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Structural basis for alternating access of a eukaryotic calcium/proton exchanger

Abstract

Eukaryotic Ca2+ regulation involves sequestration into intracellular organelles, and expeditious Ca2+ release into the cytosol is a hallmark of key signalling transduction pathways. Bulk removal of Ca2+ after such signalling events is accomplished by members of the Ca2+:cation (CaCA) superfamily1,2,3,4,5. The CaCA superfamily includes the Na+/Ca2+ (NCX) and Ca2+/H+ (CAX) antiporters, and in mammals the NCX and related proteins constitute families SLC8 and SLC24, and are responsible for the re-establishment of Ca2+ resting potential in muscle cells, neuronal signalling and Ca2+ reabsorption in the kidney1,6. The CAX family members maintain cytosolic Ca2+ homeostasis in plants and fungi during steep rises in intracellular Ca2+ due to environmental changes, or following signal transduction caused by events such as hyperosmotic shock, hormone response and response to mating pheromones7,8,9,10,11,12,13. The cytosol-facing conformations within the CaCA superfamily are unknown, and the transport mechanism remains speculative. Here we determine a crystal structure of the Saccharomyces cerevisiae vacuolar Ca2+/H+ exchanger (Vcx1) at 2.3 Å resolution in a cytosol-facing, substrate-bound conformation. Vcx1 is the first structure, to our knowledge, within the CAX family, and it describes the key cytosol-facing conformation of the CaCA superfamily, providing the structural basis for a novel alternating access mechanism by which the CaCA superfamily performs high-throughput Ca2+ transport across membranes.

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Figure 1: Topology and fold of the Vcx1 protein.
Figure 2: Calcium binding sites in the Vcx1 crystal structure.
Figure 3: The cytoplasmic vestibule.
Figure 4: Transport cycle of Vcx1 and structural comparison to mjNCX.

Accession codes

Accessions

Protein Data Bank

Data deposits

Coordinates and structure factors have been deposited in the Protein Data Bank with the accession number 4K1C.

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Acknowledgements

We thank J. Holton, G. Meigs, C. Ogata, N. Venugopalan and T. Doukov for assistance with synchrotron data collection at Advanced Light Source, Advanced Photon Source and Stanford Synchrotron Radiation Lightsource; and C. Waddling and P. Wassam for technical assistance. B.P.P. was supported by a postdoctoral fellowship from the Carlsberg Foundation and later by a fellowship from the Danish Cancer Society; A.S. by NIH grants U54 GM094625 and U01 GM61390; R.M.S. by NIH grants U54 GM094625, GM24485 and GM073210.

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Contributions

A.B.W. optimized the yeast expression system, performed expression, purification and crystallization experiments, collected and processed the data, determined, refined and analysed the structure, and performed reconstitution and transport assays. B.P.P. performed data collection and assisted with structure solution and structural analysis. B.H.C. and A.J.R. assisted in cell collection, membrane preparation and purification experiments. B.H.C. and Z.R.-Z. did cloning and expression tests. A.Sc. constructed Vcx1 comparative models as well as performed bioinformatics and distance plot analysis. M.B. performed molecular dynamics simulations and distance plot analysis. A.B.W., B.P.P. and R.M.S. wrote the paper with input from A.Sc., M.B. and A.Sa.

Corresponding author

Correspondence to Robert M. Stroud.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-9, Supplementary Table 1, Supplementary Methods and Supplementary References. (PDF 14435 kb)

The two-stroke mechanism of alternating access for the VCX1 protein

Interpolation between the VCX1 crystal structure (cytosol-facing) and the VCX1 comparative model based on the mjNCX structure (vacuole-facing) describe the proposed mechanism of alternating access. Translational movements in the M1/M6 'piston' are coordinated with M2a and M7a to cover and uncover the active site to alternating sides of the vacuolar membrane. The proton gradient across the vacuole provides energy which results in a conformational change to the cytosol-facing state. Under conditions of high cytosolic calcium concentrations, Ca2+ ions are coordinated by the acidic helix and Ca2+ can reach the active site. Glu106 coordination to the active site Ca2+ initiates M2a straightening and M1/M6 translation to expose the active site Ca2+ to the vacuole. The vacuole-facing conformation, in combination with acidic interior of the vacuole allows for lowered affinity and release of the bound Ca2+. (MP4 15399 kb)

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Waight, A., Pedersen, B., Schlessinger, A. et al. Structural basis for alternating access of a eukaryotic calcium/proton exchanger. Nature 499, 107–110 (2013). https://doi.org/10.1038/nature12233

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