Structural evidence for a sequential release mechanism for activation of heterotrimeric G proteins.
Publication Type:Journal Article
Source:J Mol Biol, Volume 393, Issue 4, p.882-97 (2009)
Keywords:Amino Acid Substitution, Animals, Catalytic Domain, Cell Line, Crystallography, X-Ray, Enzyme Activation, GTP-Binding Protein alpha Subunits, Gi-Go, Guanosine Diphosphate, Heterotrimeric GTP-Binding Proteins, Humans, Magnesium, Models, Molecular, Protein Structure, Tertiary, Rats, Signal Transduction, Thermodynamics
<p>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 alpha-subunit is not well understood. Amino acid substitutions in the conserved alpha5 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 Galpha(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 Galpha(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 alpha5 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.</p>