Mutational investigation of the specificity determining region of the Src SH2 domain

SH2 domains are protein modules which bind tyrosine phosphorylated sequence s in many signaling pathways. These domains contain two regions with specia lized functions: residues in one region form a deep pocket into which the p hosphotyrosine of the target inserts, while the other region contains the s o-called "specificity determining residues" which interact with the three r esidues C-terminal to the phosphotyrosine in the target. Here, titration ca lorimetry and site-directed mutagenesis have been used to probe the importa nce of eight specificity determining residues of the SH2 domain of the Src kinase involved in contacts with its tyrosine phosphorylated consensus pept ide target (sequence pYEEI where pY indicates a phosphotyrosine). Mutating six of these eight residues to Ala individually, resulted in a threefold or less loss in binding affinity; hence the majority of the residues in the s pecificity determining region are by themselves of minimal importance for b inding. Two residues were found to have significant effects on binding: Tyr beta D5 and Lys beta D3. Tyr beta D5 was the most crucial residue as evide nced by the 30-fold loss in affinity when Tyr beta D5 is mutated to he. How ever, while this mutation eliminated the specificity of the Src SH2 domain for the pYEEI peptide sequence, it was not sufficient to switch the specifi city of the Src SH2 domain to that of a related SH2 domain which has an lie at the beta D5 position. Mutation of Lys beta D3 to an Ala residue resulte d in a modest reduction in binding affinity (sevenfold). It is interesting that this mutation resulted in a change of specificity affecting the select ion of the +1 position residue C-terminal to the phosphotyrosine. Except fo r the Lys beta D3 - +1 Glu interaction which is significantly coupled, only weak energetic coupling was observed across the binding interface, as asse ssed using double mutant cycles. The results of this study suggest that int eractions involving the specificity determining region of SH2 domains may b e insufficient by themselves to target single SH2 domains to particular pho sphorylated sites. (C) 2000 Academic Press.