Calmodulin (CaM) engages in Ca²⁺-dependent interactions with numerous proteins, including human Na⁺/H⁺-exchanger NHE1. Using nuclear magnetic resonance (NMR) spectroscopy, isothermal titration calorimetry, and fibroblasts expressing wildtype and mutant NHE1, we discovered multiple accessible states of this important complex existing in different NHE1:CaM stoichiometries and structures. We solved the NMR solution structure of a ternary complex in which CaM links two NHE1 cytosolic tails. In vitro, stoichiometries and affinities were tunable by variations in NHE1:CaM ratio and calcium ([Ca²⁺]) and by phosphorylation of S648 in the first CaM-binding α-helix. In cells, Ca²⁺-CaM-induced NHE1 activity was reduced by mimicking S648 phosphorylation or mutating the first CaM-binding helix, whereas Ca²⁺-induced NHE1 activity was unaffected by inhibition of Akt, one of several kinases phosphorylating S648. Our results reveal the diversity of NHE1:CaM interactions and suggest that CaM may contribute to NHE1 dimerization. We propose that similar structural diversity is relevant to other CaM complexes.

中文翻译：

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改变化学计量和结构的动态NHE1-钙调蛋白复合物调节Ca2 +依赖性NHE1活化

钙调蛋白（CaM）参与Ca ²⁺依赖性与众多蛋白质的相互作用，包括人类Na ⁺ / H ⁺交换子NHE1。使用核磁共振（NMR）光谱，等温滴定量热法和表达野生型和突变NHE1的成纤维细胞，我们发现了存在于不同NHE1：CaM化学计量和结构中的这一重要复合物的多个可及状态。我们解决了其中CaM连接两个NHE1胞质尾巴的三元复合物的NMR溶液结构。在体外，通过改变NHE1：CaM比和钙（[Ca ²⁺ ]）以及通过第一个CaM结合α-螺旋中S648的磷酸化，可调节化学计量和亲和力。在细胞中，Ca ²⁺-CaM诱导的NHE1活性通过模仿S648磷酸化或突变第一个与CaM结合的螺旋而降低，而Ca ²⁺诱导的NHE1活性不受Akt（几种磷酸化S648的激酶之一）的抑制作用所影响。我们的结果揭示了NHE1：CaM相互作用的多样性，并暗示CaM可能有助于NHE1二聚化。我们建议类似的结构多样性与其他CaM复杂。