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Lumenal gating mechanism revealed in calcium pump crystal structures with phosphate analogues

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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Figure 1: Front views (parallel to the membrane (x–y) plane) of Ca2+-ATPase in three different states and a simplified reaction scheme (showing only the forward direction), in which different colours correspond to the respective structures presented here.
Figure 2: Movements of the cytoplasmic domains.
Figure 3: Movements in the transmembrane domain.
Figure 4: Movements in the transmembrane domain viewed approximately normal to the membrane plane from the cytoplasmic side.
Figure 5: Details of the phosphorylation site in E2·MgF42-.
Figure 6

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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 We are grateful to D. B. McIntosh for help in improving the manuscript and G. Inesi for communicating unpublished results to us.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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Correspondence to Chikashi Toyoshima.

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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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