Perlman J H, Laakkonen L J, Guarnieri F, Osman R, Gershengorn M C
Department of Medicine, Cornell University Medical College, New York, New York 10021, USA.
Biochemistry. 1996 Jun 18;35(24):7643-50. doi: 10.1021/bi952202r.
Seven transmembrane (TM) spanning, G protein-coupled receptors (GPCRs) appear to bind large glycoprotein hormones predominantly within their extracellular domains, small nonpeptidic ligands within the TM helical bundle, and peptide ligands within the extracellular domains and TM bundle. The tripeptide thyrotropin-releasing hormone (TRH, pyroGlu-His-ProNH2) may bind entirely within the TM bundle of the TRH receptor (TRH-R). We have previously demonstrated direct binding contacts between the pyroGlu of TRH and two residues in TM helix 3 (TM-3) of TRH-R and proposed a model of the binding pocket of TRH-R [Perlman, J. H., Laakkonen, L., Osman, R., & Gershengorn, M. C. (1994) J. Biol. Chem. 269, 23383-23386]. Here, we provide evidence for two additional direct interactions between TRH and TRH-R. One interaction is between the aromatic ring of Tyr 282 of TM-6 and His of TRH. This is based on a large increase in the half-maximally effective concentration (EC50) of TRH for stimulation of inositol phosphate formation by Y282A TRH-R and a loss of selectivity of this mutant receptor for TRH analogs substituted at His. We provide evidence for another interaction between Arg 306 of TM-7 and the terminal carboxamide of TRH. Using four direct interactions as anchors, a refined model of the TRH-R binding pocket was constructed using geometry optimization through energy minimization. A novel method for modeling GPCRs based on Monte Carlo and stochastic dynamics simulations is presented in the accompanying paper [Laakkonen, L. J., Guarnieri, F., Perlman, J. H., Gershengorn, M. C., & Osman, R. (1996) Biochemistry 35, 7651-7663].
七跨膜（TM）的G蛋白偶联受体（GPCRs）似乎主要在其细胞外结构域结合大的糖蛋白激素，在TM螺旋束内结合小的非肽配体，在细胞外结构域和TM束内结合肽配体。三肽促甲状腺激素释放激素（TRH，焦谷氨酸-组氨酸-脯氨酰胺）可能完全结合在TRH受体（TRH-R）的TM束内。我们之前已经证明了TRH的焦谷氨酸与TRH-R的TM螺旋3（TM-3）中的两个残基之间的直接结合接触，并提出了TRH-R结合口袋的模型[Perlman, J. H., Laakkonen, L., Osman, R., & Gershengorn, M. C. (1994) J. Biol. Chem. 269, 23383-23386]。在这里，我们提供了TRH与TRH-R之间另外两个直接相互作用的证据。一种相互作用是TM-6的Tyr 282的芳香环与TRH的His之间的相互作用。这是基于Y282A TRH-R刺激肌醇磷酸形成时TRH的半数最大有效浓度（EC50）大幅增加，以及该突变受体对His处被取代的TRH类似物的选择性丧失。我们提供了TM-7的Arg 306与TRH的末端羧酰胺之间另一种相互作用的证据。以四个直接相互作用为锚点，通过能量最小化的几何优化构建了TRH-R结合口袋的精细模型。随附论文[Laakkonen, L. J., Guarnieri, F., Perlman, J. H., Gershengorn, M. C., & Osman, R. (1996) Biochemistry 35, 7651-7663]中提出了一种基于蒙特卡罗和随机动力学模拟的GPCRs建模新方法。
