and packing accompanying folding. Finally, the interactions between the SH3 domains of CIN85 and ubiquitin are discussed. All three are shown to bind ubiquitin. The structure of the SH3-C domain in complex with ubiquitin is presented and the effect of disruption of ubiquitin binding on ubiquitination of CIN85 and EGFR in vivo is discussed. SH3 domains are easily amendable to a wide range of NMR approaches and provide a good system for development and testing of novel methods. Through the use of these approaches significant insights into details of SH3 domain structure, stability, mechanisms of folding and cellular function have been gained.

Due to their simplicity, viruses require the assistance of host factors for various aspects of their replication cycle. This study investigated the interaction of one of the two non-structural replicase proteins of rubella virus (RUBV), P150, with cell proteins. RUBV forms replication complexes for replicating its RNA in association with membranes of endosomes and lysosomes; the thusly modified endosomes/lysosomes are termed cytopathic vacuoles or CPVs. In the first study, a RUBV expressing a FLAG epitope-tagged P150 was used to co-immunoprecipitate putative interacting cell proteins from an infected cell lysate fraction enriched for CPVs using differential centrifugation. However, the only interacting protein identified was the companion RUBV replicase protein P90. Thus, cell proteins do not bind with either sufficient affinity or in stoichiometric amounts to be detected by this method and may not be a component of the virus holoenzyme. In the second study, a proline-rich region within P150 with three PxxPxR consensus SH3 domain-binding motifs was investigated for its ability to bind cell proteins. Substitution mutations (to alanine) of the two prolines were made in each of these motifs with the finding that mutations in the first two motifs led to lower viral titers and a small plaque phenotype with reversion to the wt sequence within one passage. Mutations in the third motif had a wt phenotype and did not revert. However, these mutations did not affect viral RNA synthesis, suggesting that the importance of these motifs is in a later stage of viral life cycle, e.g. virion assembly and release. To extend these findings, the proline hinge region with either the wt or mutant sequence was expressed as a GST-fusion in human cells. Pulldown experiments revealed specific binding with human p32 protein (gC1qR), which was previously shown to interact with the RUBV capsid protein. Binding of p32 with P150 was confirmed. The function of p32 in the RUBV replication cycle is unclear, but could involve virion assembly and release or induction of apoptosis.

SH3 domains are excellent models for probing folding and protein interactions. This thesis describes NMR studies of several SH3 domains, including the N-terminal SH3 domain of the Drosophila adaptor protein Drk (drkN SH3 domain), the SH3 domain of the proto-oncogene tyrosine-kinase Fyn, and the SH3 domains of the human adaptor protein CIN85, involved in Cbl-mediated downregulation of epidermal growth factor receptor (EGFR) and other receptor tyrosine kinases (RTKs). The drkN SH3 domain is an ideal system for studying disordered states. The unique quality of this isolated domain is that it exists in an approximately 50/50 equilibrium between its folded and unfolded states under non-denaturating buffer conditions. Interestingly, the single T22G mutation dramatically stabilizes the domain. Here the NMR structures of the drkN SH3 domain and its T22G mutant are determined and compared in order to illuminate the causes of the marginal stability of the domain. Solvent exposure of the folded and the unfolded drkN SH3 domains are probed and compared with a novel NMR technique using molecular oxygen dissolved in solution as a paramagnetic probe. The changes in partial molar volume along the folding trajectories of the drkN SH3 and Fyn SH3 domains are also studied and analyzed here in terms of changes in protein hydration and packing accompanying folding. Finally, the interactions between the SH3 domains of CIN85 and ubiquitin are discussed. All three are shown to bind ubiquitin. The structure of the SH3-C domain in complex with ubiquitin is presented and the effect of disruption of ubiquitin binding on ubiquitination of CIN85 and EGFR in vivo is discussed. SH3 domains are easily amendable to a wide range of NMR approaches and provide a good system for development and testing of novel methods. Through the use of these approaches significant insights into details of SH3 domain structure, stability, mechanisms of folding and cellular function have been gained.

Protooncogene Crk was found to be upregulated in tumours with aggressive and invasive potential. The adaptor protein Crk has an important role in cell signaling: it integrates signals from activated integrins and growth factors receptors via its SH2 domain and transmits the signal to its SH3 domain binding partners that activate the small GTPases Rac1, Rap1 and Ras. This leads to regulation of cell migration, proliferation and survival. The aim of this thesis project was to inhibit the Crk dependent signaling by a competitive inhibition of the Crk SH3 domain, using a high affinity CrkSH3 binding peptoid. Binding of the inhibitor to the Crk SH3 domain prevents binding of cellular Crk SH3 interaction partners and the corresponding signal transmission is impaired. In this thesis project the effect of the Crk SH3 inhibition on the invasiveness of cancer cells was analyzed. The observed inhibitory effect on cell invasion as well as on anchorage independent growth provides a proof of therapeutical relevance of targeting CrkSH3N domain by peptoide-based inhibitors. Powered by TCPDF (www.tcpdf.org)