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

Abstract

Stoichiometric exchange of GTP for GDP on heterotrimeric G protein alpha (Galpha) subunits is essential to most hormone and neurotransmitter initiated signal transduction. Galphas are stably activated in a Mg2+ complex with GTPgammaS, a nonhydrolyzable GTP analogue that is reported to bind Galpha, 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) Galpha-state that is intermediary to GDP/GTP exchange at a single binding site. We find Galpha native secondary and tertiary structure to persist during formation of the irreversibly inactivatable state of transducin. MT Galpha 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 GTPgammaS concentrations. Because of the weak preequilibrium, quantitative protection against Galpha 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 Galpha refolding step (isomerization) necessary for GTP sequestration after its weak preequilibrium binding. Of the two slowly interconverting Galpha x 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 Galpha x 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. Galpha x GDP and Galpha x GTP isomerization reactions are proposed as the dual target for GPCR catalysis of nucleotide exchange.

Rapid-mix flow cytometry measurements of subsecond regulation of G protein-coupled receptor ternary complex dynamics by guanine nucleotides

“…The loss in intensity is consistent with the release of Gα i3 •GDP•AlF 4 − (curve a in Figure 2B). Interestingly the fast component decay is comparable to the result obtained by Sarvazyan et al [26], while the slow dissociation was comparable to the measurements of Zelent et al [27]. Our data appear to suggest two distinct populations of G proteins that display different levels of reactivity.…”

“…Figure 1A shows the binding of fluorescein-labeled α i3 subunits to βγ subunits on beads (K D ≈ 32nM). This value is within the range of affinity constants for the interaction between α and βγ previously reported to vary from subnanomolar to tens of nM depending on experimental conditions and type of subunit [26,27]. Figure 1B shows the comparative influence of α i2 and α i3 subunits with respect to the equilibrium binding of the ligand activated FPR.…”

A Transient Interaction between the Phosphate Binding Loop and Switch I Contributes to the Allosteric Network between Receptor and Nucleotide in Gαi1

“…extent. Subsequent biophysical investigations suggest that the nucleotide-free form of the G protein is the most conformationally dynamic and is destabilized relative to the GDP-or GTP-bound forms; however, it retains an intact fold competent for signaling (62)(63)(64)(65).…”

A Dominant-negative Gα Mutant That Traps a Stable Rhodopsin-Gα-GTP-βγ Complex

“…We postulate that the ␣ T *(S43N) mutant stabilizes the ternary complex, such that it persists even following GDP-GTP exchange on ␣ T *(S43N). It has been shown that the binding of Mg 2ϩ is essential for the enhancement in intrinsic trytophan fluorescence that accompanies the conformational change in Switch 2 that occurs upon the activation of retinal ␣ T (44). In the case of the ␣ T *(S43N) mutant, we observe changes in its intrinsic tryptophan fluorescence upon binding GTP␥S, but these changes are substantially smaller than those for wildtype ␣ T *.…”