Calmodulin (CaM) serves as an important Ca²⁺ signaling hub that regulates many protein signaling pathways. Recently, it was demonstrated that plant CaM homologues can regulate mammalian targets, often in a manner that opposes the impact of the mammalian CaM (mCaM). However, the molecular basis of how CaM homologue mutations differentially impact target activation is unclear. To understand these mechanisms, we examined two CaM isoforms found in soybean plants that differentially regulate a mammalian target, calcineurin (CaN). These CaM isoforms, sCaM-1 and sCaM-4, share >90 and ∼78% identity with the mCaM, respectively, and activate CaN with comparable or reduced activity relative to mCaM. We used molecular dynamics (MD) simulations and fluorometric assays of CaN-dependent dephosphorylation of MUF-P to probe whether calcium and protein–protein binding interactions are altered by plant CaMs relative to mCaM as a basis for differential CaN regulation. In the presence of CaN, we found that the two sCaMs’ Ca²⁺ binding properties, such as their predicted coordination of Ca²⁺ and experimentally measured EC₅₀ [Ca²⁺] values are comparable to mCaM. Furthermore, the binding of CaM to the CaM binding region (CaMBR) in CaN is comparable among the three CaMs, as evidenced by MD-predicted binding energies and experimentally measured EC₅₀ [CaM] values. However, mCaM and sCaM-1 exhibited binding with a secondary region of CaN’s regulatory domain that is weakened for sCaM-4. We speculate that this secondary interaction affects the turnover rate (k_cat) of CaN based on our modeling of enzyme activity, which is consistent with our experimental data. Together, our data describe how plant-derived CaM variants alter CaN activity through enlisting interactions other than those directly influencing Ca²⁺ binding and canonical CaMBR binding, which may additionally play a role in the differential regulation of other mammalian targets.

中文翻译：

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钙调蛋白和钙调神经磷酸酶之间的非典型相互作用有助于植物源性钙调蛋白对钙调神经磷酸酶的差异调节

钙调素 (CaM) 是一种重要的 Ca ²⁺调节许多蛋白质信号通路的信号枢纽。最近，证明植物 CaM 同源物可以调节哺乳动物靶标，通常以一种反对哺乳动物 CaM (mCaM) 影响的方式。然而，CaM 同源物突变如何不同地影响靶激活的分子基础尚不清楚。为了了解这些机制，我们检查了在大豆植物中发现的两种 CaM 亚型，它们对哺乳动物靶标钙调神经磷酸酶 (CaN) 进行了不同的调节。这些 CaM 亚型 sCaM-1 和 sCaM-4 分别与 mCaM 共享 >90% 和 ∼78% 的同一性，并以与 mCaM 相当或降低的活性激活 CaN。我们使用 MUF-P 的 CaN 依赖性去磷酸化的分子动力学 (MD) 模拟和荧光测定来探测植物 CaM 相对于 mCaM 是否改变了钙和蛋白质-蛋白质结合相互作用作为差异 CaN 调节的基础。在存在 CaN 的情况下，我们发现两个 sCaMs 的 Ca²⁺结合特性，例如预测的Ca ²⁺配位和实验测量的EC ₅₀ [Ca ²⁺ ] 值与mCaM 相当。此外，CaM 与 CaN 中的 CaM 结合区 (CaMBR) 的结合在三种 CaM 中具有可比性，如 MD 预测的结合能和实验测量的 EC ₅₀ [CaM] 值所证明的那样。然而，mCaM 和 sCaM-1 表现出与 CaN 调节域的二级区域的结合，该二级区域对 sCaM-4 减弱。我们推测这种二次交互会影响周转率（k _cat）基于我们对酶活性的建模，这与我们的实验数据一致。总之，我们的数据描述了植物衍生的 CaM 变体如何通过招募相互作用来改变 CaN 活性，而不是直接影响 Ca ²⁺结合和经典 CaMBR 结合的相互作用，这可能在其他哺乳动物靶标的差异调节中发挥作用。