Functional Analysis of Single SH2 Domain Binding

Signaling cascades involving SH2 domain-containing proteins are initiated when SH2 domains engage sites of tyrosine phosphorylation on receptors and cellular proteins. Hence, the integrity of signaling pathways critically depends on the specific recognition of the phosphorylated state of tyrosine residues by SH2 domains. The question of how SH2 domains accomplish this task is the subject of this section. 

Nearly all SH2 domains require tyrosine phosphorylation of their targets for recognition (for the exception, see Section V). This has been established from numerous studies which have compared the binding of phosphorylated and dephosphorylated peptides to SH2 domains (Bibbins et al., 1993 Domchek et al., 1992 Gilmer et al., 1994 Lemmon and Ladbury, 1994 Mayer et al., 1992 Piccione et al., 1993). These binding experiments have established that high-affinity tyrosyl 

Investigations which have probed the binding of just the amino acid pTyr (or close variants thereof) to SH2 domains have underscored the dominant role played by just this single amino acid in SH2 domain binding. It was initially qualitatively observed that pTyr weakly associates with SH2 domains (Lemmon and Ladbury, 1994 Mayer et al., 1992). Competition binding experiments then revealed that pTyr-like mimetics have measurable affinity for the Src and N-p85 SH2 domains at a Kd of 1 mM (Burke et al., 1995). These findings have been confirmed by direct calorimetric titrations to measure binding of pTyr alone to the Src SH2 domain, which indicated that pTyr binds with an affinity of 333 pM at 25°C (Bradshaw et al., 1999). 

The pTyr of tyrosyl phosphopeptides is a negatively charged amino acid whose phosphate group carries a —2 charge at neutral pH. Experiments monitoring the pH dependence of SH2 domain binding have probed the importance of the phosphate s charge and have shown that 

Studies of SH2 domain mutants have established which side chains are required for coordination of the pTyr. It has been determined that Arg (3B5, the universally conserved residue which forms the base of the pTyr binding cavity, is by far the most important residue for recognition. Mutation of this residue in vivo abrogates SH2 domain attachment to pTyr-containing targets (Bibbins et al., 1993). In vitro, an Arg (3B5 to Ala mutation in the Src SH2 domain showed a 500-fold decrease in tyrosyl phosphopeptide affinity compared to wild type (Bradshaw et al., 1999). Mutation of Arg (3B5 in other SH2 domains results in either an insoluble protein (Lemmon and Ladbury, 1994) or the complete elimination of detectable binding to tyrosine phosphorylated targets (Mayer a/., 1992). 

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