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CALX-CBD1 Ca²⁺-Binding Cooperativity Studied by NMR Spectroscopy and ITC with Bayesian Statistics.

ABSTRACT: 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.

SUBMITTER: Cardoso MVC

PROVIDER: S-EPMC7376077 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Calcium ion complexation in aqueous solutions is of paramount importance in biology as it is related to cell signaling, muscle contraction, or biomineralization. However, Ca2+-complexes are dynamic soluble entities challenging to describe at the molecular level. Nuclear magnetic resonance appears as a method of choice to probe Ca2+-complexes. However, 43Ca NMR exhibits severe limitations arising from the low natural abundance coupled to the low gyromagnetic ratio and the quadrupolar nature of 43Ca, which overall make it a very unreceptive nucleus. Here, we show that 43Ca dynamic nuclear polarization (DNP) NMR of 43Ca-labeled frozen solutions is an efficient approach to enhance the NMR receptivity of 43Ca and to obtain structural insights about calcium ions complexed with representative ligands including water molecules, ethylenediaminetetraacetic acid (EDTA), and l-aspartic acid (l-Asp). In these conditions and in combination with numerical simulations and calculations, we show that 43Ca nuclei belonging to Ca2+ complexed to the investigated ligands exhibit rather low quadrupolar couplings (with CQ typically ranging from 0.6 to 1 MHz) due to high symmetrical environments and potential residual dynamics in vitrified solutions at a temperature of 100 K. As a consequence, when 1H→43Ca cross-polarization (CP) is used to observe 43Ca central transition, "high-power" νRF(43Ca) conditions, typically used to detect spin 1/2 nuclei, provide ∼120 times larger sensitivity than "low-power" conditions usually employed for detection of quadrupolar nuclei. These "high-power" CPMAS conditions allow two-dimensional (2D) 1H-43Ca HetCor spectra to be readily recorded, highlighting various Ca2+-ligand interactions in solution. This significant increase in 43Ca NMR sensitivity results from the combination of distinct advantages: (i) an efficient 1H-mediated polarization transfer from DNP, resembling the case of low-natural-abundance spin 1/2 nuclei, (ii) a reduced dynamics, allowing the use of CP as a sensitivity enhancement technique, and (iii) the presence of a relatively highly symmetrical Ca environment, which, combined to residual dynamics, leads to the averaging of the quadrupolar interaction and hence to efficient high-power CP conditions. Interestingly, these results indicate that the use of high-power CP conditions is an effective way of selecting symmetrical and/or dynamic 43Ca environments of calcium-containing frozen solution, capable of filtering out more rigid and/or anisotropic 43Ca sites characterized by larger quadrupolar constants. This approach could open the way to the atomic-level investigation of calcium environments in more complex, heterogeneous frozen solutions, such as those encountered at the early stages of calcium phosphate or calcium carbonate biomineralization events.

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CALX-CBD1 Ca²⁺-Binding Cooperativity Studied by NMR Spectroscopy and ITC with Bayesian Statistics.

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CALX-CBD1 Ca2+-Binding Cooperativity Studied by NMR Spectroscopy and ITC with Bayesian Statistics.

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OmicsDI is part of the ELIXIR infrastructure

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CALX-CBD1 Ca²⁺-Binding Cooperativity Studied by NMR Spectroscopy and ITC with Bayesian Statistics.