A set of single-tryptophan mutants of the Na(+)/K(+)-ATPase isolated, large cytoplasmic loop connecting transmembrane helices M4 and M5 (C45) was prepared to monitor effects of the natural cytoplasmic ligands (i.e., Mg(2+) and/or ATP) binding. We introduced a novel method for the monitoring of the changes in the electrostatic surface potential (ESP) induced by ligand binding, using the quenching of the intrinsic tryptophan fluorescence by acrylamide or iodide. This approach opens a new way to understanding the interactions within the proteins. Our experiments revealed that the C45 conformation in the presence of the ATP (without magnesium) substantially differed from the conformation in the presence of Mg(2+) or MgATP or in the absence of any ligand not only in the sense of geometry but also in the sense of the ESP. Notably, the set of ESP-sensitive residues was different from the set of geometry-sensitive residues. Moreover, our data indicate that the effect of the ligand binding is not restricted only to the close environment of the binding site and that the information is in fact transmitted also to the distal parts of the molecule. This property could be important for the communication between the cytoplasmic headpiece and the cation binding sites located within the transmembrane domain.

Laboratory of Biophysics Faculty of Science Palacký University Olomouc Czech Republic

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Benkovic S.J., Hammes-Schiffer S. A perspective on enzyme catalysis. Science. 2003;301:1196–1202. PubMed

Kantarci-Carsibasi N., Haliloglu T., Doruker P. Conformational transition pathways explored by Monte Carlo simulation integrated with collective modes. Biophys. J. 2008;95:5862–5873. PubMed PMC

Wen P.C., Tajkhorshid E. Dimer opening of the nucleotide binding domains of ABC transporters after ATP hydrolysis. Biophys. J. 2008;95:5100–5110. PubMed PMC

Jorgensen P.L., Hakansson K.O., Karlish S.J.D. Structure and mechanism of Na,K-ATPase: Functional sites and their interactions. Annu. Rev. Physiol. 2003;65:817–849. PubMed

Therien A.G., Deber C.M. Oligomerization of a peptide derived from the transmembrane region of the sodium pump gamma subunit: effect of the pathological mutation G41R. J. Mol. Biol. 2002;322:583–590. PubMed

Yatime L., Buch-Pedersen M.J., Musgaard M., Morth J.P., Winther A.M.L. P-type ATPases as drug targets: tools for medicine and science. Biochim. Biophys. Acta. 2009;1787:207–220. PubMed

Moller J.V., Juul B., leMaire M. Structural organization, ion transport, and energy transduction of P-type ATPases. Biochim. Biophys. Acta. 1996;1286:1–51. PubMed

Skou J.C. Further investigations on a Mg++ + Na+-activated adenosinetriphosphatase, possibly related to the active, linked transport of Na+ and K+ across the nerve membrane. Biochim. Biophys. Acta. 1960;42:6–23.

Toyoshima C., Nakasako M., Nomura H., Ogawa H. Crystal structure of the calcium pump of sarcoplasmic reticulum at 2.6 angstrom resolution. Nature. 2000;405:647–655. PubMed

Toyoshima C., Nomura H. Structural changes in the calcium pump accompanying the dissociation of calcium. Nature. 2002;418:605–611. PubMed

Toyoshima C., Nomura H., Tsuda T. Lumenal gating mechanism revealed in calcium pump crystal structures with phosphate analogues. Nature. 2004;432:361–368. PubMed

Toyoshima C., Mizutani T. Crystal structure of the calcium pump with a bound ATP analogue. Nature. 2004;430:529–535. PubMed

Sorensen T.L.M., Moller J.V., Nissen P. Phosphoryl transfer and calcium ion occlusion in the calcium pump. Science. 2004;304:1672–1675. PubMed

Sorensen T.L.M., Olesen C., Jensen A.M.L., Moller J.V., Nissen P. Crystals of sarcoplasmic reticulum Ca2+-ATPase. J. Biotechnol. 2006;124:704–716. PubMed

Olesen C., Sorensen T.L.M., Nielsen R.C., Moller J.V., Nissen P. Dephosphorylation of the calcium pump coupled to counterion occlusion. Science. 2004;306:2251–2255. PubMed

Jensen A.M.L., Sorensen T.L.M., Olesen C., Moller J.V., Nissen P. Modulatory and catalytic modes of ATP binding by the calcium pump. EMBO J. 2006;25:2305–2314. PubMed PMC

Morth J.P., Pedersen B.P., Toustrup-Jensen M.S., Sorensen T.L.M., Petersen J. Crystal structure of the sodium-potassium pump. Nature. 2007;450:1043–1046. PubMed

Lupfert C., Grell E., Pintschovius V., Apell H.J., Cornelius F. Rate limitation of the Na+,K+-ATPase pump cycle. Biophys. J. 2001;81:2069–2081. PubMed PMC

Capieaux E., Rapin C., Thines D., Dupont Y., Goffeau A. Overexpression in Escherichia coli and purification of an ATP-binding peptide from the yeast plasma-membrane H+-ATPase. J. Biol. Chem. 1993;268:21895–21900. PubMed

Gatto C., Wang A.X., Kaplan J.H. The M4M5 cytoplasmic loop of the Na,K-ATPase, overexpressed in Escherichia coli, binds nucleoside triphosphates with the same selectivity as the intact native protein. J. Biol. Chem. 1998;273:10578–10585. PubMed

Kubala M., Hofbauerova K., Ettrich R., Kopecky V., Krumscheid R. Phe(475) and Glu(446) but not Ser(445) participate in ATP-binding to the alpha-subunit of Na+/K+-ATPase. Biochem. Biophys. Res. Commun. 2002;297:154–159. PubMed

