Structural Analysis and Stochastic Modelling Suggest a Mechanism for Calmodulin Trapping by CaMKII

Activation of CaMKII by calmodulin and the subsequent maintenance of constitutive activity through autophosphorylation at threonine residue 286 (Thr286) are thought to play a major role in synaptic plasticity. One of the effects of autophosphorylation at Thr286 is to increase the apparent affinity of CaMKII for calmodulin, a phenomenon known as “calmodulin trapping”. It has previously been suggested that two binding sites for calmodulin exist on CaMKII, with high and low affinities, respectively. We built structural models of calmodulin bound to both of these sites. Molecular dynamics simulation showed that while binding of calmodulin to the supposed low-affinity binding site on CaMKII is compatible with closing (and hence, inactivation) of the kinase, and could even favour it, binding to the high-affinity site is not. Stochastic simulations of a biochemical model showed that the existence of two such binding sites, one of them accessible only in the active, open conformation, would be sufficient to explain calmodulin trapping by CaMKII. We can explain the effect of CaMKII autophosphorylation at Thr286 on calmodulin trapping: It stabilises the active state and therefore makes the high-affinity binding site accessible. Crucially, a model with only one binding site where calmodulin binding and CaMKII inactivation are strictly mutually exclusive cannot reproduce calmodulin trapping. One of the predictions of our study is that calmodulin binding in itself is not sufficient for CaMKII activation, although high-affinity binding of calmodulin is.钙调蛋白（calmodulin）对钙/钙调蛋白依赖性蛋白激酶II（CaMKII）的激活，以及后续通过苏氨酸残基286（Thr286）位点自磷酸化维持组成型活性的过程，被认为在突触可塑性中发挥关键作用。Thr286位点自磷酸化的效应之一，是增强CaMKII对钙调蛋白的表观亲和力，这一现象被称为“钙调蛋白捕获”。此前已有研究提出，CaMKII上存在两个钙调蛋白结合位点，分别具备高、低两种亲和力。我们构建了分别结合这两个位点的钙调蛋白结构模型。分子动力学模拟结果显示，尽管钙调蛋白结合至CaMKII上推测的低亲和力结合位点时，激酶处于关闭（即失活）状态是可行的，甚至可能促进该状态，但结合至高亲和力位点时则并非如此。基于生化模型的随机模拟表明，若存在两个此类结合位点，其中仅在活性开放构象下才可被结合，便足以解释CaMKII介导的钙调蛋白捕获现象。我们可阐明CaMKII在Thr286位点的自磷酸化对钙调蛋白捕获的调控机制：该过程会稳定激酶的活性构象，进而使高亲和力结合位点得以暴露。尤为关键的是，仅包含单个结合位点且钙调蛋白结合与CaMKII失活严格互斥的模型，无法复现钙调蛋白捕获现象。本研究的一项预测是，钙调蛋白结合本身并不足以激活CaMKII，尽管高亲和力的钙调蛋白结合足以激活该酶。