Structural Basis of the Ca2+ Inhibitory Mechanism of Drosophila Na+/Ca2+ Exchanger CALX and Its Modification by Alternative Splicing

Abstract

Summary The Na+/Ca2+ exchanger CALX promotes Ca2+ efflux in Drosophila sensory neuronal cells to facilitate light-mediated Ca2+ homeostasis. CALX activity is negatively regulated by specific Ca2+ interaction within its two intracellular Ca2+ regulatory domains CBD1 and CBD2, yet how the Ca2+ binding is converted to molecular motion to operate the exchanger is unknown. Here we report crystal structures of the entire Ca2+ regulatory domain CBD12 from two alternative splicing isoforms, CALX, 1.1 and 1.2, exhibiting distinct regulatory Ca2+-dependency. The structures show an open V-shaped conformation with four Ca2+ ions bound on the CBD domain interface, confirmed by LRET analysis. The structures together with Ca2+ binding analysis support that the Ca2+ inhibition of CALX is achieved by interdomain conformational changes induced by Ca2+ binding at CBD1. The conformational difference between the two isoforms also indicates that alternative splicing adjusts the interdomain orientation angle to modify the Ca2+ regulatory property of the exchangers.

Population Shift Underlies Ca2+-induced Regulatory Transitions in the Sodium-Calcium Exchanger (NCX)

“…NMR studies reported a similarly extended shape for both the apo and bound states of CBD12, whereas Ca 2ϩ binding resulted in rigidified motions of CBDs (24). Moreover, x-ray crystallography revealed a nearly identical interdomain angle in the Ca 2ϩ -bound CBD12-NCX1-AD (18) and CBD12-CALX1.1 (22), meaning that the phenotype differences in the Ca 2ϩ -dependent activation or inhibition, exhibited by these diverse orthologs, cannot be explained by interdomain angle or distance (18).…”

“…Ca 2ϩ binding is associated with CBD tethering and slow dissociation of "occluded" Ca 2ϩ from the Ca3-Ca4 sites (18). The structural properties of this interdomain module are very similar (if not identical) among diverse NCX orthologs and splice variants, exhibiting either positive, negative or no response to regulatory Ca 2ϩ (18,22). A slow dissociation of occluded Ca 2ϩ from regulatory sites may represent a physiologically relevant slow inactivation of NCX variants (I 2 state), observed in patch clamp experiments (12,23).…”

“…CALX1.1 is regulated negatively by Ca 2ϩ whereas CALX1.2 is not Ca 2ϩ regulated (22). We performed EOM analyses of the Ca 2ϩ -bound CBD12 variants of CALX to assess whether their conformations differ in solution (Fig.…”

“…Recent crystal structures of CBD12, derived from NCX (18) and CALX (22), reveal three regions, two hydrophilic and one hydrophobic, at the interface of CBDs. The amino acids contributing to the interface are highly conserved among NCX orthologs and splice variants (18).…”

“…This network is the principal linchpin holding CBDs together upon Ca 2ϩ binding to Ca3-Ca4 sites either in NCX (18) or CALX (22). Because CBD12-BD does not bind Ca 2ϩ at CBD2 (8) and the low affinity sites of CBD1 are altered in CBD12-AD-E454K (18,25), the population shift induced by Ca 2ϩ can be attributed to the high affinity Ca3-Ca4 sites of CBD1 (Figs.…”