title: Structural evidence for a sequential release mechanism for activation of heterotrimeric G proteins.
abstract: 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.
authors.affiliation: Laboratory of Biochemistry and Molecular Biology, Rockefeller University, 1230 York Avenue, New York, NY 10065, USA.
authors.forename: Neeraj
authors.initials: N
authors.lastname: Kapoor
authors.forename: Santosh T
authors.initials: ST
authors.lastname: Menon
authors.forename: Radha
authors.initials: R
authors.lastname: Chauhan
authors.forename: Pallavi
authors.initials: P
authors.lastname: Sachdev
authors.forename: Thomas P
authors.initials: TP
authors.lastname: Sakmar
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D020134:Catalytic Domain; D002460:Cell Line; D018360:Crystallography, X-Ray; D004789:Enzyme Activation; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000737:chemistry / Q000378:metabolism*; D020962:Heterotrimeric GTP-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008274:Magnesium / Q000737:chemistry; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D051381:Rats; D015398:Signal Transduction / Q000502:physiology*; D013816:Thermodynamics
chemical_list: D006153:Guanosine Diphosphate; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D020962:Heterotrimeric GTP-Binding Proteins; D008274:Magnesium