Institut des Biomolécules Max Mousseron (I.B.M.M.), CNRS UMR5247 - Université Montpellier 1 - Université Montpellier 2, Faculté de Pharmacie, 15 avenue Charles Flahault, B.P. 14 491, 34093 Montpellier Cedex 5, France.
J Mol Biol. 2010 Jan 29;395(4):769-84. doi: 10.1016/j.jmb.2009.09.051. Epub 2009 Sep 25.
Three homology models of the human ghrelin receptor (GHS-R1a) have been generated from the available X-ray structures of rhodopsin (RHO model), opsin (OPS model) and beta-2 adrenergic receptor (B2 model). The latter was used as a starting point for combined molecular dynamics simulation (MDS) and full atom normal modes analysis (NMA). A low-frequency normal mode (mode 16) perfectly reproduced the intracellular motions observed between B2 and RHO models; in the opposite direction along the same mode, the generated structures are closer to the OPS model, suggesting a direct link with GHS-R1a activation. This was in agreement with motions of the seven transmembranous segments, increase of the solvent accessibility of the 140-ERY-142 sequence, and flip of the Trp276 (C WLP) residue, some features related to GPCRs activation. According to our model, His280 was proposed to stabilize Trp276 in the active state; this was verified by site-directed mutagenesis and biochemical characterization of the resulting H280A and H280S mutants, which were fully functional but sharing an important decrease of their basal activities. Docking performed with short ghrelin derivatives Gly-Ser-Ser ([octa])-Phe-NH (2) and Gly-Ser-Ser ([octa])-Phe-Leu-NH (2) allowed the identification of a robust position of these peptides in the active site of the receptor. This model was refined by MDS and validated by docking experiments performed on a set of 55 ghrelin receptor ligands based on the 1,2,4- triazole scaffold. Finally, NMA performed on the obtained peptide-receptor complex suggested stabilization of the Trp276 residue and of the whole receptor in the active state, preventing the motion observed along mode 16 computed for the unbound receptor. Our results show that NMA offers a powerful approach to study the conformational diversity and the activation mechanism of GPCRs.
已经从现有 X 射线结构的视紫红质（RHO 模型）、视蛋白（OPS 模型）和β-2 肾上腺素能受体（B2 模型）生成了三种人类胃饥饿素受体（GHS-R1a）的同源模型。后者被用作组合分子动力学模拟（MDS）和全原子正常模式分析（NMA）的起点。一个低频正常模式（模式 16）完美地再现了在 B2 和 RHO 模型之间观察到的细胞内运动；沿着同一模式的相反方向，生成的结构更接近 OPS 模型，这表明与 GHS-R1a 的激活有直接联系。这与七跨膜片段的运动一致，140-ERY-142 序列的溶剂可及性增加，以及 Trp276（C WLP）残基的翻转，这些都是与 GPCR 激活相关的一些特征。根据我们的模型，提出 His280 稳定活性状态下的 Trp276；这通过定点突变和所得 H280A 和 H280S 突变体的生化特性得到了验证，这些突变体完全有功能，但它们的基础活性有重要的降低。与短胃饥饿素衍生物 Gly-Ser-Ser([octa])-Phe-NH（2）和 Gly-Ser-Ser([octa])-Phe-Leu-NH（2）进行对接，确定了这些肽在受体活性部位的稳定位置。该模型通过 MDS 进行了细化，并通过基于 1,2,4-三唑支架的 55 种胃饥饿素受体配体的对接实验进行了验证。最后，对获得的肽-受体复合物进行的 NMA 表明，在活性状态下，Trp276 残基和整个受体得到稳定，防止了在计算未结合受体时沿着模式 16 观察到的运动。我们的结果表明，NMA 提供了一种强大的方法来研究 GPCR 的构象多样性和激活机制。
