Structural basis of activity and subunit recognition in G protein heterotrimers.

BACKGROUND: Inactive heterotrimeric G proteins are composed of a GDP-bound alpha subunit (Galpha) and a stable heterodimer of Gbeta and Ggamma subunits. Upon stimulation by a receptor, Galpha subunits exchange GDP for GTP and dissociate from Gbetagamma, both Galpha and Gbetagamma then interact with downstream effectors. Isoforms of Galpha, Gbeta and Ggamma potentially give rise to many heterotrimeric combinations, limited in part by amino acid sequence differences that lead to selective interactions. The mechanism by which GTP promotes Gbetagamma dissociation is incompletely understood. The Gly203-->Ala mutant of Gialpha1 binds and hydrolyzes GTP normally but does not dissociate from Gbetagamma, demonstrating that GTP binding and activation can be uncoupled. Structural data are therefore important for understanding activation and subunit recognition in G protein heterotrimers.
RESULTS: The structures of the native (Gialpha1beta1gamma2) heterotrimer and that formed with Gly203-->AlaGialpha1 have been determined to resolutions of 2.3 A and 2.4 A, respectively, and reveal previously unobserved segments at the Ggamma2 C terminus. The Gly203-->Ala mutation alters the conformation of the N terminus of the switch II region (Val201-Ala203), but not the global structure of the heterotrimer. The N termini of Gbeta and Ggamma form a rigid coiled coil that packs at varying angles against the beta propeller of Gbeta. Conformational differences in the CD loop of beta blade 2 of Gbeta mediate isoform-specific contacts with Galpha.
CONCLUSIONS: The Gly203-->Ala mutation in Gialpha1 blocks the conformational changes in switch II that are required to release Gbetagamma upon binding GTP. The interface between the ras-like domain of Galpha and the beta propeller of Gbeta appears to be conserved in all G protein heterotrimers. Sequence variation at the Gbeta-Galpha interface between the N-terminal helix of Galpha and the CD loop of beta blade 2 of Gbeta1 (residues 127-135) could mediate isoform-specific contacts. The specificity of Gbeta and Ggamma interactions is largely determined by sequence variation in the contact region between helix 2 of Ggamma and the surface of Gbeta.