Dysferlin is a large membrane protein found most prominently in striated muscle. Loss of dysferlin activity is associated with reduced exocytosis, abnormal intracellular Ca2+ and the muscle diseases limb-girdle muscular dystrophy and Miyoshi myopathy. The cytosolic region of dysferlin consists of seven C2 domains with mutations in the C2A domain at the N-terminus resulting in pathology. Despite the importance of Ca2+ and membrane binding activities of the C2A domain for dysferlin function, the mechanism of the domain remains poorly characterized. In this study we find that the C2A domain preferentially binds membranes containing PI(4,5)P2 through an interaction mediated by residues Y23, K32, K33, and R77 on the concave face of the domain. We also found that subsequent to membrane binding, the C2A domain inserts residues on the Ca2+ binding loops into the membrane. Analysis of solution NMR measurements indicate that the domain inhabits two distinct structural states, with Ca2+ shifting the population between states towards a more rigid structure with greater affinity for PI(4,5)P2. Based on our results, we propose a mechanism where Ca²⁺ converts C2A from a structurally dynamic, low PI(4,5)P2 affinity state to a high affinity state that targets dysferlin to PI(4,5)P2 enriched membranes through interaction with Tyr23, K32, K33, and R77. Binding also involves changes in lipid packing and insertion by the third Ca2+ binding loop of the C2 domain into the membrane, which would contribute to dysferlin function in exocytosis and Ca2+ regulation.

Dysferlin 是一种大型膜蛋白，主要存在于横纹肌中。 Dysferlin 活性的丧失与胞吐作用减少、细胞内 Ca2+ 异常以及肌肉疾病肢带型肌营养不良症和三好肌病有关。 Dysferlin 的胞质区域由 7 个 C2 结构域组成，N 末端 C2A 结构域发生突变，导致病理。尽管 Ca2+ 和 C2A 结构域的膜结合活性对于 Dysferlin 功能非常重要，但该结构域的机制仍知之甚少。在本研究中，我们发现 C2A 结构域通过结构域凹面上残基 Y23、K32、K33 和 R77 介导的相互作用优先结合含有 PI(4,5)P2 的膜。我们还发现，膜结合后，C2A 结构域将 Ca2+ 结合环上的残基插入膜中。溶液 NMR 测量分析表明，该结构域处于两种不同的结构状态，Ca2+ 将状态之间的群体转变为更刚性的结构，对 PI(4,5)P2 具有更大的亲和力。根据我们的结果，我们提出了一种机制，其中 Ca ²⁺将 C2A 从结构动态的低 PI(4,5)P2 亲和力状态转化为高亲和力状态，通过相互作用将 Dysferlin 靶向富含 PI(4,5)P2 的膜与 Tyr23、K32、K33 和 R77 一起。结合还涉及脂质堆积的变化以及 C2 结构域的第三个 Ca2+ 结合环插入膜的变化，这将有助于 Dysferlin 在胞吐作用和 Ca2+ 调节中的功能。