Chemical modification of bovine transducin: probing the GTP-binding site with affinity analogs

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Abstract
The structure of the GTP-binding sites of transducin, a signal-transducing G-protein involved in the visual excitation process, was studied by affinity labeling. Radioactive GTP analogues with reactive groups attached to different moieties of the GTP molecule were obtained and included 8-azido-GTP, P3-(4-azidoanilino)-P1-5''-GTP (AA-GTP), 5''-[p-(fluorosulfonyl)benzoyl]guanosine (FSBG), 3''-O-{3-[N-(4-azido-2-nitrophenyl)amino]propionyl}-GTP (ANPAP-GTP), the 2'',3''-dialdehyde derivative of GTP (oGTP), and a bifunctional cross-linking analogue, 8-azido-P3-(4-azidoanilino)-P1-5''-GTP (8-azido-AA-GTP). With the exception of FSBG, all of the analogues were found to bind to transducin specifically and serve as a cofactor to activate the retinal cGMP cascade or act as a competitive inhibitor for the GTPase activity of transducin. The labeling sites of these analogues were localized by tryptic peptide mapping. ANPAP-GTP and oGTP were unable to covalently modify transducin, suggesting that the 2''- and 3''-hydroxy groups on the ribose ring of GTP are not in direct contact with the protein. AA-GTP only labeled the T.alpha. subunit of transducin and was localized on the 21-kDa tryptic fragment of T.alpha.. This indicates that the phosphate moiety of the bound GTP is in direct contact with this peptide. On the other hand, 8-azido-GTP labeled both the T.alpha. and T.beta..gamma. subunits of transducin. The labeling on T.alpha. was on the 12-kDa tryptic fragment, suggesting that the guanine ring binding site is composed of a different peptide fragment than the phosphate binding region. Treatment with the bifunctional analogue 8-azido-AA-GTP generated the cross-linked products of T.alpha. and T.beta..gamma.. This observation implies that the guanine ring of the bound GTP on T.alpha. could be in close proximity with T.beta..gamma.. The overall result of mapping the nucleotide binding site of transducin with affinity labeling is in complete agreement with the proposed model of T.alpha. [Hingorani, V. N., and Ho, Y.-K. (1987) FEBS Lett. 220, 15-22] based on the crystal structure of the GTP-binding site of elongation factor Tu [Jurnak, F. (1985) Science 230, 32-36; la Cour, T. F. M., Nyborg, J., Thirup, S., and Clark, B. F. C. (1985) EMBO J. 4, 2385-2388].