Na+/Ca2+ exchanger (NCX) proteins mediate Ca2+-fluxes across the cell membrane to maintain Ca2+ homeostasis in many cell types. Eukaryotic NCX contains Ca2+-binding regulatory domains, CBD1 and CBD2. Ca2+ binding to a primary sensor (Ca3-Ca4 sites) on CBD1 activates mammalian NCXs, whereas CALX, a Drosophila NCX ortholog, displays an inhibitory response to regulatory Ca2+. To further elucidate the underlying regulatory mechanisms, we determined the 2.7 Å crystal structure of mammalian CBD12-E454K, a two-domain construct that retains wild-type properties. In conjunction with stopped-flow kinetics and SAXS (small-angle X-ray scattering) analyses of CBD12 mutants, we show that Ca2+ binding to Ca3-Ca4 sites tethers the domains via a network of interdomain salt-bridges. This Ca2+-driven interdomain switch controls slow dissociation of “occluded” Ca2+ from the primary sensor and thus dictates Ca2+ sensing dynamics. In the Ca2+-bound conformation, the interdomain angle of CBD12 is very similar in NCX and CALX, meaning that the interdomain distances cannot account for regulatory diversity in NCX and CALX. Since the two-domain interface is nearly identical among eukaryotic NCXs, including CALX, we suggest that the Ca2+-driven interdomain switch described here represents a general mechanism for initial conduction of regulatory signals in NCX variants.

No abstract

No abstract

Gem, a member of the Rad,Gem/Kir subfamily of small G-proteins, has unique sequence features. We report here the crystallographic structure determination of the Gem G-domain in complex with nucleotide to 2.4 Å resolution. Although the basic Ras protein fold is maintained, the Gem switch regions emphatically differ from the Ras paradigm. Our ensuing biochemical characterization indicates that Gem G-domain markedly prefers GDP over GTP. Two known functions of Gem are distinctly affected by spatially separated clusters of mutations.

Background: Calmodulin and calcium-binding protein 1 (CaBP1) oppositely regulate inactivation of Ca V 1.2 channels. Results: Quantitative titration of purified proteins in intact cells suggests competition between CaM and CaBP1 for the Ca V 1.2 C terminus and an additional non-competitive action of CaBP1. Conclusion: CaBP1 counteracts CaM actions by a dual mechanism. Significance: Our approach provides new insights into mechanisms of Ca 2ϩ channel inactivation.