Nuclear Magnetic Resonance Solution Structure of the Growth Factor Receptor-Bound Protein 2 Src Homology 2 Domain

Abstract

A family of NMR solution structures of the growth factor receptor-bound protein 2 (Grb2) SH2 domain has been determined by heteronuclear multidimensional NMR. Proton, nitrogen, and carbon chemical shift assignments have been made for the SH2 domain of Grb2. Assignments were made from a combination of homonuclear two-dimensional and 15N- and 13C-edited three-dimensional spectra at pH 6.2 and 298 K. Structure-induced proton and carbon secondary shifts were calculated and used to facilitate the spectral assignment process. NOE, scalar coupling, secondary chemical shift, and amide proton exchange data were used to characterize the secondary structural elements and hydrogen-bonding network in the Grb2 SH2 domain. The three-dimensional structure of the Grb2 SH2 domain was calculated using 1112 restraints obtained from NOE, coupling constant, and amide proton exchange data. The rmsd for the 24 calculated structures to the mean structure of the Grb2 SH2 domain was 0.75 A for backbone and 1.28 A for all heavy atoms. The three-dimensional fold of the Grb2 SH2 domain is similar to that observed for other SH2 domains and consists of two alpha-helical segments and eight beta-strands, six strands that make up two contiguous antiparallel beta-sheets, and two strands that form two short parallel beta-sheets. The structure of the phosphotyrosine binding pocket of Grb2 is similar to that observed for other SH2 domains. The hydrophobic binding pocket of Grb2 is similar to that observed for Src with the exception that tryptophan 121 of Grb2 occupies part of the pY+3 binding pocket. Structural implications for the Grb2 SH2 domain selectivity at the pY+2 and pY+3 sites are discussed.

Structure-based Design of Compounds Inhibiting Grb2-SH2 Mediated Protein-protein Interactions in Signal Transduction Pathways

“…These studies together with the nuclear magnetic resonance (NMR) structure of the SH2 domain in solution [22] revealed that the general fold of the Grb2-SH2 domain ( Fig. (2)) is similar to that of the previously described SH2 domains, although substantial differences appear in the loop regions.…”

An NMR strategy for fragment-based ligand screening utilizing a paramagnetic lanthanide probe

“…The backbone amide signals of LBT-Grb2 containing Lu 3+ were assigned based on the assignments reported previously (Wang et al 1996; Thornton et al 1996; Ogura et al 2008). 1 H– 15 N HSQC spectra of the 15 N-labeled LBT-Grb2 were recorded in the presence of 1 equivalent of lanthanide ions: Lu 3+ , Tb 3+ , Dy 3+ , Er 3+ and Tm 3+ (Fig.…”

α2-Chimaerin, a Cdc42/Rac1 Regulator, Is Selectively Expressed in the Rat Embryonic Nervous System and Is Involved in Neuritogenesis in N1E-115 Neuroblastoma Cells

“…Interestingly, ␣2-chimaerin N94H SH2 no longer binds Crmp-2 (Ferrari, 1999), although in vitro phosphotyrosine binding is unaffected. Predicted chimaerin SH2 domain structure modeled on Grb-2 SH2 (Thornton et al, 1996) indicates that the N94H substitution could displace an aspartate residue (D60), locally altering surface charge in the region adjacent to the phosphotyrosine binding pocket, potentially disrupting target interactions. Relocation and/or lipid activation of GAP activity could be responsible for the morphological effects (cell rounding) of ␣2-chimaerin N94H SH2 in N1E-115 cells, because mutation of its GAP domain promoted microspikes and neurite outgrowth.…”