Rapid Irreversible G Protein Alpha Subunit Misfolding Due to Intramolecular Kinetic Bottleneck that Precedes Mg2+ “Lock” after GTP/GDP Exchange

Stoichiometric exchange of GTP for GDP on heterotrimeric G protein α (Gα) subunits is essential to most hormone and neurotransmitter initiated signal transduction. Gαs are stably activated in a Mg2+ complex with GTPγS, a nonhydrolyzable GTP analogue that is reported to bind Gα with very high affinity. Yet, it is common to find that substantial amounts (30−90%) of purified G proteins cannot be activated. Inactivatable G protein has heretofore been thought to have become “denatured” during formation of the obligatory nucleotide-free or empty (MT) Gα-state that is intermediary to GDP/GTP exchange at a single binding site. We find Gα native secondary and tertiary structure to persist during formation of the irreversibly inactivatable state of transducin. MT Gα is therefore irreversibly misfolded rather than denatured. Inactivation by misfolding is found to compete kinetically with protective but weak preequilibrium nucleotide binding at micromolar ambient GTPγS concentrations. Because of the weak preequilibrium, quantitative protection against Gα aggregation is only achieved at free nucleotide concentrations 10−100 times higher than those commonly employed in G protein radio-nucleotide binding studies. Initial GTP protection is also poor because of the extreme slowness of an intramolecular Gα refolding step (isomerization) necessary for GTP sequestration after its weak preequilibrium binding. Of the two slowly interconverting Gα·GTP isomers described here, only the second can bind Mg2+, “locking” GTP in place with a large net rise in GTP binding affinity. A companion Gα·GDP isomerization reaction is identified as the cause of the very slow spontaneous GDP dissociation that characterizes G protein nucleotide exchange and low spontaneous background activity in the absence of GPCR activation. Gα·GDP and Gα·GTP isomerization reactions are proposed as the dual target for GPCR catalysis of nucleotide exchange.