Kubala M., Plasek J., Amler E. Limitations in linearized analyses of binding equilibria: binding of TNP-ATP to the H-4-H-5 loop of Na/K-ATPase. Eur. Biophys. J. Biophys. Lett. 2003;32:363–369. PubMed

Kubala M., Plasek J., Amler E. Fluorescence competition assay for the assessment of ATP binding to an isolated domain of Na+,K+-ATPase. Physiol. Res. 2004;53:109–113. PubMed

Kubala M., Teisinger J., Ettrich R., Hofbauerova K., Kopecky V. Eight amino acids form the ATP recognition site of Na+/K+-ATPase. Biochemistry. 2003;42:6446–6452. PubMed

Lansky Z., Kubala M., Ettrich R., Kuty M., Plasek J. The hydrogen bonds between Arg(423) and Glu(472) and other key residues, Asp(443), Ser(477), and Pro(489), are responsible for the formation and a different positioning of TNP-ATP and ATP within the nucleotide-binding site of Na+/K+-ATPase. Biochemistry. 2004;43:8303–8311. PubMed

Grycova L., Sklenovsky P., Lansky Z., Janovska M., Otyepka M. ATP and magnesium drive conformational changes of the Na+/K+-ATPase cytoplasmic headpiece. Biochim. Biophys. Acta. 2009;1788:1081–1091. PubMed

Garciamoreno B., Chen L.X., March K.L., Gurd R.S., Gurd F.R.N. Electrostatic interactions in sperm whale myoglobin—site specificity, roles in structural elements, and external electrostatic potential distributions. J. Biol. Chem. 1985;260:4070–4082. PubMed

Warshel A., Sharma P.K., Kato M., Parson W.W. Modeling electrostatic effects in proteins. Biochem. Biophys. Acta. 2006;1764:1647–1676. PubMed

Lakowicz J.R. Kluwer/Plenum; New York: 1999. Principles of Fluorescence Spectroscopy.

Albani J.R. Elsevier; Amsterdam, The Netherlands: 2004. Structure and Dynamics of Macromolecules: Absorption and Fluorescence Studies.

Arras, K. O. 1998. An introduction to error propagation: derivation, meaning and examples of equation Cy=FxCxFxT. In Technical Report of the Autonomous Systems Lab. Swiss Federal Institute of Technology Lausanne, EPFL-ASL-TR-98–01 R3.

Baker N.A., Sept D., Joseph S., Holst M.J., McCammon J.A. Electrostatics of nanosystems: application to microtubules and the ribosome. Proc. Natl. Acad. Sci. USA. 2001;98:10037–10041. PubMed PMC

Sanner M.F. Python: a programming language for software integration and development. J. Mol. Graph. 1999;17:57–61. PubMed

Kubala M. ATP-binding to P-type ATPases as revealed by biochemical, spectroscopic, and crystallographic experiments. Proteins. 2006;64:1–12. PubMed

Pratap P.R., Dediu O., Nienhaus G.U. FTIR study of ATP-induced changes in Na+/K+-ATPase from duck supraorbital glands. Biophys. J. 2003;85:3707–3717. PubMed PMC

Picard M., Toyoshima C., Champeil P. The average conformation at micromolar [Ca2+] of Ca2+-ATPase with bound nucleotide differs from that adopted with the transition state analog ADP center dot AlFx or with AMPPCP under crystallization conditions at millimolar. J. Biol. Chem. 2005;280:18745–18754. PubMed

Mead-Savery F.C., Wang R., Tanna-Topan B., Chen S.R.W., Welch W. Changes in negative charge at the luminal mouth of the pore alter ion handling and gating in the cardiac ryanodine-receptor. Biophys. J. 2009;96:1374–1387. PubMed PMC

Ledvina P.S., Yao N.H., Choudhary A., Quiocho F.A. Negative electrostatic surface potential of protein sites specific for anionic ligands. Proc. Natl. Acad. Sci. USA. 1996;93:6786–6791. PubMed PMC

Clausen J.D., Andersen J.P. Functional consequences of alterations to Thr(247), Pro(248), Glu(340), Asp(813), Arg(819), and Arg(822) at the interfaces between domain P, M3, and L6–7 of sarcoplasmic reticulum Ca2+-ATPase—roles in Ca2+ interaction and phosphoenzyme processing. J. Biol. Chem. 2004;279:54426–54437. PubMed

Xu G.Y., Kane D.J., Faller L.D., Farley R.A. The role of loop 6/7 in folding and functional performance of Na,K-ATPase. J. Biol. Chem. 2004;279:45594–45602. PubMed

Zhang Z.S., Lewis D., Sumbilla C., Inesi G., Toyoshima C. The role of the M6–M7 loop (L67) in stabilization of the phosphorylation and Ca2+ binding domains of the sarcoplasmic reticulum Ca2+-ATPase (SERCA) J. Biol. Chem. 2001;276:15232–15239. PubMed

Lenoir G., Picard M., Moller J.V., le Maire M., Champeil P. Involvement of the L6–7 loop in SERCA1a Ca2+-ATPase activation by Ca2+ (or Sr2+) and ATP. J. Biol. Chem. 2004;279:32125–32133. PubMed

Corre F., Jaxel C., Fuentes J., Menguy T., Falson P. Involvement of the cytoplasmic loop L6–7 in the entry mechanism for transport of Ca2+ through the sarcoplasmic reticulum Ca2+-ATPase. J. Biol. Chem. 2002;277:13016–13028. PubMed

Identification of cisplatin-binding sites on the large cytoplasmic loop of the Na+/K+-ATPase

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