Structural and biochemical characterization of the GTPgammaS-, GDP.Pi-, and GDP-bound forms of a GTPase-deficient Gly42 --> Val mutant of Gialpha1.
The Gly42 --> Val mutant of Gialpha1 was characterized structurally and biochemically to elucidate two important features of Gialpha1-catalyzed GTP hydrolysis. The crystal structure of the GTPgammaS-bound G42VGialpha1 protein demonstrates that the steric bulk of Val42 pushes the Gln204 residue into a catalytically incompetent conformation, providing a rationale for the diminished GTPase activity of this mutant. The same phenomenon may also account for the diminished GTPase activity of the homologous transforming Gly42 --> Val mutation in p21(ras). Similarly, the steric bulk of the unique Ser42 residue in Gzalpha may account for the comparatively slower rate of GTP hydrolysis by this Galpha subunit. The G42VGialpha1 subunit was also characterized structurally in its GDP.Pi- and GDP-bound states, providing a unique opportunity to view three "snapshots" of GTP hydrolysis. Hydrolysis of GTP to a transient GDP.Pi-bound intermediate is associated with substantial conformational changes in the switch II segment of the protein. Eventual release of Pi results in further removal of switch I from the active site and a highly mobile switch II segment. Despite their disparate biochemical properties, the structural similarity of G42VGialpha1 to the G203AGialpha1 mutant in the GDP.Pi-bound form suggests that both mutations stabilize a conformation of the GDP. Pi-bound protein that occurs only transiently in the wild-type protein. The structures of the GDP-bound forms of the wild-type and mutant proteins are similar.
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Department of Pharmacology, and Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas 75235, USA.
Coleman
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Gilman
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Sprang
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D000818:Animals; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006860:Hydrogen Bonding; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D016296:Mutagenesis; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D051381:Rats; D014357:Trypsin / Q000378:metabolism
D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D014357:Trypsin; D020558:GTP Phosphohydrolases; D019206:GTP-Binding Protein alpha Subunits, Gi-Go