Stereochemistry of the guanyl nucleotide binding site of transducin probed by phosphorothioate analogs of GTP and GDP

Abstract

The stereochemistry of the guanyl nucleotide binding site of transducin from bovine retinal rod outer segments was probed with phosphorothioate analogues of GTP and GDP. Transducing has markedly different affinities for the five thio analogues of GTP, as measured by their effectiveness in inhibiting GTPase activity, competing with GTP for entry into transducin, and displacing GDP bound to transducin. The order of binding affinities is GTP.gamma.S = (Sp)-GTP.alpha.S > (Rp)-GTP.alpha.S > (Sp)-GTP.beta.S .mchgt. (Rp)-GTP.beta.S. The affinity of transducin for GTP.gamma.S is > 104 higher than that for (Rp)-GTP.beta.S. These five analogues have the same relative potencies in eliciting the release of transducin from the membrane and in activating the phosphodiesterase. Transducin hydrolyzes (Sp)-GTP.alpha.S with a 1/e time of 55 s, compared with 28 s for GTP. In contrast, (Rp)-GTP.alpha.S, like GTP.gamma.S, is not hydrolyzed on the time scale of several hours. The order of effectiveness of thio analogues of GDP in displacing bound GDP is (Sp)-GDP.alpha.S > GDP > (Rp)-GDP.alpha.S > GDP.beta.S. The affinity of transducin for (Sp)-GDP.alpha.S is about 10-fold higher than that for GDP.beta.S. Mg2+ is required for the binding of GTP and GDP to transducin. Cd2+ does not lead to a reversal of stereospecificity at either the .alpha.- or .beta.-phosphorus atom of GTP. These results lead to the following conclusions: (1) The pro-R oxygen atom at the .alpha.-phosphorus of GTP does not bind Mg2+ but instead interacts with the protein. (2) The pro-S oxygen at the .alpha.-phosphorus does not appear to be involved in a critical interaction with transducin. (3) In contrast, both the pro-R and pro-S oxygen atoms at the .beta.-phosphorus of GTP are involved in key interactions with the protein or Mg2+. (4). The .alpha.-phosphoryl groups of GDP and GTP bind similarly to transducin. Guanyl nucleotide binding proteins exhibit many paterns of stereoselectivity in their uptake and hydrolysis of thio analogues of GTP. Transducin resembles elongation factor Tu [Leupold, C.M., Goody, R.S., and Wittinghofer, A. (1983) Eur. J. Biochem. 135, 237-241], another protein that undergoes translocations coupled to interconversions between GTP and GDP forms, in its stereospecificity at the .alpha.-phosphorus of GTP but may differ at the .beta.-phosphorus atom. An attractive prospect is that thio analogues can be used to probe the evolutionary kinship of guanyl nucleotide binding proteins.