Structural and Biochemical Characterization of the GTPγS-, GDP‚Pi-, and GDP-Bound Forms of a GTPase-Deficient Gly42 f Val Mutant of GiR1

The Gly42 f Val mutant of GiR1 was characterized structurally and biochemically to elucidate two important features of GiR1-catalyzed GTP hydrolysis. The crystal structure of the GTPγS-bound GVGiR1 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 f Val mutation in p21ras. Similarly, the steric bulk of the unique Ser42 residue in GzR may account for the comparatively slower rate of GTP hydrolysis by this GR subunit. The GVGiR1 subunit was also characterized structurally in its GDP‚Piand 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 GVGiR1 to the GAGiR1 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 GDPbound forms of the wild-type and mutant proteins are similar. Transient, high-affinity interactions between GTP and the R subunits of heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins)1 activate the downstream signaling reactions that are initiated by these molecules (1-5). Binding of GTP to GR is catalyzed by an appropriate agonist-receptor complex, which facilitates dissociation of tightly bound GDP from the G protein heterotrimer. GTP, present in cells at much higher concentrations than GDP, fills the empty nucleotide binding site and causes conformational changes that result in dissociation of GR(GTP) from a high-affinity dimer of â and γ subunits (6). The intrinsic GTPase activity of GR eventually regenerates GR(GDP), and subsequent binding of Gâγ completes the cycle. The GTPase reaction catalyzed by a variety of regulatory GTPases has been studied extensively because of its intrinsic biological importance, revealed in part by the consequences of its inhibition. Thus, cholera toxin-catalyzed ADPribosylation of the GR protein responsible for stimulation of adenylyl cyclase (GsR) inhibits its GTPase activity (7). Resultant persistent activation of the pathway causes uncontrolled synthesis of cyclic AMP and life-threatening diarrheal disease. Mutations in p21ras that inactivate the GTPase activity of this protein (and render it unsusceptible to the activity of GTPase-activating proteins or GAPs) cause a large number of human malignancies; the Gly12 f Val mutation of p21ras is the most common of these alterations (8, 9). Gly12 in p21ras is located in the so-called P loop (GXXXXGK sequence motif), which is involved in binding the Rand â-phosphates of nucleotides in many proteins (Table 1). Despite crystallographic studies that have documented the proximity of Gly12 to the catalytic Gln61 residue in p21ras, the explanation for the catalytic inactivity of Val12-p21ras remains unclear because the so-called switch II segment that includes Gln61 is poorly ordered (10-12). Most GR proteins also have P-loops with a conserved Gly residue that is homologous to Gly12 of p21ras. However, we originally felt that study of heterotrimeric G protein R subunits had little to contribute to the question of the importance of this Gly residue because the GTPase activity of the equivalent Gly49 f Val mutant of GsR was only modestly diminished (13). We have reexamined this issue † This work was supported by NIH Grant DK46371 and Welch Foundation Grant I-1229 (to S.R.S.) and NIH Grant GM34497 and Welch Foundation Grant I-1271 (to A.G.G.). A.G.G. also acknowledges support from the Raymond and Ellen Willie Distinguished Chair in Molecular Neuropharmacology. * To whom correspondence should be addressed. ‡ Department of Pharmacology. § Department of Biochemistry. | Howard Hughes Medical Institute, Department of Biochemistry. X Abstract published in AdVance ACS Abstracts, December 1, 1997. 1 Abbreviations: G proteins, heterotrimeric guanine nucleotidebinding proteins; GAP, GTPase-activating protein; GTPγS, guanosine 5′-(3-O-thiotriphosphate); C12E10, poly(oxyethylene) 10-lauryl ether. Mutant proteins are designated by the wild-type amino acid residue, the position of this residue, and the residue used for replacement. Table 1: Conserved Phosphate-Binding Loop Motifa a The number preceding the sequence corresponds to the first residue number of the motif. 15660 Biochemistry 1997, 36, 15660-15669 S0006-2960(97)01912-0 CCC: $14.00 © 1997 American Chemical Society for several reasons. (1) The switch II segment in at least certain GTPγS-bound GR proteins is well ordered compared to that of p21ras (14, 15). (2) GzR, a member of the Gi subfamily of GR subunits, is distinct because of very low GTPase activity (100-fold lower than GsR or GiR) and a unique TSN sequence in the P-loop rather than the consensus AGE in other GR proteins (16). (3) The Gly203 f Ala mutant of GiR1 (an unrelated Gly residue), a conformationally constrained protein, can be crystallized in an intermediate GR(GDP‚Pi) state but not in its active GTPγS-bound form (17). Wild-type GiR1 cannot be crystallized in its GDP‚Pibound form. (4) Gly42 f Val GiR1 (analogous to Gly12 f Val p21ras), which has severely impaired GTPase activity (see below), can be crystallized in all three GTPγS-, GDP-, and GDP‚Pi-bound forms. Hence, this protein offers a unique opportunity to obtain three “snapshots” of guanine nucleotide binding and hydrolytic reactions as well as insights into the consequences of mutation at Gly42. MATERIALS AND METHODS Proteins and Plasmids. A cDNA containing the entire coding sequence of rat GiR1 was ligated into the EcoRI site of M13mp19. Mutagenesis was performed by the method of Kunkel et al. (18). The complete coding sequences of the G42V and G203A mutants were confirmed by dideoxynucleotide sequencing. The complete coding sequences (NcoI-HindIII fragments) of the two mutants and the wildtype protein were subcloned into the NcoI and HindIII sites of plasmid pQE-6. Other P-loop mutants were subcloned into the NcoI and HindIII sites of plasmid H6pQE-6 to generate proteins with hexahistidine sequences at their amino termini. These plasmids were transformed together with plasmid pREP4 into Escherichia coli BL21 (DE3) cells. Proteins were expressed and purified as previously described