STRUCTURAL AND DYNAMIC CHARACTERIZATION OF THE PHOSPHOTYROSINE BINDING REGION OF A SRC HOMOLOGY-2 DOMAIN-PHOSPHOPEPTIDE COMPLEX BY NMR RELAXATION, PROTON-EXCHANGE, AND CHEMICAL-SHIFT APPROACHES

Arginine side chains are often involved in protein-protein and protein -nucleic acid interactions. Due to a number of factors, resonance assi gnment and detection of NOEs involving the arginine side chains via st andard NMR techniques can be difficult. We present here an approach to characterization of the interaction between a phosphopeptide (pY1021) and four arginine residues that line the phosphotyrosine-binding pock et of the C-terminal SH2 domain of phospholipase C-gamma 1 (PLCC SH2). Previously published [Pascal, S. M., et al. (1994) Cell 77, 461] NOE data provide a partial description of this interaction, including cont acts between the aliphatic region of Arg 59 and the phosphotyrosine (p Tyr) aromatic ring. Further characterization has now been accomplished by using N-15 and C-13 NMR relaxation studies of arginine NE and C-ze ta spins, respectively, and proton exchange rates of arginine H-epsilo n nuclei. Differences between the chemical shifts of the arginine guan idino groups of the free SH2 domain in imidazole and phosphate buffers or in complex with pY1021 have provided insight into specific interac tions with the phosphate and the aromatic ring of the pTyr. The result ing data are consistent with the most stable hydrogen bonds to phospha te donated by the Arg 39 epsilon-NH and the two Arg 37 eta-NH2 groups and with pTyr aromatic ring interactions involving the Arg 39 and poss ibly the Arg 18 guanidino groups.