Carpenter Andrew P, Khuu Patricia, Weidner Tobias, Johnson Colin P, Roeters Steven J, Baio Joe E
The School of Chemical, Biological, and Environmental Engineering, Oregon State University, Corvallis, Oregon97331, United States.
Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon97331, United States.
J Phys Chem B. 2023 Jan 19;127(2):577-589. doi: 10.1021/acs.jpcb.2c06716. Epub 2023 Jan 6.
Dysferlin is a 230 kD protein that plays a critical function in the active resealing of micron-sized injuries to the muscle sarcolemma by recruiting vesicles to patch the injured site via vesicle fusion. Muscular dystrophy is observed in humans when mutations disrupt this repair process or dysferlin is absent. While lipid binding by dysferlin's C2A domain (dysC2A) is considered fundamental to the membrane resealing process, the molecular mechanism of this interaction is not fully understood. By applying nonlinear surface-specific vibrational spectroscopy, we have successfully demonstrated that dysferlin's N-terminal C2A domain (dysC2A) alters its binding orientation in response to a membrane's lipid composition. These experiments reveal that dysC2A utilizes a generic electrostatic binding interaction to bind to most anionic lipid surfaces, inserting its calcium binding loops into the lipid surface while orienting its β-sheets 30-40° from surface normal. However, at lipid surfaces, where PI(4,5)P2 is present, dysC2A tilts its β-sheets more than 60° from surface normal to expose a polybasic face, while it binds to the PI(4,5)P2 surface. Both lipid binding mechanisms are shown to occur alongside dysC2A-induced lipid clustering. These different binding mechanisms suggest that dysC2A could provide a molecular cue to the larger dysferlin protein as to signal whether it is bound to the sarcolemma or another lipid surface.
肌膜蛋白是一种230千道尔顿的蛋白质，它在通过募集囊泡以通过囊泡融合来修补损伤部位从而对微米级大小的肌肉肌膜损伤进行主动修复的过程中发挥关键作用。当突变破坏这一修复过程或肌膜蛋白缺失时，人类就会出现肌肉萎缩症。虽然肌膜蛋白的C2A结构域（dysC2A）与脂质的结合被认为是膜修复过程的基础，但其相互作用的分子机制尚未完全了解。通过应用非线性表面特异性振动光谱，我们成功证明了肌膜蛋白的N端C2A结构域（dysC2A）会根据膜的脂质组成改变其结合方向。这些实验表明，dysC2A利用一种通用的静电结合相互作用与大多数阴离子脂质表面结合，将其钙结合环插入脂质表面，同时使其β折叠与表面法线成30 - 40°角。然而，在存在磷脂酰肌醇 - 4,5 - 二磷酸（PI(4,5)P2）的脂质表面，dysC2A会使其β折叠与表面法线倾斜超过60°以暴露出一个多碱性表面，同时它与PI(4,5)P2表面结合。两种脂质结合机制均显示与dysC2A诱导的脂质聚集同时发生。这些不同的结合机制表明，dysC2A可能为更大的肌膜蛋白提供分子线索，以表明它是否与肌膜或另一种脂质表面结合。
