title: 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.
abstract: Arginine side chains are often involved in protein--protein and protein--nucleic acid interactions. Due to a number of factors, resonance assignment and detection of NOEs involving the arginine side chains via standard 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 pocket 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 contacts between the aliphatic region of Arg 59 and the phosphotyrosine (pTyr) aromatic ring. Further characterization has now been accomplished by using 15N and 13C NMR relaxation studies of arginine N episilon and C zeta spins, respectively, and proton exchange rates of arginine H episilon nuclei. Differences between the chemical shifts of the arginine guanidino groups of the free SH2 domain in imidazole and phosphate buffers or in complex with pY1021 have provided insight into specific interactions with the phosphate and the aromatic ring of the pTyr. The resulting data are consistent with the most stable hydrogen bonds to phosphate 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 possibly the Arg 18 guanidino groups.
authors.affiliation: Biochemistry Research Division, Hospital for Sick Children, Toronto, Ontario, Canada.
authors.forename: S M
authors.initials: SM
authors.lastname: Pascal
authors.forename: T
authors.initials: T
authors.lastname: Yamazaki
authors.forename: A U
authors.initials: AU
authors.lastname: Singer
authors.forename: L E
authors.initials: LE
authors.lastname: Kay
authors.forename: J D
authors.initials: JD
authors.lastname: Forman-Kay
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000378:metabolism*; D019000:Phosphotyrosine / Q000378:metabolism*; D011522:Protons; D010738:Type C Phospholipases / Q000737:chemistry / Q000378:metabolism*; D018909:src Homology Domains
chemical_list: D010748:Phosphopeptides; D011522:Protons; D019000:Phosphotyrosine; D010738:Type C Phospholipases