ABSTRACT
Grb2 is an adaptor protein composed of a single SH2 domain flanked by two SH3 domains. Grb2 functions as an important evolutionary conserved link between a variety of cell membrane receptors and the Ras/MAP kinase-signaling cascade. Here, we describe the solution structure of Grb2 as revealed by NMR and small angle X-ray scattering measurements. We demonstrate that Grb2 is a flexible protein in which the C-terminal SH3 domain is connected to the SH2 domain via a flexible linker. This is in contrast to the previously described Grb2 crystal structure, which showed a compact structure with intramolecular contact between two SH3 domains. Binding experiments on Grb2 and peptides containing two different proline-rich sequences indicate that Grb2 adapts the relative position and orientation of the two SH3 domains to bind bivalently to the target peptide sequences.
Subject(s)
Adaptor Proteins, Signal Transducing , Proteins/chemistry , Proteins/metabolism , src Homology Domains , Amino Acid Sequence , Computer Simulation , GRB2 Adaptor Protein , Humans , Ligands , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Sequence Data , Mutation , Pliability , Proline/genetics , Proline/metabolism , Protein Binding , Protein Structure, Secondary , Proteins/genetics , Sequence Alignment , Software , Solutions , Substrate Specificity , X-Ray DiffractionABSTRACT
The solution structure of growth factor receptor-bound protein 2 (Grb2) SH2 complexed with a Shc-derived phosphotyrosine (pTyr)-containing peptide was determined by nuclear magnetic resonance (NMR) spectroscopy. The pTyr binding site of Grb2 SH2 was similar to those of other SH2 domains. In contrast, the amino acid residues C-terminal to pTyr did not form an extended structure because of steric hindrance caused by a bulky side-chain of Trp121 (EF1). As a result, the peptide formed a turn-structure on the surface of Grb2 SH2. The asparagine residue at the pTyr+2 position of the Shc-peptide interacted with the main-chain carbonyl groups of Lys109 and Leu120. The present solution structure was similar to the crystal structure reported for Grb2 SH2 complexed with a BCR-Abl-derived phosphotyrosine-containing peptide. Finally, the structure of Grb2 SH2 domain was compared with those of the complexes of Src and phospholipase C-gamma1 with their cognate peptides, showing that the specific conformation of the peptide was required for binding to the SH2 domains.
Subject(s)
Adaptor Proteins, Signal Transducing , ErbB Receptors/chemistry , Peptide Fragments/chemistry , Proteins/chemistry , src Homology Domains , Binding Sites , ErbB Receptors/metabolism , GRB10 Adaptor Protein , GRB2 Adaptor Protein , Isoenzymes/chemistry , Isoenzymes/metabolism , Magnetic Resonance Spectroscopy , Models, Molecular , Peptide Fragments/metabolism , Phospholipase C gamma , Phosphotyrosine/chemistry , Protein Conformation , Proteins/metabolism , Type C Phospholipases/chemistry , Type C Phospholipases/metabolism , ValineABSTRACT
We have determined the structure of an Shc-derived phosphotyrosine-containing peptide complexed with Grb2 SH2 based on intra- and intermolecular NOE correlations observed by a series of isotope-filtered NMR experiments using a PFG z-filter. In contrast to an extended conformation of phosphotyrosine-containing peptides bound to Src, Syp and PLC gamma SH2s, the Shc-derived peptide formed a turn at the +1 and +2 positions next to the phosphotyrosine residue. Trp121, located at the EF1 site of Grb2 SH2, blocked the peptide binding in an extended conformation. The present study confirms that each phosphotyrosine-containing peptide binds to the cognate SH2 with a specific conformation, which gives the structural basis for the binding specificity between SH2s and target proteins.
