Structures of Active Conformations of Giα1 and the Mechanism of GTP Hydrolysis

Mechanisms of guanosine triphosphate (GTP) hydrolysis by members of the G protein α subunit-p21^ras superfamily of guanosine triphosphatases have been studied extensively but have not been well understood. High-resolution x-ray structures of the GTPλS and GDP\cdotAIF_4^- complexes formed by the G protein g_iα1 demonstrate specific roles in transition-state stabilization for two highly conserved residues Glutamine²⁰⁴ (Gln⁶¹ in p21^ras) stabilizes and orients the hydrolytic water in the trigonal-bipyramidal transition state. Arginine 178 stabilizes the negative charge at the equatorial oxygen atoms of the pentacoordinate phosphate intermediate. Conserved only in the G_α family, this residue may account for the higher hydrolytic rate of G_α proteins relative to those of the p21^ras family members. The fold of G_iα1 differs from that of the homologous G_tα subunit in the conformation of a helix-loop sequence located in the α-helical domain that is characteristic of these proteins; this site may participate in effector binding. The amino-terminal 33 residues are disordered in GTPλS-G_iα1, suggesting a mechanism that may promote release of the βλ subunit complex when the α subunit is activated by GTP.