Mapping of effector binding sites of transducin alpha-subunit using G alpha(t)/G alpha(i1) chimeras

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 G alpha(t)/G alpha(i1) chimeras differing at as few as 11 positions from native G alpha(t). Ten chimeras are properly folded, contain GDP, can assume an AlF4--induced activated conformation, and interact with beta gamma(t) and light-activated rhodopsin. They differ dramatically in their affinity for GDP, from G(i)-like (initial rates 225 mu mol/mol s) to G(t)-like (initial rates 4.9 mu mol/mol s). We have used these chimeras to define contact sites on G alpha(t) with the effector enzyme cGMP phosphodiesterase. G alpha(t)GTP but not G alpha(t)GDP activates it by removing the phosphodiesterase (PDE) gamma inhibitory subunit, In solution, G alpha(t)GTP interacts with PDE gamma (K-d 12 nM), while G alpha(t)GDP binds PDE gamma more weakly (K-d 0.88 mu M). The interaction of G alpha(i)GDP with PDE gamma is undetectable, but G alpha(i)GDP-AlF4- interacts weakly with PDE gamma (K-d 2.4 mu M) Using defined G alpha(t)/G alpha(i) chimeras, we have individuated the regions on G alpha(t) most important for interaction with PDE gamma in the basal and activated states, The G alpha(t), sequence encompassing alpha helix 3 and the alpha 3/beta 5 loop contributes most binding energy to interaction with PDE gamma. Another composite P gamma interaction site is the conserved switch, through which the GTP-bound G alpha(t) as well as G alpha(i1) interact with P gamma. Competition studies between PDE gamma and truncated regions of PDE gamma provide evidence for the point-to point interactions between the two proteins, The amino-terminal 1-45 segment containing the central polycationic region binds to G alpha(t)'s alpha 3 helix and alpha 3/beta 5 loop, while the COOH-terminal region of P gamma, 63-87, binds in concert to the conserved switch regions. The first interaction provides specific interaction with both the GDP- and GTP-liganded G alpha(t), while the second one is conserved between G alpha(t) and G alpha(i1) and dependent on the activated conformation.