P-type ATPases are ATP-powered ion pumps that establish ion concentration gradients across biological membranes, and are distinct from other ATPases in that the reaction cycle includes an autophosphorylation step. The best studied is Ca2+-ATPase from muscle sarcoplasmic reticulum (SERCA1a), a Ca2+ pump that relaxes muscle cells after contraction, and crystal structures have been determined for most of the reaction intermediates1,2. An important outstanding structure is that of the E1 intermediate, which has empty high-affinity Ca2+-binding sites ready to accept new cytosolic Ca2+. In the absence of Ca2+ and at pH 7 or higher, the ATPase is predominantly in E1, not in E2 (low affinity for Ca2+)3, and if millimolar Mg2+ is present, one Mg2+ is expected to occupy one of the Ca2+-binding sites with a millimolar dissociation constant4,5. This Mg2+ accelerates the reaction cycle4, not permitting phosphorylation without Ca2+ binding. Here we describe the crystal structure of native SERCA1a (from rabbit) in this E1·Mg2+ state at 3.0 Å resolution in addition to crystal structures of SERCA1a in E2 free from exogenous inhibitors, and address the structural basis of the activation signal for phosphoryl transfer. Unexpectedly, sarcolipin6, a small regulatory membrane protein of Ca2+-ATPase7, is bound, stabilizing the E1·Mg2+ state. Sarcolipin is a close homologue of phospholamban, which is a critical mediator of β-adrenergic signal in Ca2+ regulation in heart (for reviews, see, for example, refs 8–10), and seems to play an important role in muscle-based thermogenesis11. We also determined the crystal structure of recombinant SERCA1a devoid of sarcolipin, and describe the structural basis of inhibition by sarcolipin/phospholamban. Thus, the crystal structures reported here fill a gap in the structural elucidation of the reaction cycle and provide a solid basis for understanding the physiological regulation of the calcium pump.
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Protein Data Bank
Atomic coordinates and structure factors for the reported crystal structures are deposited in the Protein Data Bank under accession codes 3W5D (E2+Pi), 3W5C (E2), 3W5A (E1•Mg2+ (native enzyme with bound sarcolipin)) and 3W5B (E1•Mg2+ (recombinant enzyme devoid of sarcolipin)).
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We thank H. Suzuki and S. Danko (Asahikawa Medical College) for providing us with much unpublished information. Thanks are also due to S. Hasegawa (JASRI) and H. Mimura for data collection at BL41XU of SPring-8, S. Yonekura for refinement of the E2 crystals and Y. Norimatsu for molecular dynamics simulations. We are grateful to D. B. McIntosh for help in improving the manuscript. This work is part of a long-term project (2009B0025) at SPring-8, and was supported by a Specially Promoted Project Grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to C.T.).
The authors declare no competing financial interests.
This file contains Supplementary Table 1 and Supplementary Figures 1-14. (PDF 2306 kb)
SLN appears as a transparent cylinder in E2 and E1•2Ca2+ in the same position as in E1•Mg2+ to show mismatch between SLN and the binding cavity. Animation derived from Figure 3b. When this article was originally published, only the download link for the video worked. The title link has now been updated to work correctly. (MOV 8399 kb)
Van der Waals surface of the transmembrane region around the SLN binding cavity. Animation derived from Supplementary Figure 14. When this article was originally published, only the download link for the video worked. The title link has now been updated to work correctly. (MOV 3695 kb)
Aligned with the P domain and viewed approximately perpendicular to the membrane. D351 is the phosphorylation residue. Animation derived from Figure 4a. The direct link to this video is currently out of action. To view this video please use the "download" link (MOV 1320 kb)
Cytoplasmic domains are aligned with the P7 helix and viewed approximately parallel to the membrane. Animation derived from Figure 4b. (MOV 5667 kb)
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Toyoshima, C., Iwasawa, S., Ogawa, H. et al. Crystal structures of the calcium pump and sarcolipin in the Mg2+-bound E1 state. Nature 495, 260–264 (2013). https://doi.org/10.1038/nature11899
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