title: The second Ca2+-binding domain of the Na+ Ca2+ exchanger is essential for regulation: crystal structures and mutational analysis.
abstract: The Na(+)-Ca(2+) exchanger plays a central role in cardiac contractility by maintaining Ca(2+) homeostasis. Two Ca(2+)-binding domains, CBD1 and CBD2, located in a large intracellular loop, regulate activity of the exchanger. Ca(2+) binding to these regulatory domains activates the transport of Ca(2+) across the plasma membrane. Previously, we solved the structure of CBD1, revealing four Ca(2+) ions arranged in a tight planar cluster. Here, we present structures of CBD2 in the Ca(2+)-bound (1.7-A resolution) and -free (1.4-A resolution) conformations. Like CBD1, CBD2 has a classical Ig fold but coordinates only two Ca(2+) ions in primary and secondary Ca(2+) sites. In the absence of Ca(2+), Lys(585) stabilizes the structure by coordinating two acidic residues (Asp(552) and Glu(648)), one from each of the Ca(2+)-binding sites, and prevents a substantial protein unfolding. We have mutated all of the acidic residues that coordinate the Ca(2+) ions and have examined the effects of these mutations on regulation of exchange activity. Three mutations (E516L, D578V, and E648L) at the primary Ca(2+) site completely remove Ca(2+) regulation, placing the exchanger into a constitutively active state. These are the first data defining the role of CBD2 as a regulatory domain in the Na(+)-Ca(2+) exchanger.
authors.affiliation: Department of Physiology, Cardiovascular Research Laboratories, and U.S. Department of Energy Institute for Genomics and Proteomics, University of California, Los Angeles, CA 90095, USA.
authors.forename: Gabriel Mercado
authors.initials: GM
authors.lastname: Besserer
authors.forename: Michela
authors.initials: M
authors.lastname: Ottolia
authors.forename: Debora A
authors.initials: DA
authors.lastname: Nicoll
authors.forename: Vincent
authors.initials: V
authors.lastname: Chaptal
authors.forename: Duilio
authors.initials: D
authors.lastname: Cascio
authors.forename: Kenneth D
authors.initials: KD
authors.lastname: Philipson
authors.forename: Jeff
authors.initials: J
authors.lastname: Abramson
mesh_terms: D001665:Binding Sites; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism*; D002412:Cations / Q000737:chemistry; D018360:Crystallography, X-Ray; D008024:Ligands; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D017434:Protein Structure, Tertiary; D019831:Sodium-Calcium Exchanger / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D002412:Cations; D008024:Ligands; D019831:Sodium-Calcium Exchanger; D002118:Calcium