AFFINITY AND SPECIFICITY REQUIREMENTS FOR THE FIRST SRC HOMOLOGY-3 DOMAIN OF THE CRK PROTEINS

The specificity of SH3 domain complex formation plays an important rol e in determining signal transduction events, We have previously identi fied a highly specific interaction between the first CrkSH3 domain [Cr kSH3(1)] and proline-rich sequences in the guanine nucleotide exchange factor C3G. A 10 amino acid peptide derived from the first proline-ri ch sequence (P(3)P(4)P(5)A(6)L(7)P(8)P(9)K(10)K(11)R(12)) bound with a K-d of 1.89 +/- 0.06 mu M and fully retained the high affinity and un ique selectivity for the CrKSH3(1) domain. Mutational analysis showed that P-5, P-8, L(7) and K-10 are critical for high affinity binding. A conservative mutation, K10R, significantly decreased the affinity for the CrkSH3(1) domain while increasing the affinity for Grb2. Comparat ive binding studies with the K10R and K10A mutant peptides to c-Crk an d v-Crk further suggested that K-10 binds via a charge-dependent and a charge-independent interaction to the RT loop of the CrkSH3(1) domain , Besides determining important structural features necessary for high affinity and specificity binding to the CrkSH3(1) domain, our results also demonstrate that a conservative mutation in a single amino acid can significantly alter the specificity of an SH3 binding peptide.