Crystal structure of Ca ²⁺ /H ⁺ antiporter protein YfkE reveals the mechanisms of Ca ²⁺ efflux and its pH regulation

Abstract

CaCAs are integral membrane proteins containing 10-11 predicted transmembrane helices (TMs) (1) (SI Appendix, Fig. S1). Despite the divergence in cation driving force, the superfamily is defined by the presence of two highly conserved α-repeat motifs in TMs 2-3 (α-1) and TMs 7-8 (α-2). The importance of these conserved motifs for Ca 2+ /cation exchange has been well documented in all characterized CaCA proteins (8-10), implying the conservation of the Ca 2+ /cation translocation mechanism in the CaCA superfamily. Recently, Liao et al. reported the crystal structure of the NCX_Mj protein (6). The structure shows a large cavity opening on the periplasmic surface (outward-facing conformation) with both Ca 2+ and Na + bound at the α-repeat regions, suggesting the following hypotheses: (i) a substrate alternating access mechanism in which Ca 2+ and the counter transported cation access the ion-binding sites from each side of the membrane surface; and (ii) transition between outward-facing and inward-facing conformations is required to achieve Ca 2+ /cation exchange. Although the structure of the outward-facing conformation of NCX_Mj is available, structural evidence for the conformational change essential for alternating substrate access has not yet been obtained for any CaCA member. In addition, how the CaCA superfamily proteins modify their Ca 2+ -translocation pathways to use the different cation-gradient driving forces, H + vs. Na + , remains a fundamental and interesting question.To address these important questions, here, we report the crystal structure of an inward-facing conformation of the YfkE protein. This structure, together with protein kinetic analysis, not only provides an important structural characterization of the mechanism of Ca 2+ efflux across the cell membrane but also sheds light on the different modes of energy coupling used by members of the CaCA protein superfamily. Results Ca 2+-Transport Specificity and Structural Determination of YfkE. YfkE catalyzes a H+ -coupled Ca 2+ influx in everted vesicles (SI Appendix, Fig. S2A This article is a PNAS Direct Submission.Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 4KJR and 4KJS).

CAX‐ing a wide net: Cation/H+ transporters in metal remediation and abiotic stress signalling

“…2013; Wu et al . 2013). Such structural information allows a much more detailed insight into the structure–function relationships of CAX proteins and the mechanisms of Ca 2+ /H + exchange.…”

“…(2013) and Wu et al . (2013). The M2/M3 and M7/M8 helices (from At CAX 1) that derive the conserved α1‐ and α2‐repeat regions, respectively, together make up the cation binding pocket.…”

“…2013; Wu et al . 2013). CAXs from plants have previously been reported to form homomeric or indeed heteromeric oligomers, with potential implications for functional activity (Zhao et al .…”

Structure-Functional Basis of Ion Transport in Sodium–Calcium Exchanger (NCX) Proteins

“…TM1 and TM6 form the gating bundle, and the sliding of this two-helix cluster was suggested to be a major conformational change associated with the alternating-access during the transport cycle turnover [24]. This hypothesis is strongly supported by the X-ray structures of the H + /Ca 2+ (CAX) exchangers that were solved after NCX_Mj [32,33,34], although the dynamic aspects of underlying transitions remain unresolved. Interestingly, to date, all H + /Ca 2+ exchangers have crystallized in the inward-facing conformation.…”

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“…These repeats play vital role during ion-selectivity, binding and transportation by various groups of CaCA proteins (Kamiya and Maeshima, 2004; Ottolia et al, 2005; Shigaki et al, 2005; Nicoll et al, 2007). Recent crystallography analyses of certain CaCA proteins from archaea, bacteria and yeast provided detail insight into the structure (Liao et al, 2012; Nishizawa et al, 2013; Waight et al, 2013; Wu et al, 2013). The length of CaCA superfamily proteins varies from 300 to 1000 amino acid (AA) residues with almost similar topological structure.…”