Molecular basis of G protein-coupled receptor function

GPCR同源二聚体的结构与功能研究 大多数GPCRs属于GPCRs的A亚家族，由670个人类成员组成。A类GPCRs能够形成二聚体或寡聚体复合体。越来越多的证据表明，这种复合体的形成可以调节GPCR功能的各个方面，包括(但不限于)受体-G蛋白偶联的效率和选择性。因此，为了更好地了解A类GPCRs如何在分子水平上发挥作用，确定控制这类受体二聚化/寡聚化的结构元件是至关重要的。 在过去的几年里，我们已经使用M3M受体(M3R)作为模型系统来探索A类GPCR二聚体的各个方面。最近，我们开始使用二硫键交联策略来捕获存在于天然脂环境中的各种M3R二聚体物种(来自转基因COS-7细胞的膜)。我们对一个在M3R区细胞质表面含有单半胱氨酸(Cys)取代的大突变体M3Rs进行了二硫键交联研究。我们获得的交联链模式，加上分子模拟研究，强烈表明存在多个M3R/M3R界面。我们还注意到，涉及螺旋8(H8)的二硫键交联物干扰了M3R-G蛋白的生产性偶联。这一观察结果支持了受体介导的G蛋白激活需要涉及H8的构象变化的概念。M3R与大多数其他A类GPCR显示出高度的结构同源性。因此，上述总结的调查结果应具有相当大的普遍意义。 (胡静，等人)对M3 M受体二聚体/低聚物形成的结构和功能的新见解。《生物化学》2013年第288期，34777-90期) 激动剂激活M2受体(M2R)的结构 最近，多个实验室的合作努力导致确定了激动剂(Iperoxo)结合的人类M2R的活性状态的结构。Iperoxo是一种正构体激动剂，在M2R上显示出非常高的效力。M2R活性构象的关键特征是TM6的细胞质末端显著向外移动，TM5的C末端部分向外移动较小，以及高度保守的NPXXY(TM7)和Dry(TM3的细胞质末端)基序的重排。视紫红质和β2肾上腺素能受体的活化态构象也有类似的构象变化。 Iperoxo与M2R结合导致正位立位结合部位的结构发生显着变化。具体地说，Iperoxo与M2R的结合导致正位立位结合部位的显著收缩，从而完全封闭了激动剂配体与溶剂。在活性M2R构象中，TM5、TM6和TM7向内朝向Iperoxo配体移动，TM3绕其轴发生轻微旋转。最重要的是，TM6的内移允许N4046.52的侧链与异恶唑啉环形成氢键。TM6外表面部分的内移导致Y4036.51、Y1043.33和Y4267.39之间形成一个氢网络，导致激动剂配体上方的酪氨酸盖关闭。几乎无一例外，接触激动剂配体的M2R氨基酸侧链也在M受体拮抗剂/反向激动剂QNB与非活性M2R的结合中发挥重要作用。 (Kruse AC，et al.M碱型乙酰胆碱受体的激活和变构调节。《自然》2013年第504期，101-6期) 变构调节剂与M2R的结合方式 M2R已成为研究小分子变构调节剂调节GPCR功能的良好模型系统。不活跃的M2R和拮抗剂结合的M3R一样，有一个很大的细胞外前庭，据预测它参与了变构M受体的结合。引人注目的是，激动剂激活M2R会触发这个外腔的明显收缩，主要是由于TM6细胞外部分的内移。 我们解决了过氧基M2R与正变构体LY2119620形成的络合物的结构。LY2119620与伊罗昔布具有很强的正协同作用，并选择性地增强了正构体激动剂对M2R的亲和力。在M2R-iperoxo-LY2119620络合物中，变构调节剂位于正构激动剂的正上方。LY2119620参与了与细胞外前庭的广泛相互作用。这种接触包括芳香族堆积、氢键和电荷相互作用。有趣的是，M2RiperoxoLY2119620复合体的结构与M2Iperoxo复合体的结构非常相似，这表明LY2119620的结合位点在很大程度上是在正构体激动剂Iperoxo结合后预先形成的。与M2R的非活动状态相比，细胞外前庭的过氧物稳定收缩使LY2119620能够与这个外部感受器腔进行更广泛的相互作用。这些发现支持LY2119620和其他毒扁豆碱类正变构调节剂通过稳定受体的活性构象和减缓激动剂从正构体结合袋中解离来增强正构体激动剂的受体亲和力的概念。这些数据提供了类药物变构配体如何与GPCR结合的第一个结构视图。这一新的结构信息应该指导具有高度选择性的不同mAChR亚型的新型毒扁豆碱类药物的开发。 (Kruse AC，et al.M碱型乙酰胆碱受体的激活和变构调节。《自然》2013年第504期，101-6期)

GPCR homodimers: structure-function studies Most GPCRs belong to the class A subfamily of GPCRs which consists of 670 members in human. Class A GPCRs are able to form dimeric or oligomeric complexes. Accumulating evidence suggests that the formation of such complexes can modulate various aspects of GPCR function, including (but not limited to) receptor-G protein coupling efficiency and selectivity. Thus, in order to better understand how class A GPCRs function at the molecular level, it is critical to identify the structural elements governing the dimerization/oligomerization of this class of receptors. During the past few years, we have used the M3 muscarinic acetylcholine receptor (M3R) as a model system to explore various aspects of class A GPCR dimerization. Recently, we started to employ a disulfide cross-linking strategy to trap various M3R dimeric species present in a native lipid environment (membranes from transfected COS-7 cells). We carried out disulfide cross-linking studies with a large mutant M3Rs containing single cysteine (Cys) substitutions on the cytoplasmic surface of the M3R regions. The pattern of cross-links that we obtained, together with molecular modeling studies, strongly suggested the existence of multiple M3R/M3R interfaces. We also noted that disulfide cross-links involving helix 8 (H8) interfered with productive M3R-G protein coupling. This observation supports the concept that receptor-mediated G protein activation requires conformational changes that involve H8. The M3R shows a high degree of structural homology with most other class A GPCRs. For this reason, the findings summarized above should be of considerable general interest. (Hu J, et al. Novel structural and functional insights into M3 muscarinic receptor dimer/oligomer formation. J Biol Chem 288, 34777-90, 2013) Structure of the agonist-activated M2 muscarinic receptor (M2R) Recently, a multi-lab collaborative effort resulted in the