Abstract
P-type ion transporting ATPases are ATP-powered ion pumps that establish ion concentration gradients across biological membranes. Transfer of bound cations to the lumenal or extracellular side occurs while the ATPase is phosphorylated. Here we report at 2.3 Å resolution the structure of the calcium-ATPase of skeletal muscle sarcoplasmic reticulum, a representative P-type ATPase that is crystallized in the absence of Ca2+ but in the presence of magnesium fluoride, a stable phosphate analogue. This and other crystal structures determined previously provide atomic models for all four principal states in the reaction cycle. These structures show that the three cytoplasmic domains rearrange to move six out of ten transmembrane helices, thereby changing the affinity of the Ca2+-binding sites and the gating of the ion pathway. Release of ADP triggers the opening of the lumenal gate and release of phosphate its closure, effected mainly through movement of the A-domain, the actuator of transmembrane gates.
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References
Møller, J. V., Juul, B. & le Maire, M. Structural organization, ion transport, and energy transduction of P-type ATPases. Biochim. Biophys. Acta 1286, 1–51 (1996)
Kühlbrandt, W. Biology, structure and mechanism of P-type ATPases. Nature Rev. Mol. Cell Biol. 5, 282–295 (2004)
Albers, R. W. Biochemical aspects of active transport. Annu. Rev. Biochem. 36, 727–756 (1967)
Post, R. L., Hegyvary, C. & Kume, S. Activation by adenosine triphosphate in the phosphorylation kinetics of sodium and potassium ion transport adenosine triphosphatase. J. Biol. Chem. 247, 6530–6540 (1972)
de Meis, L. & Vianna, A. L. Energy interconversion by the Ca2+-dependent ATPase of the sarcoplasmic reticulum. Annu. Rev. Biochem. 48, 275–292 (1979)
Brandl, C. J., deLeon, S., Martin, D. R. & MacLennan, D. H. Adult forms of the Ca2+ ATPase of sarcoplasmic reticulum. Expression in developing skeletal muscle. J. Biol. Chem. 262, 3768–3774 (1987)
MacLennan, D. H., Brandl, C. J., Korczak, B. & Green, N. M. Amino-acid sequence of a Ca2+ + Mg2+-dependent ATPase from rabbit muscle sarcoplasmic reticulum, deduced from its complementary DNA sequence. Nature 316, 696–700 (1985)
Toyoshima, C., Nakasako, M., Nomura, H. & Ogawa, H. Crystal structure of the calcium pump of sarcoplasmic reticulum at 2.6 Å resolution. Nature 405, 647–655 (2000)
Toyoshima, C. & Inesi, G. Structural basis of ion pumping by Ca2+-ATPase of the sarcoplasmic reticulum. Annu. Rev. Biochem. 73, 269–292 (2004)
Toyoshima, C. & Nomura, H. Structural changes in the calcium pump accompanying the dissociation of calcium. Nature 418, 605–611 (2002)
Toyoshima, C. & Mizutani, T. Crystal structure of the calcium pump with a bound ATP analogue. Nature 430, 529–535 (2004)
Sørensen, T. L., Møller, J. V. & Nissen, P. Phosphoryl transfer and calcium ion occlusion in the calcium pump. Science 304, 1672–1675 (2004)
Troullier, A., Girardet, J. L. & Dupont, Y. Fluoroaluminate complexes are bifunctional analogues of phosphate in sarcoplasmic reticulum Ca2+-ATPase. J. Biol. Chem. 267, 22821–22829 (1992)
Murphy, A. J. & Coll, R. J. Fluoride is a slow, tight-binding inhibitor of the calcium ATPase of sarcoplasmic reticulum. J. Biol. Chem. 267, 5229–5235 (1992)
Danko, S. et al. ADP-insensitive phosphoenzyme intermediate of sarcoplasmic reticulum Ca2+-ATPase has a compact conformation resistant to proteinase K, V8 protease and trypsin. FEBS Lett. 489, 277–282 (2001)
Danko, S., Yamasaki, K., Daiho, T. & Suzuki, H. Distinct natures of beryllium fluoride-bound, aluminum fluoride-bound, and magnesium fluoride-bound stable analogues of an ADP-insensitive phosphoenzyme intermediate of sarcoplasmic reticulum Ca2+-ATPase: changes in catalytic and transport sites during phosphoenzyme hydrolysis. J. Biol. Chem. 279, 14991–14998 (2004)
Danko, S., Yamasaki, K., Daiho, T., Suzuki, H. & Toyoshima, C. Organization of cytoplasmic domains of sarcoplasmic reticulum Ca2+-ATPase in E1P and E1ATP states: a limited proteolysis study. FEBS Lett. 505, 129–135 (2001)
Brünger, A. T. Extension of molecular replacement: a new search strategy based on Patterson correlation refinement. Acta Crystallogr. A 46, 46–57 (1990)
