Proline-rich segments in the guanine nucleotide exchange factor C3G bind much more strongly to the N-terminal Src homology 3 domain (SH3-N) of the proto-oncogene product c-Crk than to other SH3 domains. The presence of a lysine instead of an arginine in the peptides derived from C3G appears to be crucial for this specificity towards c-Crk.In order to understand the chemical basis of this specificity we have determined the crystal structure of Crk SH3-N in complex with a high affinity peptide from C3G (PPPALPPKKR, Kd approximately 2 microM) at 1.5 A resolution. The peptide adopts a polyproline type II helix that binds, as dictated by electrostatic complementarity, in reversed orientation relative to the orientation seen in the earliest structures of SH3-peptide complexes. A lysine in the C3G peptide is tightly coordinated by three acidic residues in the SH3 domain. In contrast, the co-crystal structure of c-Crk SH3-N and a peptide containing an arginine at the equivalent position (determined at 1.9 A resolution) reveals non-optimal geometry for the arginine and increased disorder.The c-Crk SH3 domain engages in an unusual lysine-specific interaction that is rarely seen in protein structures, and which appears to be a key determinant of its unique ability to bind the C3G peptides with high affinity.