Structures of Active Conformations of G$_{i\alpha1}$ 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 GTPγS and GDP·AIF$_4^-$ complexes formed by the G protein g$_{i\alpha1}$ demonstrate specific roles in transition-state stabilization for two highly conserved residues Glutamine$^{204}$ (Gln$^{61}$ 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$_\alpha$ family, this residue may account for the higher hydrolytic rate of $G_\alpha$ proteins relative to those of the p21$^{ras}$ family members. The fold of G$_{i\alpha1}$ differs from that of the homologous G$_{t\alpha}$ 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 GTP$\lambda$S-G$_{i\alpha1}$, suggesting a mechanism that may promote release of the βγ subunit complex when the α subunit is activated by GTP.