Yamasaki, K., Daiho, T. & Suzuki, H. Remarkable stability of solubilized and delipidated sarcoplasmic reticulum Ca2+-ATPase with tightly bound fluoride and magnesium against detergent-induced denaturation. J. Biol. Chem. 277, 13615–13619 (2002)
Zhang, P., Toyoshima, C., Yonekura, K., Green, N. M. & Stokes, D. L. Structure of the calcium pump from sarcoplasmic reticulum at 8-Å resolution. Nature 392, 835–839 (1998)
Graham, D. L. et al. MgF3- as a transition state analog of phosphoryl transfer. Chem. Biol. 9, 375–381 (2002)
Coll, R. J. & Murphy, A. J. Fluoride-inhibited calcium ATPase of sarcoplasmic reticulum. Magnesium and fluoride stoichiometry. J. Biol. Chem. 267, 21584–21587 (1992)
Daiho, T., Kubota, T. & Kanazawa, T. Stoichiometry of tight binding of magnesium and fluoride to phosphorylation and high-affinity binding of ATP, vanadate, and calcium in the sarcoplasmic reticulum Ca2+-ATPase. Biochemistry 32, 10021–10026 (1993)
Coll, R. J. & Murphy, A. J. Purification of the CaATPase of sarcoplasmic reticulum by affinity chromatography. J. Biol. Chem. 259, 14249–14254 (1984)
Stokes, D. L. & Green, N. M. Three-dimensional crystals of CaATPase from sarcoplasmic reticulum. Symmetry and molecular packing. Biophys. J. 57, 1–14 (1990)
Palmgren, M. G. & Axelsen, K. B. Evolution of P-type ATPases. Biochim. Biophys. Acta 1365, 37–45 (1998)
Clarke, D. M., Loo, T. W. & MacLennan, D. H. Functional consequences of mutations of conserved amino acids in the β-strand domain of the Ca2+-ATPase of sarcoplasmic reticulum. J. Biol. Chem. 265, 14088–14092 (1990)
Ogurusu, T., Wakabayashi, S. & Shigekawa, M. Activation of sarcoplasmic reticulum Ca2+-ATPase by Mn2+: a Mn2+ binding study. J. Biochem. 109, 472–476 (1991)
Lewis, R. J., Brannigan, J. A., Muchova, K., Barak, I. & Wilkinson, A. J. Phosphorylated aspartate in the structure of a response regulator protein. J. Mol. Biol. 294, 9–15 (1999)
Ridder, I. S. & Dijkstra, B. W. Identification of the Mg2+-binding site in the P-type ATPase and phosphatase members of the HAD (haloacid dehalogenase) superfamily by structural similarity to the response regulator protein CheY. Biochem. J. 339, 223–226 (1999)
Clausen, J. D., Vilsen, B., McIntosh, D. B., Einholm, A. P. & Andersen, J. P. Glutamate-183 in the conserved TGES motif of domain A of sarcoplasmic reticulum Ca2+-ATPase assists in catalysis of E2/E2P partial reactions. Proc. Natl Acad. Sci. USA 101, 2776–2781 (2004)
Maruyama, K. et al. Functional consequences of alterations to amino acids located in the catalytic center (isoleucine 348 to threonine 357) and nucleotide-binding domain of the Ca2+-ATPase of sarcoplasmic reticulum. J. Biol. Chem. 264, 13038–13042 (1989)
Clausen, J. D., McIntosh, D. B., Woolley, D. G. & Andersen, J. P. Importance of Thr-353 of the conserved phosphorylation loop of the sarcoplasmic reticulum Ca2+-ATPase in MgATP binding and catalytic activity. J. Biol. Chem. 276, 35741–35750 (2001)
McIntosh, D. B. & Boyer, P. D. Adenosine 5′-triphosphate modulation of catalytic intermediates of calcium ion activated adenosinetriphosphatase of sarcoplasmic reticulum subsequent to enzyme phosphorylation. Biochemistry 22, 2867–2875 (1983)
Champeil, P. et al. ATP regulation of sarcoplasmic reticulum Ca2+-ATPase. Metal-free ATP and 8-bromo-ATP bind with high affinity to the catalytic site of phosphorylated ATPase and accelerate dephosphorylation. J. Biol. Chem. 263, 12288–12294 (1988)
Kato, S. et al. Val 200 residue in Lys 189-Lys 205 outermost loop on the A domain of sarcoplasmic reticulum Ca2+-ATPase is critical for rapid processing of phosphoenzyme intermediate after loss of ADP sensitivity. J. Biol. Chem. 278, 9624–9629 (2003)
Lenoir, G. et al. Functional properties of sarcoplasmic reticulum Ca2+-ATPase after proteolytic cleavage at Leu 119-Lys 120, close to the A-domain. J. Biol. Chem. 279, 9156–9166 (2004)
Yamasaki, K., Daiho, T., Danko, S. & Suzuki, H. Multiple and distinct effects of mutations of Tyr 122, Glu 123, Arg 324, and Arg 334 involved in interactions between the top part of second and fourth transmembrane helices in sarcoplasmic reticulum Ca2+-ATPase: changes in cytoplasmic domain organization during isometric transition of phosphoenzyme intermediate and subsequent Ca2+ release. J. Biol. Chem. 279, 2202–2210 (2004)
