CALX-CBD1 Ca2+-Binding Cooperativity Studied by NMR Spectroscopy and ITC with Bayesian Statistics
CALX-CBD1 Ca2+-Binding Cooperativity Studied by NMR Spectroscopy and ITC with Bayesian Statistics
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DOI：
10.1016/j.bpj.2020.05.031
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发表时间：
2020-07-21
影响因子：
3.4
通讯作者：
Salinas, Roberto K.
中科院分区：
文献类型：
--
作者：
Cardoso, Marcus V. C.;Rivera, Jose D.;Salinas, Roberto K.
果蝇的 Na+/Ca2+ 交换器 CALX 是嗅觉感觉神经元和感光细胞中主要的 Ca2+ 排出机制。 Na+/Ca2+ 交换器有两个 Ca2+ 传感器域：CBD1 和 CBD2。与哺乳动物同源物相反，CALX 受到 Ca2+ 与 CALX-CBD1 结合的抑制，而 CALX-CBD2 在生理浓度下不结合 Ca2+。 CALX-CBD1 由 β 三明治组成，并在结构域顶端显示四个 Ca2+ 结合位点。在本研究中，我们使用核磁共振波谱和等温滴定量热法 (ITC) 来研究 Ca2+ 与 CALX-CBD1 结合的协同性。我们观察到该结构域在 NMR 化学位移时间尺度上以缓慢交换方式结合 Ca2+。 Ca2+ 结合限制了 Ca2+ 结合区域的动力学。 N-15 化学交换饱和转移和 N-15 R(2 ) 分散实验允许确定 Ca2+ 解离速率（类似于 30 s(-1)）。由于化学交换对横向磁化弛豫率 R-2 的贡献，Ca2+ 结合区域中残基的 NMR 滴定曲线呈 S 形。因此，据我们所知，提出了一种分析 NMR 滴定曲线的新颖方法。假设两种不同的化学计量结合模型并使用贝叶斯方法进行数据分析，检查了 Ca2+ 结合协同性。 NMR 和 ITC 结合曲线与 Hill 模型的拟合产生类似于 2.9 的 n(Hill )，接近最大协同性 (n(Hill) = 4）。通过假设逐步模型来解释 ITC 数据，我们发现结合 2 到 4 个 Ca2+ 的概率比结合单个 Ca2+ 的概率高出大约三个数量级。因此，四个 Ca2+ 离子几乎同时与 CALX-CBD1 结合。协同 Ca2+ 结合是使该交换器能够有效响应感觉神经元细胞内 Ca2+ 浓度变化的关键。
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 beta-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 N-15 chemical exchange saturation transfer and N-15 R(2 )dispersion allowed the determination of Ca2+ dissociation rates (similar to 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, R-2. 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 )similar to 2.9, near maximal cooperativity (n(Hill) = 4). By assuming a step-wise 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.