title: Crystal Structure of the Vanadate-Inhibited Ca(2+)-ATPase.
abstract: Vanadate is the hallmark inhibitor of the P-type ATPase family; however, structural details of its inhibitory mechanism have remained unresolved. We have determined the crystal structure of sarcoplasmic reticulum Ca(2+)-ATPase with bound vanadate in the absence of Ca(2+). Vanadate is bound at the catalytic site as a planar VO3(-) in complex with water and Mg(2+) in a dephosphorylation transition-state-like conformation. Validating bound VO3(-) by anomalous difference Fourier maps using long-wavelength data we also identify a hitherto undescribed Cl(-) site near the dephosphorylation site. Crystallization was facilitated by trinitrophenyl (TNP)-derivatized nucleotides that bind with the TNP moiety occupying the binding pocket that normally accommodates the adenine of ATP, rationalizing their remarkably high affinity for E2P-like conformations of the Ca(2+)-ATPase. A comparison of the configurations of bound nucleotide analogs in the E2·VO3(-) structure with that in E2·BeF3(-) (E2P ground state analog) reveals multiple binding modes to the Ca(2+)-ATPase.
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, 8000 Aarhus, Denmark; Centre for Membrane Pumps in Cells and Disease - PUMPKIN, Danish National Research Foundation, Aarhus University, 8000 Aarhus, Denmark; Department of Biomedicine, Aarhus University, 8000 Aarhus, Denmark.
authors.forename: Johannes D
authors.initials: JD
authors.lastname: Clausen
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, 8000 Aarhus, Denmark; Centre for Membrane Pumps in Cells and Disease - PUMPKIN, Danish National Research Foundation, Aarhus University, 8000 Aarhus, Denmark.
authors.forename: Maike
authors.initials: M
authors.lastname: Bublitz
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, 8000 Aarhus, Denmark; Centre for Membrane Pumps in Cells and Disease - PUMPKIN, Danish National Research Foundation, Aarhus University, 8000 Aarhus, Denmark.
authors.forename: Bertrand
authors.initials: B
authors.lastname: Arnou
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, 8000 Aarhus, Denmark; Centre for Membrane Pumps in Cells and Disease - PUMPKIN, Danish National Research Foundation, Aarhus University, 8000 Aarhus, Denmark; Department of Biomedicine, Aarhus University, 8000 Aarhus, Denmark.
authors.forename: Claus
authors.initials: C
authors.lastname: Olesen
authors.affiliation: Department of Biomedicine, Aarhus University, 8000 Aarhus, Denmark.
authors.forename: Jens Peter
authors.initials: JP
authors.lastname: Andersen
authors.affiliation: Centre for Membrane Pumps in Cells and Disease - PUMPKIN, Danish National Research Foundation, Aarhus University, 8000 Aarhus, Denmark; Department of Biomedicine, Aarhus University, 8000 Aarhus, Denmark.
authors.forename: Jesper Vuust
authors.initials: JV
authors.lastname: Møller
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, 8000 Aarhus, Denmark; Centre for Membrane Pumps in Cells and Disease - PUMPKIN, Danish National Research Foundation, Aarhus University, 8000 Aarhus, Denmark; Danish Research Institute of Translational Neuroscience - DANDRITE, Nordic EMBL Partnership for Molecular Medicine, Aarhus University, 8000 Aarhus, Denmark. Electronic address: pn@mbg.au.dk.
authors.forename: Poul
authors.initials: P
authors.lastname: Nissen
mesh_terms: D000818:Animals; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D010766:Phosphorylation; D011487:Protein Conformation; D011817:Rabbits; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism*; D014638:Vanadates / Q000494:pharmacology*
chemical_list: D014638:Vanadates; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases