G-protein alpha o subunit: mutation of conserved cysteines identifies a subunit contact surface and alters GDP affinity.
The reversible association of alpha and beta gamma subunits of GTP-binding proteins is important for signal transmission from a variety of cell-surface receptors to intracellular effectors. Previous work showed that 1,6-bis(maleimido)hexane, which crosslinks cysteine residues, crosslinks alpha o and alpha i-1 to beta gamma. These crosslinks are likely to form through a conserved cysteine because 1,6-bis(maleimido)hexane can also crosslink alpha i-2, alpha 1, alpha s and Drosophila alpha 1 to give products of the same apparent molecular weight as crosslinked alpha o beta gamma and alpha i-1 beta gamma. These proteins have only two cysteines in common. Therefore, we mutated each of the two conserved cysteines of alpha o to alanines. Mutation of Cys215 prevents crosslinking to beta gamma, but does not affect binding of guanosine 5'-[gamma-thio]triphosphate or the ability of the mutated alpha subunit to bind beta gamma. In models of the alpha subunit based on the crystal structure of p21ras, Cys215 is located on the face opposite to the GTP-binding site and near an area that changes conformation depending on the nucleotide bound. This surface on the alpha subunit overlaps a putative effector binding region, raising important questions about the spatial organization of the proteins as they form ternary complexes. Mutation of Cys325 has no effect on crosslinking but, surprisingly, decreases by a factor of 10 the affinity of the mutated protein for GDP, relative to wild type, without changing the affinity for guanosine 5'-[gamma-thio]triphosphate. This mutation falls within a region thought to contact receptors and may represent a site through which receptors enhance the release of GDP.
Thomas
T C
TC
Department of Medicine, Brigham and Women's Hospital, Boston, MA.
Schmidt
C J
CJ
Neer
E J
EJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D003432:Cross-Linking Reagents; D003545:Cysteine; D017931:DNA Primers; D004330:Drosophila / Q000235:genetics / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008301:Maleimides; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary
D003432:Cross-Linking Reagents; D017931:DNA Primers; D006150:Guanine Nucleotides; D046911:Macromolecular Substances; D008301:Maleimides; D006153:Guanosine Diphosphate; C075594:1,6-bismaleimidohexane; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D003545:Cysteine