CALX-CBD1 Ca2+-binding cooperativity studied by NMR spectroscopy and ITC with Bayesian statistics

The Na+/Ca2+ exchanger of Drosophila melanogaster, CALX, is the main Ca2+-extrusion mechanism in olfactory sensory neurons and photoreceptor cells. Na+/Ca2+ exchangers have two Ca2+ sensor domains, CBD1 and CBD2. In contrast to the mammalian homologs, CALX is inhibited by Ca2+ binding to CALX-CBD1, whereas CALX-CBD2 does not bind Ca2+ at physiological concentrations. CALX-CBD1 consists of a β-sandwich and displays four Ca2+-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 Ca2+ binding to CALX-CBD1. We observed that this domain binds Ca2+ in the slow exchange regime at the NMR chemical shift timescale. Ca2+ binding restricts the dynamics in the Ca2+-binding region. Experiments of 15N chemical exchange saturation transfer and 15N R2 dispersion allowed the determination of Ca2+ dissociation rates (∼30 s-1). NMR titration curves of residues in the Ca2+-binding region were sigmoidal because of the contribution of chemical exchange to transverse magnetization relaxation rates, R2. Hence, a novel, to our knowledge, approach to analyze NMR titration curves was proposed. Ca2+-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 nHill ∼2.9, near maximal cooperativity (nHill = 4). By assuming a stepwise model to interpret the ITC data, we found that the probability of binding from 2 up to 4 Ca2+ is approximately three orders of magnitude higher than that of binding a single Ca2+. Hence, four Ca2+ ions bind almost simultaneously to CALX-CBD1. Cooperative Ca2+ binding is key to enable this exchanger to efficiently respond to changes in the intracellular Ca2+ concentration in sensory neuronal cells.