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 A1F4(-)-induced activated conformation, and interact with beta gamma t and light-activated rhodopsin. They differ dramatically in their affinity for GDP, from Gi-like (initial rates 225 mumol/mol s) to Gt-like (initial rates 4.9 mumol/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 (Kd 12 nM), while G alpha t GDP binds PDE gamma more weakly (Kd 0.88 microM). The interaction of G alpha i GDP with PDE gamma is undetectable, but G alpha i GDP-A1F4- interacts weakly with PDE gamma (Kd 2.4 microM). 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.
Skiba
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Department of Physiology and Biophysics, College of Medicine, University of Illinois, Chicago 60612-7342, USA.
Bae
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Hamm
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D000595:Amino Acid Sequence; D001665:Binding Sites; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D015293:Transducin / Q000737:chemistry / Q000378:metabolism*
D010726:Phosphodiesterase Inhibitors; D011993:Recombinant Fusion Proteins; D010727:Phosphoric Diester Hydrolases; D019204:GTP-Binding Proteins; D015293:Transducin