Massink Arnault, Gutiérrez-de-Terán Hugo, Lenselink Eelke B, Ortiz Zacarías Natalia V, Xia Lizi, Heitman Laura H, Katritch Vsevolod, Stevens Raymond C, IJzerman Adriaan P
Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research, Leiden University, Leiden, The Netherlands (A.M., E.B.L., N.V.O.Z., L.X., L.H.H., A.P.IJ.); Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden (H.G.T.); and Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, California (V.K., R.C.S.).
Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research, Leiden University, Leiden, The Netherlands (A.M., E.B.L., N.V.O.Z., L.X., L.H.H., A.P.IJ.); Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden (H.G.T.); and Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, California (V.K., R.C.S.)
Mol Pharmacol. 2015 Feb;87(2):305-13. doi: 10.1124/mol.114.095737. Epub 2014 Dec 3.
Recently we identified a sodium ion binding pocket in a high-resolution structure of the human adenosine A2A receptor. In the present study we explored this binding site through site-directed mutagenesis and molecular dynamics simulations. Amino acids in the pocket were mutated to alanine, and their influence on agonist and antagonist affinity, allosterism by sodium ions and amilorides, and receptor functionality was explored. Mutation of the polar residues in the Na(+) pocket were shown to either abrogate (D52A(2.50) and N284A(7.49)) or reduce (S91A(3.39), W246A(6.48), and N280A(7.45)) the negative allosteric effect of sodium ions on agonist binding. Mutations D52A(2.50) and N284A(7.49) completely abolished receptor signaling, whereas mutations S91A(3.39) and N280A(7.45) elevated basal activity and mutations S91A(3.39), W246A(6.48), and N280A(7.45) decreased agonist-stimulated receptor signaling. In molecular dynamics simulations D52A(2.50) directly affected the mobility of sodium ions, which readily migrated to another pocket formed by Glu13(1.39) and His278(7.43). The D52A(2.50) mutation also decreased the potency of amiloride with respect to ligand displacement but did not change orthosteric ligand affinity. In contrast, W246A(6.48) increased some of the allosteric effects of sodium ions and amiloride, whereas orthosteric ligand binding was decreased. These new findings suggest that the sodium ion in the allosteric binding pocket not only impacts ligand affinity but also plays a vital role in receptor signaling. Because the sodium ion binding pocket is highly conserved in other class A G protein-coupled receptors, our findings may have a general relevance for these receptors and may guide the design of novel synthetic allosteric modulators or bitopic ligands.
最近，我们在人腺苷A2A受体的高分辨率结构中确定了一个钠离子结合口袋。在本研究中，我们通过定点诱变和分子动力学模拟对该结合位点进行了探索。将口袋中的氨基酸突变为丙氨酸，并研究了它们对激动剂和拮抗剂亲和力、钠离子和阿米洛利的变构作用以及受体功能的影响。结果表明，Na(+)口袋中极性残基的突变要么消除（D52A(2.50)和N284A(7.49)），要么降低（S91A(3.39)、W246A(6.48)和N280A(7.45)）钠离子对激动剂结合的负变构效应。突变D52A(2.50)和N284A(7.49)完全消除了受体信号传导，而突变S91A(3.39)和N280A(7.45)提高了基础活性，突变S91A(3.39)、W246A(6.48)和N280A(7.45)降低了激动剂刺激的受体信号传导。在分子动力学模拟中，D52A(2.50)直接影响钠离子的迁移率，钠离子很容易迁移到由Glu13(1.39)和His278(7.43)形成的另一个口袋中。D52A(2.50)突变还降低了阿米洛利在配体置换方面的效力，但没有改变正构配体亲和力。相比之下，W246A(6.48)增加了钠离子和阿米洛利的一些变构效应，而正构配体结合减少。这些新发现表明，变构结合口袋中的钠离子不仅影响配体亲和力，而且在受体信号传导中起着至关重要的作用。由于钠离子结合口袋在其他A类G蛋白偶联受体中高度保守，我们的发现可能对这些受体具有普遍意义，并可能指导新型合成变构调节剂或双位点配体的设计。
