Kahlert M, König B, Hofmann K P
Institut für Biophysik und Strahlenbiologie, Albert-Ludwigs-Universität, Freiburg, Federal Republic of Germany.
J Biol Chem. 1990 Nov 5;265(31):18928-32.
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.
我们研究了鸟苷二磷酸（GDP）及其类似物鸟苷 - 5'-硫代磷酸酯（GDPβS）对视紫红质与转导蛋白（Gt）之间相互作用的影响。结合的Gt对视紫红质光诱导的活性中间体变视紫红质II（MII）的稳定作用（额外MII效应）监测了蛋白质之间的催化相互作用。在（4℃，pH 8，7.5 nM光活化视紫红质）条件下，GDP可完全消除额外MII效应，10μM时出现半抑制。与GTP诱导的复合物解离不同，GDP的作用不依赖于二价阳离子。GDP类似物GDPβS虽然与MII - Gt复合物结合时亲和力仅低三倍，但并不影响额外MII效应（GDPβS可逆转GDP的效应）。然而，GDPβS显著提高了合成视紫红质肽竞争形成额外MII的效率。GDP和GDPβS减缓了Gt的活化速率（通过动态光散射监测），且相对效率相同。因此，我们推测GDP、GDPβS和GTP结合在同一位点。我们讨论了一种广义的诱导契合机制，其中MII诱导Gt核苷酸位点开放并释放GDP，而这反过来又是建立稳定MII的视紫红质 - Gt三环相互作用所必需的（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）。从结合的GDP干扰与视紫红质两个结合位点的相互作用以及GDPβS仅干扰其中一个结合位点的角度讨论了GDPβS / GDP的差异。从机制上讲，我们的结果表明β - 磷酸与核苷酸结合位点的相互作用在GDP诱导的Gt转化中起关键作用。
