Engineering thermodynamic stability and peptide binding properties of the Abp1p SH3 domain

This thesis investigates protein-protein interactions using the SH3 domain as a model system. The interaction between the SH3 domain of the yeast Actin binding protein 1 (Abp1p) and ligands derived from its biological target proteins is studied. Information derived from an SH3 domain sequence alignment and SH3 domain structural alignments was used to predict functional Abp1p SH3 domain residues. Eight unusual Abp1p SH3 domain residues were identified. Replacement of three of these residues significantly stabilized the domain, increasing its Tm from 60°C to greater than 90°C. These residues were not important for the in vitro binding activity of the Abp1p SH3 domain, but their location on the SH3 domain surface and role in modulating stability suggests potential roles for their function in vivo. Investigation of the functional roles of certain other unusual residues via mutagenesis experiments, in vitro peptide binding assays, and NMR spectroscopy revealed that they are involved in ligand recognition at a binding surface of the Abp1p SH3 domain that differs from the typical interaction surface. The level of Abp1p SH3 domain binding affinity for its in vivo binding sites ranged from 2 muM to 0.03 muM, and residues at either end of the ligand sequences were shown to be required for high affinity. Substitution of conserved residues on the typical interaction surface of the SH3 domain indicated that certain hydrogen bonds across this interface contribute more than others to binding energy. Replacement of Asn 53 was found to reduce binding affinity in a target-specific manner. Mutagenesis experiments indicated that this effect is related to the structural propensity of a single residue in the XP-X-XP motif of the Abp1p SH3 domain ligands, and may result from a change in binding kinetics. These studies reveal that SH3 domains can use an additional binding surface to interact with target sequences, and indicates that certain hydrogen bonds in SH3 domain interaction interfaces control binding affinity and kinetics. The utility of sequence alignment analysis in identifying residues that are important for the stability and function of SH3 domains is also demonstrated.