title: Circular dichroic investigation of the native and non-native conformational states of the growth factor receptor-binding protein 2 N-terminal src homology domain 3: effect of binding to a proline-rich peptide from guanine nucleotide exchange factor.
abstract: SH3 (src homology domain 3) domains are small protein modules that interact with proline-rich peptides. The structure of the N-terminal SH3 domain from growth factor receptor-binding protein 2 (Grb2), an adapter protein in the intracellular signaling pathway to Ras, was investigated by circular dichroic (CD) spectroscopy. The compact native beta-barrel conformation, previously elucidated by NMR spectroscopy, was largely predominant at pH = 4.8, in the absence of salt. From the structural changes induced by varying pH, ionic strength, temperature, or hydrophobicity of the environment, evidence for the existence of distinct nonnative conformations was obtained in the far- and near-UV domains. Along the free energy scale, these appear to distribute into two conformational ensembles, depending on the extent of structural and thermodynamic differences compared to the native conformation. The first ensemble consists of non-native conformations with a nativelike secondary structure, and the second is composed of partially unfolded conformations having short alpha-helical fragments or turnlike motifs in their nonnative secondary structure. Most of the observed nonnative conformations exist in mild or nondenaturing conditions. They probably have distinct compactness of their inner structure, depending on the strength of nonlocal interactions, but only the native all-beta conformation possesses a condensed protein exterior, appropriate for the binding to the VPPPVPPRRR decapeptide from Sos. Upon binding, the native conformation undergoes a local tertiary structure change in a hydrophobic pocket at the binding site. This is accompanied by the PP-II helix folding of the proline-rich peptide. Interestingly, in the near-UV domain, a significant change in the spectral contribution of an aromatic exciton was observed, thus allowing quantitative tracking of the binding process.
authors.affiliation: Pharmacologie et Physico-Chimie des Interactions Cellulaires et Moléculaires, UMR CNRS 7034, Faculté de Pharmacie de Strasbourg, Université Louis Pasteur, BP 24, F-67401 Illkirch, France. bousquet@pharma.u-strasbg.fr
authors.forename: J A
authors.initials: JA
authors.lastname: Bousquet
authors.forename: C
authors.initials: C
authors.lastname: Garbay
authors.forename: B P
authors.initials: BP
authors.lastname: Roques
authors.forename: Y
authors.initials: Y
authors.lastname: Mély
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D002942:Circular Dichroism; D051380:GRB2 Adaptor Protein; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation; D009994:Osmolar Concentration; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D055232:Proline-Rich Protein Domains; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D014466:Ultraviolet Rays; D018909:src Homology Domains / Q000502:physiology*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; D020662:Guanine Nucleotide Exchange Factors; D010455:Peptides; D011506:Proteins