STRUCTURAL BASIS OF ACTIVITY AND SUBUNIT RECOGNITION IN G-PROTEIN HETEROTRIMERS

Background: Inactive heterotrimeric G proteins are composed of a GDP-b ound alpha subunit (G(alpha)) and a stable heterodimer of G(beta) and G(gamma) subunits, Upon stimulation by a receptor, G(alpha) subunits e xchange GDP for GTP and dissociate from G(beta gamma), both G(alpha) a nd G(beta gamma) then interact with downstream effecters. Isoforms of G(alpha), G(beta) and G(gamma) potentially give rise to many heterotri meric combinations, limited in part by amino acid sequence differences that lead to selective interactions. The mechanism by which GTP promo tes G(beta gamma) dissociation is incompletely understood. The Gly203- ->Ala mutant of G(i alpha 1) binds and hydrolyzes GTP normally but doe s not dissociate from G(beta gamma), demonstrating that GTP binding an d activation can be uncoupled. Structural data are therefore important for understanding activation and subunit recognition in G protein het erotrimers, Results: The structures of the native (G(i alpha 1 beta 1 gamma 2)) heterotrimer and that formed with Gly203-->AlaG(i alpha 1) h ave been determined to resolutions of 2.3 Angstrom and 2.4 Angstrom, r espectively, and reveal previously unobserved segments at the G(gamma 2) C terminus. The Gly203-->Ala mutation alters the conformation of th e N terminus of the switch II region (Val201-Ala203), but: not the glo bal structure of the heterotrimer. The N termini of G(beta) and G(gamm a) form a rigid coiled coil that packs at varying angles against the b eta propeller of G(beta). Conformational differences in the CD loop of beta blade 2 of Gp mediate isoform-specific contacts with G(alpha). C onclusions: The Gly203-->Ala mutation in G(i alpha 1) blocks the confo rmational changes in switch II that are required to release G(beta gam ma) upon binding GTP. The interface between the ras-like domain of G(a lpha) and the beta propeller of G(beta) appears to be conserved in all G protein heterotrimers. Sequence variation at the G(beta)-G(alpha) i nterface between the N-terminal helix of G(alpha) and the CD loop of b eta blade 2 of G(beta 1) (residues 127-135) could mediate isoform-spec ific contacts. The specificity of G(beta) and G(gamma) interactions is largely determined by sequence variation in the contact region betwee n helix 2 of G(gamma) and the surface of G(beta).