Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon.
Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon.
Biophys J. 2014 Jan 21;106(2):382-9. doi: 10.1016/j.bpj.2013.11.4492.
Dysferlin is a large membrane protein involved in calcium-triggered resealing of the sarcolemma after injury. Although it is generally accepted that dysferlin is Ca(2+) sensitive, the Ca(2+) binding properties of dysferlin have not been characterized. In this study, we report an analysis of the Ca(2+) and membrane binding properties of all seven C2 domains of dysferlin as well as a multi-C2 domain construct. Isothermal titration calorimetry measurements indicate that all seven dysferlin C2 domains interact with Ca(2+) with a wide range of binding affinities. The C2A and C2C domains were determined to be the most sensitive, with Kd values in the tens of micromolar, whereas the C2D domain was least sensitive, with a near millimolar Kd value. Mutagenesis of C2A demonstrates the requirement for negatively charged residues in the loop regions for divalent ion binding. Furthermore, dysferlin displayed significantly lower binding affinity for the divalent cations magnesium and strontium. Measurement of a multidomain construct indicates that the solution binding affinity does not change when C2 domains are linked. Finally, sedimentation assays suggest all seven C2 domains bind lipid membranes, and that Ca(2+) enhances but is not required for interaction. This report reveals for the first time, to our knowledge, that all dysferlin domains bind Ca(2+) albeit with varying affinity and stoichiometry.
肌营养不良蛋白是一种大型膜蛋白,参与损伤后肌细胞膜的钙触发重封。尽管普遍认为肌营养不良蛋白对 Ca(2+)敏感,但肌营养不良蛋白的 Ca(2+)结合特性尚未得到表征。在这项研究中,我们报告了对肌营养不良蛋白所有七个 C2 结构域以及多 C2 结构域构建体的 Ca(2+)和膜结合特性的分析。等温滴定量热法测量表明,所有七个肌营养不良蛋白 C2 结构域都与 Ca(2+)相互作用,具有广泛的结合亲和力。C2A 和 C2C 结构域被确定为最敏感的结构域,其 Kd 值在数十微摩尔范围内,而 C2D 结构域则最不敏感,其 Kd 值接近毫摩尔。C2A 的突变表明,环区的负电荷残基对于二价离子结合是必需的。此外,肌营养不良蛋白对二价阳离子镁和锶的结合亲和力明显降低。对多结构域构建体的测量表明,当 C2 结构域连接时,溶液结合亲和力不会改变。最后,沉淀测定表明,所有七个 C2 结构域都与脂质膜结合,并且 Ca(2+)增强但不是相互作用所必需的。本报告首次揭示,据我们所知,所有肌营养不良蛋白结构域都结合 Ca(2+),尽管亲和力和化学计量比不同。