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Crystal structure of the calcium pump with a bound ATP analogue

Abstract

P-type ATPases are ATP-powered ion pumps that establish ion concentration gradients across cell and organelle membranes. Here, we describe the crystal structure of the Ca2+ pump of skeletal muscle sarcoplasmic reticulum, a representative member of the P-type ATPase superfamily, with an ATP analogue, a Mg2+ and two Ca2+ ions in the respective binding sites. In this state, the ATP analogue reorganizes the three cytoplasmic domains (A, N and P), which are widely separated without nucleotide, by directly bridging the N and P domains. The structure of the P-domain itself is altered by the binding of the ATP analogue and Mg2+. As a result, the A-domain is tilted so that one of the transmembrane helices moves to lock the cytoplasmic gate of the transmembrane Ca2+-binding sites. This appears to be the mechanism for occluding the bound Ca2+ ions, before releasing them into the lumen of the sarcoplasmic reticulum.

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Figure 1: Front views (parallel to the membrane (xy) plane) of Ca2+-ATPase with (E1·AMPPCP) and without (E1·2Ca2+) AMPPCP (shown in space fill), an ATP analogue, in the presence of 10 mM Ca2+.
Figure 2: Superimposition of the E1·2Ca2+ and E1·AMPPCP forms of Ca2+-ATPase fitted with the transmembrane domain. E1·2Ca2+, violet; E1·AMPPCP, cyan (A-domain and M1–M3 helices), light green (N-domain) and orange (P-domain and M4–M10 helices).
Figure 3: Transmembrane Ca2+-binding sites (I and II) and the movement of the M1 helix.
Figure 4: Omit-annealed Fo - Fc map around AMPPCP at 5σ (a) and the hydrogen-bonding network around AMPPCP (b).
Figure 5: Superposition of the P-domain in E1·2Ca2+ and E1·AMPPCP, fitted with the 15 residues at the N-terminal end of the M5 helix.
Figure 6: Water-accessible surface of the cytoplasmic domains in E1·AMPPCP showing the A–N interface.

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Acknowledgements

We thank T. Tsuda for help in many aspects of this work; M. Kawamoto and H. Sakai for data collection at BL41XU of SPring-8; N. Miyashita for making many movies; 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 We are grateful to D.B. McIntosh for help in improving the manuscript and G. Inesi for communicating unpublished results to us.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 are deposited in the PDB under accession code 1VFP.

Supplementary information

Supplementary Figure 1

Configuration of cytoplasmic domains (A, N and P) in E2(TG) and E1·AMPPCP in ribbon representation. (JPG 161 kb)

Supplementary Figure 2

Details of the interaction between the A- and P-domains, viewed from the back side. (JPG 249 kb)

Supplementary Figure 3

A solvent flattened map showing the N-domain – P-domain interface calculated from the initial model containing only the P-domain and M3-M10 helices. (JPG 329 kb)

Supplementary Figure 4

A solvent flattened map showing the electron density representing the M1 and M2 helices, calculated from the model excluding them. (JPG 313 kb)

Supplementary Figure 5

Electron density (composite omit and Fo – Fc) maps around AMPPCP calculated for the P21 crystal using the model constructed for the C2 crystal before introducing AMPPCP in the model. (JPG 188 kb)

Supplementary Movie

A movie showing the conformation changes in Ca2+-ATPase for the sequence E2 → E1·2Ca2+ → E1·ATP, made by N. Miyashita using a morphing technique. (MOV 1211 kb)

Supplementary Figure Legends

Legends to the Supplementary Figures 1-5 and Supplementary Movie. (DOC 22 kb)

Supplementary Methods

Detailed description on the structure determination by molecular replacement. Containing one Table showing the progress of refinement. (DOC 29 kb)

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Toyoshima, C., Mizutani, T. Crystal structure of the calcium pump with a bound ATP analogue. Nature 430, 529–535 (2004). https://doi.org/10.1038/nature02680

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