Møller, J. V. et al. Calcium transport by sarcoplasmic reticulum Ca2+-ATPase. Role of the A domain and its C-terminal link with the transmembrane region. J. Biol. Chem. 277, 38647–38659 (2002)
Orlowski, S. & Champeli, P. Kinetics of calcium dissociation from its high-affinity transport sites on sarcoplasmic reticulum ATPase. Biochemistry 30, 352–361 (1991)
Inesi, G., Ma, H., Lewis, D. & Xu, C. Ca2+ occlusion and gating function of Glu 309 in the ADP-fluoroaluminate analog of the Ca2+-ATPase phosphoenzyme intermediate. J. Biol. Chem. 279, 31629–31637 (2004)
Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–325 (1997)
Brünger, A. T. et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998)
Wang, W. et al. Structural characterization of the reaction pathway in phosphoserine phosphatase: crystallographic “snapshots” of intermediate states. J. Mol. Biol. 319, 421–431 (2002)
Juul, B. et al. Do transmembrane segments in proteolysed sarcoplasmic reticulum Ca2+-ATPase retain their functional Ca2+ binding properties after removal of cytoplasmic fragments by proteinase K? J. Biol. Chem. 270, 20123–20134 (1995)
Kraulis, P. J. MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallogr. 24, 946–950 (1991)
Kabsch, W. & Sander, C. Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers 22, 2577–2637 (1983)
Acknowledgements
We thank H. Suzuki for his contribution at the initial phase of this work. We acknowledge that the first crystals of E2·MgF42- were made by Y. Tsubaki. Thanks are also due to M. Kawamoto, H. Sakai and E. Yamashita for data collection at SPring-8; N. Miyashita for making many movies; M. Takahashi and J. Tsueda for preparing figures; and Y. Ohuchi for computer programs. We are grateful to D. B. McIntosh for help in improving the manuscript and G. Inesi for communicating unpublished results to us. This work was supported in part by a Creative Science Project Grant from the Ministry of Education, Culture, Sports, Science and Technology, the Japan New Energy and Industry Technology Development Organization, and the Human Frontier Science Program.
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Additional information
The atomic coordinates for E1·AlFx·ADP and E2·MgF42- are deposited in the PDB under accession codes 1WPE and 1WPG, respectively.
Supplementary information
Supplementary Figure 1
A solvent flattened map at 2.3 Å resolution around MgF42- calculated from the model built for C2 crystals without MgF42- and Mg2+ using the diffraction data from P21 crystals. (JPG 87 kb)
Supplementary Figure 2
Details around ADP in the crystals of E2·MgF42-. (JPG 94 kb)
Supplementary Figure 3
Surface representation of the transmembrane region, showing a proposed ion pathway. (JPG 74 kb)
Supplementary Figure 4
An initial solvent flattened map calculated from the model containing only the A and N domains, showing the electron density representing the P-domain. (JPG 140 kb)
Supplementary Figure 5
A solvent flattened map calculated from the model containing only the 3 cytoplasmic domains, showing the electron density around the M4 and M5 helices that were not included in the model. (JPG 153 kb)
Supplementary Movie
A movie showing the conformation changes in Ca2+-ATPase for the sequence E2 → E1·2Ca2+ → E1·ATP → E1P→ E2P, made by N. Miyashita using a morphing technique. (MOV 1528 kb)
Supplementary Legends
Legends to the Supplementary Figures 1-4 and Supplementary Movie. (DOC 23 kb)
Supplementary Methods
Detailed description on the structure determination by molecular replacement. Containing one Table showing the progress of refinement. (DOC 25 kb)
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Toyoshima, C., Nomura, H. & Tsuda, T. Lumenal gating mechanism revealed in calcium pump crystal structures with phosphate analogues. Nature 432, 361–368 (2004). https://doi.org/10.1038/nature02981
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DOI: https://doi.org/10.1038/nature02981
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