Mapping the nucleotide-dependent conformational change of human N-ras p21 in solution by heteronuclear-edited proton-observed NMR methods.
Heteronuclear-edited proton-detected NMR methods are used to study the nucleotide-dependent conformational change between GDP- and GTP gamma S-bound forms of human N-ras p21. Amide groups of Asp are used as sensitive probes. When GTP gamma S is substituted for GDP in cellular N-ras p21, the chemical shifts of resonances Asp-47, -126, -154, and Asn-172, as well as Gly-77 and -151, are not sensitive to nucleotide exchange, whereas Asp-30, -33, -38, -54, -57, -69, -92, -105, and -119 are affected. Distinct chemical shift changes of Asp-33, -38, and -69 indicate that substantial structure changes occur in the effector-binding region and the switch II region. Crystallographic studies of H-ras p21 have indicated that the conformational differences are confined to residues 32-38 and 60-76. Our observations indicate that the nucleotide-dependent structural transitions of the protein in solution may not be identical to those in the crystal. They suggest that the peptide beyond Glu-76 participates in a conformational switch, and possibly is involved in effector function. We propose that the region roughly from Asp-92 to -105, and the region of guanine base-binding motif(s), e.g., 116NKXD, are candidate sites recognized by either a GDP/GTP release factor or a GTPase-affected protein.
Hu
J S
JS
Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254.
Redfield
A G
AG
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006801:Humans; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012996:Solutions
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D012996:Solutions; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)