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A bimodular mechanism of calcium control in eukaryotes

Abstract

Calcium ions (Ca2+) have an important role as secondary messengers in numerous signal transduction processes1,2,3,4, and cells invest much energy in controlling and maintaining a steep gradient between intracellular (0.1-micromolar) and extracellular (2-millimolar) Ca2+ concentrations1. Calmodulin-stimulated calcium pumps, which include the plasma-membrane Ca2+-ATPases (PMCAs), are key regulators of intracellular Ca2+ in eukaryotes5,6,7,8. They contain a unique amino- or carboxy-terminal regulatory domain responsible for autoinhibition, and binding of calcium-loaded calmodulin to this domain releases autoinhibition and activates the pump. However, the structural basis for the activation mechanism is unknown and a key remaining question is how calmodulin-mediated PMCA regulation can cover both basal Ca2+ levels in the nanomolar range as well as micromolar-range Ca2+ transients generated by cell stimulation7. Here we present an integrated study combining the determination of the high-resolution crystal structure of a PMCA regulatory-domain/calmodulin complex with in vivo characterization and biochemical, biophysical and bioinformatics data that provide mechanistic insights into a two-step PMCA activation mechanism mediated by calcium-loaded calmodulin. The structure shows the entire PMCA regulatory domain and reveals an unexpected 2:1 stoichiometry with two calcium-loaded calmodulin molecules binding to different sites on a long helix. A multifaceted characterization of the role of both sites leads to a general structural model for calmodulin-mediated regulation of PMCAs that allows stringent, highly responsive control of intracellular calcium in eukaryotes, making it possible to maintain a stable, basal level at a threshold Ca2+ concentration, where steep activation occurs.

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Figure 1: Overall structure of the A. thaliana (Cam7) 2–Aca8R complex.
Figure 2: Physiological and biochemical characterization of wild-type Aca8 and mutants.
Figure 3: Binding of CaM to the putative second CaM-binding site of PMCA splice variants.
Figure 4: Schematic of the proposed two-step, Ca 2+-mediated CaM-activation mechanism.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors for the crystal structure of (Cam7)2–Aca8R have been deposited with the Protein Data Bank under accession code 4AQR.

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Acknowledgements

We thank members of the Nissen and Palmgren labs for discussions, and P. Gourdon for help with data collection. We thank the staff at beamlines ID23-2 at the European Radiation Synchrotron Facility, France; PX3 at the Swiss Light Source, Paul Scherrer Institute, Switzerland; and X33 at EMBL/DESY, Germany. We are grateful to K. Nagai for a plasmid expressing mammalian CaM. Support from the European Community-Research Infrastructure Action under the FP7 is acknowledged for access to EMBL/DESY. H.T. is a Junior Research Fellow at Trinity College, Cambridge, and was supported by an EMBO Long-Term Fellowship, a Marie-Curie Intra-European Fellowship and an HFSP Long-Term Fellowship. P.N. was supported by an ERC advanced grant (BIOMEMOS).

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Authors

Contributions

H.T. designed and initiated the project, designed the expression constructs and developed the co-expression strategy, initially assisted by K.L.H. Protein purification, crystallization, structure determination and refinement, and the overall analysis of the results, was performed by H.T. L.R.P. performed biochemical and genetic analyses of Aca8 and derived mutants, developed methods for measuring calcium concentrations in vitro, and analysed biochemical and yeast complementation assays, supervised by M.G.P. A.A. performed bioinformatics sequence analysis, homology modelling and docking experiments. M.K. performed mathematical modelling, supervised by C.W. P.N. designed and supervised the project, and analysed results. H.T., L.R.P., A.A., M.G.P. and P.N. wrote the paper, and all authors commented on the paper.

Corresponding authors

Correspondence to Michael G. Palmgren or Poul Nissen.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-10, Supplementary Tables 1-2 and additional references. (PDF 8599 kb)

Structural illustration of the proposed two-step, broad range Ca2+-mediated CaM-activation mechanism.

A homology model of ACA8 in surface representation color-coded based on sequence conservation (magenta = conserved / cyan = non-conserved) is shown with its regulatory domain docked against a conserved cleft only accessible in E2 conformation. With increasing Ca2+-concentration, Ca2+-CaM first binds and displaces high-affinity CaMBS1 allowing the pump to function at a basal rate (slow functional cycle) before even higher Ca2+-concentration leads to displacement of CaMBS2 from the catalytic core allowing free movement of the catalytic core as required for full ion pumping activity. Colour code of all components as in Fig. 1A. (MPG 3630 kb)

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Tidow, H., Poulsen, L., Andreeva, A. et al. A bimodular mechanism of calcium control in eukaryotes. Nature 491, 468–472 (2012). https://doi.org/10.1038/nature11539

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