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+/Ca2+ exchangers (NCX) constitute a major Ca2+ export system that facilitates the re-establishment of cytosolic Ca2+ levels in many tissues. Ca2+ interactions at its Ca2+ binding domains (CBD1 and CBD2) are essential for the allosteric regulation of Na+/Ca2+ exchange activity. The structure of the Ca2+-bound form of CBD1, the primary Ca2+ sensor from canine NCX1, but not the Ca2+-free form, has been reported, although the molecular mechanism of Ca2+ regulation remains unclear. Here, we report crystal structures for three distinct Ca2+ binding states of CBD1 from CALX, a Na+/Ca2+ exchanger found in Drosophila sensory neurons. The fully Ca2+-bound CALX-CBD1 structure shows that four Ca2+ atoms bind at identical Ca2+ binding sites as those found in NCX1 and that the partial Ca2+ occupancy and apoform structures exhibit progressive conformational transitions, indicating incremental regulation of CALX exchange by successive Ca2+ binding at CBD1. The structures also predict that the primary Ca2+ pair plays the main role in triggering functional conformational changes. Confirming this prediction, mutagenesis of Glu455, which coordinates the primary Ca2+ pair, produces dramatic reductions of the regulatory Ca2+ affinity for exchange current, whereas mutagenesis of Glu520, which coordinates the secondary Ca2+ pair, has much smaller effects. Furthermore, our structures indicate that Ca2+ binding only enhances the stability of the Ca2+ binding site of CBD1 near the hinge region while the overall structure of CBD1 remains largely unaffected, implying that the Ca2+ 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.