Displacement of rhodopsin by GDP from three-loop interaction with transducin depends critically on the diphosphate beta-position.
We have studied the effect of GDP and its analog guanyl-5'-yl thiophosphate (GDP beta S) on the interaction between rhodopsin and transducin (Gt). Stabilization of the light-induced active intermediate, metarhodopsin II (MII), by bound Gt (extra-MII effect) monitored the catalytic interaction between the proteins. Extra-MII can be completely abolished by GDP, with a half-suppression at 10 microM under the conditions (4 degrees C, pH 8, 7.5 nM photoactivated rhodopsin). The effect of GDP did not depend on divalent cations, in contrast to GTP-induced dissociation of the complex. The GDP analog GDP beta S did not affect extra-MII although it binds to the MII-Gt complex with only three times lower affinity (reversal of the GDP effect by GDP beta S). However, GDP beta S enhanced considerably the efficiency of synthetic rhodopsin peptide competition against the formation of extra-MII. GDP and GDP beta S slow the Gt activation rate (monitored by kinetic light scattering), with the same relative efficiencies. We therefore assume that GDP, GDP beta S, and GTP bind at the same site. We discuss a generalized induced fit mechanism, where MII induces opening of the Gt nucleotide site and release of GDP which in turn is obligatory to establish the MII-stabilizing rhodopsin-Gt three-loop interaction (König, B., Arendt, A., McDowell, J.H., Kahlert, M., Hargrave, P.A., and Hofmann, K.P. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6878-6882). The GDP beta S/GDP difference is discussed in terms of bound GDP disturbing the interaction with two and GDP beta S with only one of the rhodopsin binding sites. Mechanistically, our results indicate a critical role of the beta-phosphate interaction with the nucleotide binding site in the GDP-induced transformation of Gt.
Kahlert
M
M
Institut für Biophysik und Strahlenbiologie, Albert-Ludwigs-Universität, Freiburg, Federal Republic of Germany.
König
B
B
Hofmann
K P
KP
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology*; D007700:Kinetics; D008954:Models, Biological; D012243:Rhodopsin / Q000031:analogs & derivatives / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000378:metabolism; D013873:Thionucleotides / Q000494:pharmacology; D015293:Transducin / Q000378:metabolism*
D013873:Thionucleotides; D006153:Guanosine Diphosphate; C013110:metarhodopsins; C023427:guanosine 5'-O-(2-thiodiphosphate); D012243:Rhodopsin; D015293:Transducin