CALX-CBD1 Ca-Binding Cooperativity Studied by NMR Spectroscopy and ITC with Bayesian Statistics.

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