title: Phe783, Thr797, and Asp804 in transmembrane hairpin M5-M6 of Na+,K+-ATPase play a key role in ouabain binding.
abstract: Ouabain is a glycoside that binds to and inhibits the action of Na+,K+-ATPase. Little is known, however, about the specific requirements of the protein surface for glycoside binding. Using chimeras of gastric H+,K+-ATPase and Na+,K+-ATPase, we demonstrated previously that the combined presence of transmembrane hairpins M3-M4 and M5-M6 of Na+,K+-ATPase in a backbone of H+,K+-ATPase (HN34/56) is both required and sufficient for high affinity ouabain binding. Since replacement of transmembrane hairpin M3-M4 by the N terminus up to transmembrane segment 3 (HNN3/56) resulted in a low affinity ouabain binding, hairpin M5-M6 seems to be essential for ouabain binding. To assess which residues of M5-M6 are required for ouabain action, we divided this transmembrane hairpin in seven parts and individually replaced these parts by the corresponding sequences of H+,K+-ATPase in chimera HN34/56. Three of these chimeras failed to bind ouabain following expression in Xenopus laevis oocytes. Altogether, these three chimeras contained 7 amino acids that were specific for Na+,K+-ATPase. Individual replacement of these 7 amino acids by the corresponding amino acids in H+,K+-ATPase revealed a dramatic loss of ouabain binding for F783Y, T797C, and D804E. As a proof of principle, the Na+,K+-ATPase equivalents of these 3 amino acids were introduced in different combinations in chimera HN34. The presence of all 3 amino acids appeared to be required for ouabain action. Docking of ouabain onto a three-dimensional-model of Na+,K+-ATPase suggests that Asp804, in contrast to Phe783 and Thr797, does not actually form part of the ouabain-binding pocket. Most likely, the presence of this amino acid is required for adopting of the proper conformation for ouabain binding.
authors.affiliation: Department of Biochemistry, Nijmegen Center for Molecular Life Sciences, University of Nijmegen, P. O. Box 9101, 6500 HB Nijmegen, The Netherlands.
authors.forename: Li Yan
authors.initials: LY
authors.lastname: Qiu
authors.forename: Jan B
authors.initials: JB
authors.lastname: Koenderink
authors.forename: Herman G P
authors.initials: HG
authors.lastname: Swarts
authors.forename: Peter H G M
authors.initials: PH
authors.lastname: Willems
authors.forename: Jan Joep H H M
authors.initials: JJ
authors.lastname: De Pont
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001224:Aspartic Acid; D017506:H(+)-K(+)-Exchanging ATPase / Q000737:chemistry / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry; D008958:Models, Molecular; D010042:Ouabain / Q000378:metabolism*; D010649:Phenylalanine; D011485:Protein Binding; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D013912:Threonine
chemical_list: D008565:Membrane Proteins; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D013912:Threonine; D001224:Aspartic Acid; D010649:Phenylalanine; D010042:Ouabain; D017506:H(+)-K(+)-Exchanging ATPase; D000254:Sodium-Potassium-Exchanging ATPase