Keov Peter, López Laura, Devine Shane M, Valant Celine, Lane J Robert, Scammells Peter J, Sexton Patrick M, Christopoulos Arthur
From the Drug Discovery Biology Theme and Department of Pharmacology and.
the Medicinal Chemistry Theme, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, Australia.
J Biol Chem. 2014 Aug 22;289(34):23817-37. doi: 10.1074/jbc.M114.582874. Epub 2014 Jul 8.
TBPB and 77-LH-28-1 are selective agonists of the M1 muscarinic acetylcholine receptor (mAChR) that may gain their selectivity through a bitopic mechanism, interacting concomitantly with the orthosteric site and part of an allosteric site. The current study combined site-directed mutagenesis, analytical pharmacology,and molecular modeling to gain further insights into the structural basis underlying binding and signaling by these agonists. Mutations within the orthosteric binding site caused similar reductions in affinity and signaling efficacy for both selective and prototypical orthosteric ligands. In contrast, the mutation of residues within transmembrane helix (TM) 2 and the second extracellular loop (ECL2) discriminated between the different classes of ligand. In particular, ECL2 appears to be involved in the selective binding of bitopic ligands and in coordinating biased agonism between intracellular calcium mobilization and ERK1/2 phosphorylation. Molecular modeling of the interaction between TBPB and the M1 mAChR revealed a binding pose predicted to extend from the orthosteric site up toward a putative allosteric site bordered by TM2, TM3, and TM7, thus consistent with a bitopic mode of binding. Overall, these findings provide valuable structural and mechanistic insights into bitopic ligand actions and receptor activation and support a role for ECL2 in dictating the active states that can be adopted by a G protein-coupled receptor. This may enable greater selective ligand design and development for mAChRs and facilitate improved identification of bitopic ligands.
TBPB和77-LH-28-1是M1型毒蕈碱型乙酰胆碱受体（mAChR）的选择性激动剂，它们可能通过双位点机制获得选择性，同时与正构位点和部分变构位点相互作用。本研究结合定点诱变、分析药理学和分子建模，以进一步深入了解这些激动剂结合和信号传导的结构基础。正构结合位点内的突变对选择性和原型正构配体的亲和力和信号传导效能产生了类似的降低。相比之下，跨膜螺旋（TM）2和第二个细胞外环（ECL2）内残基的突变区分了不同类型的配体。特别是，ECL2似乎参与了双位点配体的选择性结合，并协调了细胞内钙动员和ERK1/2磷酸化之间的偏向激动作用。TBPB与M1 mAChR相互作用的分子建模揭示了一种预测的结合姿势，该姿势从正构位点向上延伸至由TM2、TM3和TM7界定的假定变构位点，因此与双位点结合模式一致。总体而言，这些发现为双位点配体作用和受体激活提供了有价值的结构和机制见解，并支持ECL2在决定G蛋白偶联受体可采用的活性状态方面的作用。这可能有助于为mAChRs设计和开发更具选择性的配体，并促进对双位点配体的更好识别。
