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. 2013 Jul 4;499(7456):107-10.
doi: 10.1038/nature12233. Epub 2013 May 19.

Structural Basis for Alternating Access of a Eukaryotic Calcium/Proton Exchanger

Free PMC article

Structural Basis for Alternating Access of a Eukaryotic Calcium/Proton Exchanger

Andrew B Waight et al. Nature. .
Free PMC article


Eukaryotic Ca(2+) regulation involves sequestration into intracellular organelles, and expeditious Ca(2+) release into the cytosol is a hallmark of key signalling transduction pathways. Bulk removal of Ca(2+) after such signalling events is accomplished by members of the Ca(2+):cation (CaCA) superfamily. The CaCA superfamily includes the Na(+)/Ca(2+) (NCX) and Ca(2+)/H(+) (CAX) antiporters, and in mammals the NCX and related proteins constitute families SLC8 and SLC24, and are responsible for the re-establishment of Ca(2+) resting potential in muscle cells, neuronal signalling and Ca(2+) reabsorption in the kidney. The CAX family members maintain cytosolic Ca(2+) homeostasis in plants and fungi during steep rises in intracellular Ca(2+) due to environmental changes, or following signal transduction caused by events such as hyperosmotic shock, hormone response and response to mating pheromones. 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 Ca(2+)/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 Ca(2+) transport across membranes.


Figure 1
Figure 1. Topology and fold of the VCX1 protein
The symmetrically related halves of the VCX1 monomer are colored in a double rainbow from the N to C terminus. Helices of matching color are related by symmetry. a, The VCX1 monomer as viewed in the membrane along the axis of symmetry, b, rotated by 90° and c, viewed from the vacuolar side of the membrane. d, Topology map of the VCX1 monomer; CAX family conserved residues are colored in red, α-repeat sequences are denoted by dashed circles.
Figure 2
Figure 2. Calcium binding sites in the VCX1 crystal structure
a, Overview of Site 1 and Site 2 with helices MR, M1, and M6 removed for clarity. The cytosol is on the bottom of the image and Ca2+ ions are colored in yellow. b, Active site Ca2+ substrate ion and interacting residues found in Site 1. Hydrogen bonds are shown as dashed lines; numbers denote atomic distances (Å). 2mFo-DFc map is shown contoured at 1σ (blue mesh) c, Ca2+ ions at the Acidic Helix, in Site 2 with interacting residues labeled. Hydrogen bonds are shown as dashed lines; numbers denote atomic distances (Å). 2mFo-DFc map is shown contoured at 1σ (blue mesh)
Figure 3
Figure 3. The cytoplasmic vestibule
a, The protein cavity is rendered as a surface and colored in grey, the helices M1 and M2 are colored in purple and shown from the axis of symmetry. b, View rotated by 90°. c, The cytoplasmic vestibule as oriented in panel a and depicted with a slab surface representation colored by electrostatic potential (red to blue; -10 to 10kT/e). Helices MR, M1 and M6 have been removed for clarity. Ca2+ ions (yellow spheres) pinpoint Site 1 and Site 2.
Figure 4
Figure 4. Transport cycle of VCX1 and structural comparison to mjNCX
a, Comparison of M2 and M7 and active site glutamate residues between VCX1 (purple) and mjNCX (cyan). Ca2+ ions from each model are depicted as spheres. b, Comparison of M1 and M6 between VCX1 (purple) and mjNCX (cyan). c, Schematic of VCX1 turnover. Structures are colored as in panel a. Proposed substrate movement is denoted by black arrows, and calcium by yellow circles. Red arrows show protein movement in the cytosol-facing facing state of VCX1 (left) that results in the vacuole-facing conformation on the right. Return to the cytosol-facing state presumably requires reversal of the movements denoted by the red arrows.

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