title: Sodium ion binding pocket mutations and adenosine A2A receptor function.
abstract: 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.
authors.affiliation: 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.).
authors.forename: Arnault
authors.initials: A
authors.lastname: Massink
authors.affiliation: 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.).
authors.forename: Hugo
authors.initials: H
authors.lastname: Gutiérrez-de-Terán
authors.affiliation: 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.).
authors.forename: Eelke B
authors.initials: EB
authors.lastname: Lenselink
authors.affiliation: 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.).
authors.forename: Natalia V
authors.initials: NV
authors.lastname: Ortiz Zacarías
authors.affiliation: 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.).
authors.forename: Lizi
authors.initials: L
authors.lastname: Xia
authors.affiliation: 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.).
authors.forename: Laura H
authors.initials: LH
authors.lastname: Heitman
authors.affiliation: 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.).
authors.forename: Vsevolod
authors.initials: V
authors.lastname: Katritch
authors.affiliation: 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.).
authors.forename: Raymond C
authors.initials: RC
authors.lastname: Stevens
authors.affiliation: 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.) ijzerman@lacdr.leidenuniv.nl.
authors.forename: Adriaan P
authors.initials: AP
authors.lastname: IJzerman
mesh_terms: D000494:Allosteric Regulation / Q000502:physiology; D001665:Binding Sites / Q000502:physiology; D018360:Crystallography, X-Ray; D004305:Dose-Response Relationship, Drug; D057809:HEK293 Cells; D006801:Humans; D009154:Mutation / Q000502:physiology*; D017433:Protein Structure, Secondary; D043705:Receptor, Adenosine A2A / Q000737:chemistry / Q000502:physiology*; D012964:Sodium / Q000378:metabolism*
chemical_list: D043705:Receptor, Adenosine A2A; D012964:Sodium