HETERONUCLEAR NMR-STUDIES OF THE COMBINED SRC HOMOLOGY DOMAIN-2 AND DOMAIN-3 OF PP60 C-SRC - EFFECTS OF PHOSPHOPEPTIDE BINDING

The results of heteronuclear NMR studies on the combined Src homology domains 2 and 3 (SH3-SH2) of pp60 c-Src are presented. Resonance assig nments were obtained using heteronuclear triple-resonance experiments in conjunction with N-15-separated nuclear Overhauser effect spectrosc opy (NOESY) data. A modified three-dimensional (CO)-C-13-N-15-H-1 spec tral correlation experiment [(HACA)CO(CA)NH] with improved sensitivity is presented that provided additional sequential information and reso lved several ambiguities. Chemical shifts and sequential-and medium-ra nge NOE cross peaks indicate that the structures of both the SH3 and S H2 portions of the polypeptide are very similar to those of the isolat ed SH3 and SH2 domains. Binding of a high-affinity phosphopeptide, EPQ pYEEIPIYL, induces large chemical shift changes at several locations i n the SH2 domain. Comparison with known results for peptide binding to SH2 domains shows that the residues displaying the largest effects ar e all involved in peptide binding or undergo significant conformationa l changes upon binding. However, subtle changes of both H-1 and N-15 c hemical shifts are observed for residues within the SH3 domain and the connecting linker region, indicating possible cross-domain communicat ion.