The β₂-adrenergic receptor (β₂AR) is a member of the G protein-coupled receptor (GPCR) family that is an important drug target for asthma and COPD. Clinical studies coupled with biochemical data have identified a critical receptor variant, Thr164Ile, to have a reduced response to agonist-based therapy, although the molecular mechanism underlying this seemingly “non-deleterious” substitution is not clear. Here, we couple molecular dynamics simulations with network analysis and free-energy calculations to identify the molecular determinants underlying the differential drug response. We are able to identify hydration sites in the transmembrane domain that are essential to maintain the integrity of the binding site but are absent in the variant. The loss of these hydration sites in the variant correlates with perturbations in the intra-protein interaction network and rearrangements in the orthosteric ligand binding site. In conjunction, we observe an altered binding and reduced free energy of a series of agonists, in line with experimental trends. Our work identifies a functional allosteric pathway connected by specific hydration sites in β₂AR that has not been reported before and provides insight into water-mediated networks in GPCRs in general. Overall, the work is one of the first step towards developing variant-specific potent and selective agonists.

所述β ₂ -肾上腺素能受体（β ₂AR）是G蛋白偶联受体（GPCR）家族的成员，该家族是哮喘和COPD的重要药物靶标。临床研究与生化数据相结合，已经确定了一种关键的受体变体Thr164Ile对基于激动剂的疗法的反应减少，尽管这种看似“无害”取代的分子机制尚不清楚。在这里，我们将分子动力学模拟与网络分析和自由能计算相结合，以识别差异药物反应的分子决定因素。我们能够鉴定跨膜结构域中的水合位点，这些位点对于维持结合位点的完整性必不可少，但在变体中却不存在。这些水合位点在变体中的丧失与蛋白内相互作用网络的扰动和正构配体结合位点的重排有关。结合起来，我们观察到一系列激动剂的结合改变和自由能降低，与实验趋势一致。我们的工作确定了由β中特定的水合位点连接的功能性变构途径₂以前尚未报道过的AR，可提供有关GPCR中水介导网络的一般见解。总的来说，这项工作是开发变异型有效和选择性激动剂的第一步。