Mapping of effector binding sites of transducin α-subunit using Gαt/Gαi1 chimeras

Abstract

The G protein transducin has been an often-used model for biochemical, structural, and mechanistic studies of G protein function. Experimental studies have been limited, however, by the inability to express quantities of mutants in heterologous systems with ease. In this study we have made a series of Gαt/Gαi1 chimeras differing at as few as 11 positions from native Gαt. Ten chimeras are properly folded, contain GDP, can assume an AlF4--induced activated conformation, and interact with βγt and light-activated rhodopsin. They differ dramatically in their affinity for GDP, from Gi-like (initial rates 225 μmol/mol s) to Gt-like (initial rates 4.9 μmol/ mol s). We have used these chimeras to define contact sites on Gαt with the effector enzyme cGMP phosphodiesterase. GαtGTP but not GαtGDP activates it by removing the phosphodiesterase (PDE) γ inhibitory subunit. In solution, GαtGTP interacts with PDEγ (Kd 12 nM), while GαtGDP binds PDEγ more weakly (Kd 0.88 μM). The interaction of GαiGDP with PDEγ is undetectable, but GαiGDP-AlF4- interacts weakly with PDEγ (Kd 2.4 αM). Using defined Gαt/Gαi chimeras, we have individuated the regions on Gαt most important for interaction with PDEγ in the basal and activated states. The Gαt sequence encompassing α helix 3 and the α3/β5 loop contributes most binding energy to interaction with PDEγ. Another composite Pγ interaction site is the conserved switch, through which the GTP-bound Gαt as well as Gαi1 interact with Pγ. Competition studies between PDEγ and truncated regions of PDEγ provide evidence for the point-to-point interactions between the two proteins. The amino-terminal 1-45 segment containing the central polyeationic region binds to Gαt's α3 helix and α3/β5 loop, while the COOH-terminal region of Pγ, 63-87, binds in concert to the conserved switch regions. The first interaction provides specific interaction with both the GDP- and GTP-liganded Gαt, while the second one is conserved between Gαt and Gαi1 and dependent on the activated conformation.