determination of the structure of an agonist (iperoxo)-bound, active state of the human M2R. Iperoxo is an orthosteric agonist that displays very high potency at the M2R. The key features of the active conformation of the M2R are a significant outward displacement of the cytoplasmic end of TM6, together with a smaller outward movement of the C-terminal portion of TM5 and a rearrangement of the highly conserved NPXXY (TM7) and DRY (cytoplasmic end of TM3) motifs. Similar conformational changes have been reported for the active-state conformations of rhodopsin and the beta2-adrenergic receptor. Iperoxo binding to the M2R leads to striking changes in the structure of the orthosteric binding site. Specifically, iperoxo binding to the M2R results in a significant contraction of the orthosteric binding site, which completely occludes the agonist ligand from solvent. In the active M2R conformation, TM5, TM6, and TM7 move inward toward the iperoxo ligand and TM3 undergoes a slight rotation about its axis. Most importantly, the inward movement of TM6 allows the side chain of N4046.52 to form a hydrogen bond with the isoxazoline ring of iperoxo. The inward motion of the exofacial portion of TM6 leads to the formation of a hydrogen network between Y4036.51, Y1043.33, and Y4267.39, resulting in the closure of the tyrosine lid above the agonist ligand. With almost no exception, the M2R amino acid side chains that contact the agonist ligand also play important roles in the binding of QNB, a muscarinic antagonist/inverse agonist, to the inactive M2R. (Kruse AC, et al. Activation and allosteric modulation of a muscarinic acetylcholine receptor. Nature 504, 101-6, 2013) Mode of binding of an allosteric modulator to the M2R The M2R has served as an excellent model system for studying the regulation of GPCR function by small allosteric modulators. The inactive M2R, like the antagonist-bound M3R, features a large extracellular vestibule, which is predicted to be involved in the binding of allosteric muscarinic ligands. Strikingly, agonist activation of the M2R triggers a pronounced contraction of this outer cavity, primarily due to the inward movement of the extracellular portion of TM6. We solved the structure of the iperoxo-occupied M2R in complex with LY2119620, a positive allosteric. LY2119620 shows strong positive cooperativity with iperoxo and selectively enhances the affinity of the orthosteric agonist for the M2R. In the M2R-iperoxo-LY2119620 complex, the allosteric modulator is located directly above the orthosteric agonist. LY2119620 engages in extensive interactions with the extracellular vestibule. Such contacts include aromatic stacking, hydrogen bond, and chargecharge interactions. Interestingly, the structure of the M2RiperoxoLY2119620 complex is very similar to that of the M2iperoxo complex, indicating that the binding site for LY2119620 is largely pre-formed after binding of the orthosteric agonist iperoxo. The iperoxo-stabilized contraction of the extracellular vestibule enables LY2119620 to engage in far more extensive interactions with this outer receptor cavity, as compared to the inactive state of the M2R. These findings support the concept that LY2119620 and, most likely, other muscarinic positive allosteric modulators enhance the receptor affinity of orthosteric agonists by stabilizing the active conformation of the receptor and slowing agonist dissociation from the orthosteric binding pocket. These data provide the first structural view of how a drug-like allosteric ligand binds to a GPCR. This new structural information should guide the development of novel muscarinic agents endowed with a high degree of selectivity for distinct mAChR subtypes. (Kruse AC, et al. Activation and allosteric modulation of a muscarinic acetylcholine receptor. Nature 504, 101-6, 2013)