Molecular Determinants of Allosteric Regulation in NCX Proteins
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Allosteric activation of NCX involves the binding of cytosolic Ca2+ to regulatory domains CBD1 and CBD2. Previous studies with isolated CBD12 and full-size NCX identified synergistic interactions between the two CBD domains that modify the affinity and kinetic properties of Ca2+ sensing, although it remains unclear how the Ca2+-binding signal is decoded and propagates to transmembrane domains. Biophysical analyses (X-ray, SAXS, and stopped-flow techniques) of isolated preparations of CBD1, CBD2, and CBD12 have shown that Ca2+ binding to Ca3-Ca4 sites of CBD1 results in interdomain tethering of CBDs through specific amino acids on CBD1 (Asp499 and Asp500) and CBD2 (Arg532 and Asp565). Mutant analyses of isolated CBDs suggest that the two-domain interface governs Ca2+-driven conformational alignment of CBDs, resulting in slow dissociation of Ca2+ from CBD12, and thus, it mediates Ca2+-induced conformational transitions associated with allosteric signal transmission. Specifically, occupation of Ca3-Ca4 sites by Ca2+ induces disorder-to-order transition owing to charge neutralization and coordination, thereby constraining CBD conformational freedom, rigidifying the NCX1 f-loop, and triggering allosteric signal transmission to the membrane domain. The newly found interdomain switch is highly conserved among NCX isoform/splice variants, although some additional structural motifs may shape the regulatory specificity of NCX variants.
KeywordsNCX Allosteric regulation Interdomain Ca2+ switch Disorder-to-order transition
This work was partially funded by the Israeli Ministry of Health Grant # 2010-3-6266, the USA-Israeli Binational Research Grant # 2009-334, and the Israel Science Foundation Grant # 23/10. Financial support from the Bernstein Foundation is highly appreciated.
- G.M. Besserer, M. Ottolia, D.A. Nicoll, V. Chaptal, D. Cascio, K.D. Philipson, J. Abramson, The second Ca2+-binding domain of the Na+/Ca2+ exchanger is essential for regulation: crystal structures and mutational analysis. Proc. Natl. Acad. Sci. U. S. A. 104, 18467–18472 (2007)PubMedCrossRefGoogle Scholar
- A.E. Doering, W.J. Lederer, The action of Na+ as a cofactor in the inhibition by cytoplasmic protons of the cardiac Na+-Ca2+ exchanger in the guinea-pig. J. Physiol. (Lond.) 480, 9–20 (1994)Google Scholar
- D. Khananshvili, Structure, mechanism and regulation of the cardiac sarcolemma Na+-Ca2+ exchanger. Mol. Cell. Biol. 23B, 309–356 (1998)Google Scholar
- D. Khananshvili, The SLC8 gene family of sodium-calcium exchangers (NCX) – structure, function, and regulation in health and disease. Mol. Asp. Med. (2012) in pressGoogle Scholar