Search

Na + /Ca 2+ exchangers (NCXs) are secondary active transporters that couple the translocation of Na + with the transport of Ca 2+ in the opposite direction. The exchanger is an essential Ca 2+ extrusion mechanism in excitable cells. It consists of a transmembrane domain and a large intracellular loop that contains two Ca 2+ -binding domains, CBD1 and CBD2. The two CBDs are adjacent to each other and form a two-domain Ca 2+ sensor called CBD12. Binding of intracellular Ca 2+ to CBD12 activates the NCX but inhibits the NCX of Drosophila, CALX. NMR spectroscopy and SAXS studies showed that CALX and NCX CBD12 constructs display significant interdomain flexibility in the apo state but assume rigid interdomain arrangements in the Ca 2+ -bound state. However, detailed structure information on CBD12 in the apo state is missing. Structural characterization of proteins formed by two or more domains connected by flexible linkers is notoriously challenging and requires the combination of orthogonal information from multiple sources. As an attempt to characterize the conformational ensemble of CALX-CBD12 in the apo state, we applied molecular dynamics (MD) simulations, NMR ( 1 H- 15 N residual dipolar couplings), and small-angle x-ray scattering (SAXS) data in a combined strategy to select an ensemble of conformations in agreement with the experimental data. This joint approach demonstrated that CALX-CBD12 preferentially samples closed conformations, whereas the wide-open interdomain arrangement characteristic of the Ca 2+ -bound state is less frequently sampled. These results are consistent with the view that Ca 2+ binding shifts the CBD12 conformational ensemble toward extended conformers, which could be a key step in the NCXs' allosteric regulation mechanism. This strategy, combining MD with NMR and SAXS, provides a powerful approach to select ensembles of conformations that could be applied to other flexible multidomain systems., (Copyright © 2021 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The Na + /Ca 2+ exchanger of Drosophila melanogaster, CALX, is the main Ca 2+ -extrusion mechanism in olfactory sensory neurons and photoreceptor cells. Na + /Ca 2+ exchangers have two Ca 2+ sensor domains, CBD1 and CBD2. In contrast to the mammalian homologs, CALX is inhibited by Ca 2+ binding to CALX-CBD1, whereas CALX-CBD2 does not bind Ca 2+ at physiological concentrations. CALX-CBD1 consists of a β-sandwich and displays four Ca 2+ -binding sites at the tip of the domain. In this study, we used NMR spectroscopy and isothermal titration calorimetry (ITC) to investigate the cooperativity of Ca 2+ binding to CALX-CBD1. We observed that this domain binds Ca 2+ in the slow exchange regime at the NMR chemical shift timescale. Ca 2+ binding restricts the dynamics in the Ca 2+ -binding region. Experiments of 15 N chemical exchange saturation transfer and 15 N R 2 dispersion allowed the determination of Ca 2+ dissociation rates (∼30 s -1 ). NMR titration curves of residues in the Ca 2+ -binding region were sigmoidal because of the contribution of chemical exchange to transverse magnetization relaxation rates, R 2 . Hence, a novel, to our knowledge, approach to analyze NMR titration curves was proposed. Ca 2+ -binding cooperativity was examined assuming two different stoichiometric binding models and using a Bayesian approach for data analysis. Fittings of NMR and ITC binding curves to the Hill model yielded n Hill ∼2.9, near maximal cooperativity (n Hill  = 4). By assuming a stepwise model to interpret the ITC data, we found that the probability of binding from 2 up to 4 Ca 2+ is approximately three orders of magnitude higher than that of binding a single Ca 2+ . Hence, four Ca 2+ ions bind almost simultaneously to CALX-CBD1. Cooperative Ca 2+ binding is key to enable this exchanger to efficiently respond to changes in the intracellular Ca 2+ concentration in sensory neuronal cells., (Copyright © 2020 Biophysical Society. Published by Elsevier Inc. All rights reserved.)