title: Crystal structures of progressive Ca2+ binding states of the Ca2+ sensor Ca2+ binding domain 1 (CBD1) from the CALX Na+/Ca2+ exchanger reveal incremental conformational transitions.
abstract: Na(+)/Ca(2+) exchangers (NCX) constitute a major Ca(2+) export system that facilitates the re-establishment of cytosolic Ca(2+) levels in many tissues. Ca(2+) interactions at its Ca(2+) binding domains (CBD1 and CBD2) are essential for the allosteric regulation of Na(+)/Ca(2+) exchange activity. The structure of the Ca(2+)-bound form of CBD1, the primary Ca(2+) sensor from canine NCX1, but not the Ca(2+)-free form, has been reported, although the molecular mechanism of Ca(2+) regulation remains unclear. Here, we report crystal structures for three distinct Ca(2+) binding states of CBD1 from CALX, a Na(+)/Ca(2+) exchanger found in Drosophila sensory neurons. The fully Ca(2+)-bound CALX-CBD1 structure shows that four Ca(2+) atoms bind at identical Ca(2+) binding sites as those found in NCX1 and that the partial Ca(2+) occupancy and apoform structures exhibit progressive conformational transitions, indicating incremental regulation of CALX exchange by successive Ca(2+) binding at CBD1. The structures also predict that the primary Ca(2+) pair plays the main role in triggering functional conformational changes. Confirming this prediction, mutagenesis of Glu(455), which coordinates the primary Ca(2+) pair, produces dramatic reductions of the regulatory Ca(2+) affinity for exchange current, whereas mutagenesis of Glu(520), which coordinates the secondary Ca(2+) pair, has much smaller effects. Furthermore, our structures indicate that Ca(2+) binding only enhances the stability of the Ca(2+) binding site of CBD1 near the hinge region while the overall structure of CBD1 remains largely unaffected, implying that the Ca(2+) regulatory function of CBD1, and possibly that for the entire NCX family, is mediated through domain interactions between CBD1 and the adjacent CBD2 at this hinge.
authors.affiliation: Center for Membrane Biology, Department of Biochemistry and Molecular Biology, University of Texas Medical School at Houston, Houston, Texas 77030, USA.
authors.forename: Mousheng
authors.initials: M
authors.lastname: Wu
authors.forename: Hoa Dinh
authors.initials: HD
authors.lastname: Le
authors.forename: Meitian
authors.initials: M
authors.lastname: Wang
authors.forename: Vladimir
authors.initials: V
authors.lastname: Yurkov
authors.forename: Alexander
authors.initials: A
authors.lastname: Omelchenko
authors.forename: Mark
authors.initials: M
authors.lastname: Hnatowich
authors.forename: Jay
authors.initials: J
authors.lastname: Nix
authors.forename: Larry V
authors.initials: LV
authors.lastname: Hryshko
authors.forename: Lei
authors.initials: L
authors.lastname: Zheng
mesh_terms: D000818:Animals; D017920:Antiporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D003461:Crystallography; D004330:Drosophila / Q000235:genetics / Q000502:physiology*; D029721:Drosophila Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D018408:Patch-Clamp Techniques; D054730:Protein Interaction Domains and Motifs / Q000502:physiology; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011984:Sensory Receptor Cells / Q000502:physiology; D012964:Sodium / Q000378:metabolism*
chemical_list: D017920:Antiporters; C108396:Calx protein, Drosophila; D029721:Drosophila Proteins; D008565:Membrane Proteins; D012964:Sodium; D002118:Calcium