Abstract

G-protein ? subunits consist of two domains: a Ras-like domain also called GTPase domain (GTPaseD), structurally homologous to monomeric G-proteins, and a more divergent domain, unique to heterotrimeric G-proteins, called helical domain (HD). G-protein activation, requires the exchange of bound GDP for GTP, and since the guanine nucleotide is buried in a deep cleft between both domains, it has been postulated that activation may involve a conformational change that will allow the opening of this cleft. Therefore, it has been proposed, that interdomain interactions are playing an important role in regulating the nucleotide exchange rate of the ? subunit. While constructing different Gs? quimeras, we identified a Gs? random mutant, which was very inefficient in stimulating adenylyl cyclase activity. The introduced mutation corresponded to the substitution of Ser111 for Asn (S111N), located in the carboxi terminal end of helix A of the HD, a region neither involved in AC interaction nor in the interdomain interface. In order to characterize this mutant, we expressed it in bacteria, purified it by niquel-agarose chromatography, and studied its nucleotide exchange properties. We demonstrated that the recombinant S111N Gs? was functional since it was able to undergo the characteristic conformational change upon GTP binding, detected by the acquisition of a trypsin-resistant conformation. When the biochemical properties were determined, the mutant protein exhibited a reduced GDP dissociation kinetics and as a consequence a slower GTP?S binding rate that was responsible for a diminished adenylyl cyclase activation when GTP?S was used as activator. These data provide new evidence that involves the HD as a regulator of Gs? function, in this case the ?A helix, which is not directly involved with the nucleotide binding site nor the interdomain interface. © 2002 Wiley-Liss, Inc.