Structural Evidence for a Sequential Release Mechanism for Activation of Heterotrimeric G Proteins

Heptahelical G-protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors couple to heterotrimeric G proteins to relay extracellular signals to intracellular signaling networks, but the molecular mechanism underlying guanosine 5′-diphosphate (GDP) release by the G protein α-subunit is not well understood. Amino acid substitutions in the conserved α5 helix of G_i, which extends from the C-terminal region to the nucleotide-binding pocket, cause dramatic increases in basal (receptor-independent) GDP release rates. For example, mutant Gα_i1-T329A shows an 18-fold increase in basal GDP release rate and, when expressed in culture, it causes a significant decrease in forskolin-stimulated cAMP accumulation. The crystal structure of Gα_i1-T329A·GDP shows substantial conformational rearrangement of the switch I region and additional striking alterations of side chains lining the catalytic pocket that disrupt the Mg⁺² coordination sphere and dislodge bound Mg⁺². We propose a “sequential release” mechanism whereby a transient conformational change in the α5 helix alters switch I to induce GDP release. Interestingly, this mechanistic model for heterotrimeric G protein activation is similar to that suggested for the activation of the plant small G protein Rop4 by RopGEF8.