Structure Short Article Crystal Structure of the Vanadate-Inhibited Ca 2+ -ATPase Johannes D. Clausen, 1,2,3,5,6 Maike Bublitz, 1,2,5,7 Bertrand Arnou, 1,2 Claus Olesen, 1,2,3 Jens Peter Andersen, 3 Jesper Vuust Møller, 2,3 and Poul Nissen 1,2,4, * 1 Department of Molecular Biology and Genetics, Aarhus University, 8000 Aarhus, Denmark 2 Centre for Membrane Pumps in Cells and Disease – PUMPKIN, Danish National Research Foundation, Aarhus University, 8000 Aarhus, Denmark 3 Department of Biomedicine, Aarhus University, 8000 Aarhus, Denmark 4 Danish Research Institute of Translational Neuroscience – DANDRITE, Nordic EMBL Partnership for Molecular Medicine, Aarhus University, 8000 Aarhus, Denmark 5 Co-first author 6 Present address: Pcovery ApS, Ole Maaløes Vej 3, 2200 Copenhagen N, Denmark 7 Present address: Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK *Correspondence: pn@mbg.au.dk http://dx.doi.org/10.1016/j.str.2016.02.018 SUMMARY Vanadate is the hallmark inhibitor of the P-type ATPase family; however, structural details of its inhib- itory mechanism have remained unresolved. We have determined the crystal structure of sarcoplasmic re- ticulum Ca 2+ -ATPase with bound vanadate in the absence of Ca 2+ . Vanadate is bound at the catalytic site as a planar VO 3  in complex with water and Mg 2+ in a dephosphorylation transition-state-like conformation. Validating bound VO 3  by anomalous difference Fourier maps using long-wavelength data we also identify a hitherto undescribed Cl  site near the dephosphorylation site. Crystallization was facil- itated 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 nucleo- tide analogs in the E2$VO 3  structure with that in E2$BeF 3  (E2P ground state analog) reveals multiple binding modes to the Ca 2+ -ATPase. INTRODUCTION Vanadate is a potent inhibitor of a large variety of enzymes that interact with phosphate compounds, including the P-type ATPases (Cantley et al., 1978; Dupont and Bennett, 1982; Pick, 1982). Models of the inhibitory mechanism of vanadate are generally based on its structural and chemical similarity to phosphate and the ability of vanadate to adopt a pentacoordi- nated trigonal bipyramidal geometry analogous to the transition state for phosphate ester hydrolysis of phosphoryl transfer (Crans et al., 2004; Davies and Hol, 2004). However, vanadate solutions typically exist as a complex mixture of the monomeric orthovanadate ion and various oligomeric species that also bind with high affinity to P-type ATPases and inhibit their activity (Aur- eliano, 2000; Csermely et al., 1985a), giving rise to some confu- sion about the actual chemical nature of vanadate inhibition. Decameric vanadate, favored at low pH, likely asserts its inhibi- tory effect by competing with ATP for binding (Coan et al., 1986; Csermely et al., 1985b; Hua et al., 2000). Previous X-ray crystallographic studies of E2P-like confor- mations of P-type ATPases have employed aluminum fluoride (AlF 4  ), magnesium fluoride (MgF x ), or beryllium fluoride (BeF 3  ) as phosphate analogs (Gourdon et al., 2011; Morth et al., 2007; Olesen et al., 2004, 2007; Toyoshima et al., 2004) (Figure 1A); however, neither of these are isosteric with the trigonal bipyramidal transition state of a phosphoryl transfer reac- tion. AlF 4  adopts a square planar geometry (Sorensen et al., 2004b), whereas MgF x and BeF 3  are non-planar and rather mimic a tetrahedral phosphate anion and a phosphorylated state, respectively (Olesen et al., 2007; Toyoshima et al., 2004). Vana- date is therefore thought to mimic the phosphoryl transition state more accurately than the metal-fluoride compounds (Davies and Hol, 2004). We have determined the structure of the vanadate- inhibited Ca 2+ -ATPase to obtain structural insight into the phos- phoryl transfer reaction during the autophosphatase activity of the Ca 2+ -ATPase (Figure 1A) and to clarify the differential effects of vanadate inhibition of transport activity. RESULTS AND DISCUSSION Vanadate Stabilizes the Ca 2+ -ATPase in an E2$P Transition-State-like Conformation Ca 2+ -ATPase, pre-equilibrated with orthovanadate (VO 4 3 ) and Mg 2+ (a critical co-factor for orthovanadate binding; Dupont and Bennett, 1982) in the absence of Ca 2+ to accumulate the orthovanadate-reactive E2 form (Pick, 1982), was crystallized and analyzed by X-ray diffraction (Figure S1 and Table S1). Crystals grown in the presence of 2 0 ,3 0 -O-(2,4,6-trinitrophenyl)- b,g-methyleneadenosine 5 0 -triphosphate (TNP-AMPPCP) and the Ca 2+ -ATPase-specific membrane-bound inhibitor thapsigar- gin diffracted to 3.05 A ˚ resolution, and data were collected at Structure 24, 617–623, April 5, 2016 ª2016 Elsevier Ltd All rights reserved 617