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 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 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.