Drp1 Structure: Regulatory Domains and Conformational Flexibility

项目总结/摘要 线粒体在细胞中形成动态网络，具有调节的裂变和融合周期以满足能量需求。 从心力衰竭到癌症，这个周期的不平衡与很多疾病都有关系。 动力蛋白相关蛋白1（Drp 1）是裂变的主要调节因子，其活性通过几个 途径，包括自组装调节，翻译后修饰，伴侣蛋白相互作用， 线粒体外膜接触部位富含不饱和心磷脂。本项目旨在了解 Drp 1二聚体（功能单元）的结构，以及螺旋结构所需的构象重排。 在脂质模板上组装这些二聚体。我建议使用冷冻EM单粒子方法来解决 WT二聚体和组装缺陷突变体的结构。这些结构将确定二聚体界面的 溶液中的蛋白质和结构的区域有助于采取宽的 裂变所需的几何形状范围。虽然冷冻EM将识别Drp 1二聚体的不同状态，但分子 动态模拟将用于阐明通过中间或过渡状态的构象运动 需要在线粒体膜上构建更大的复合体。这些计算机模拟研究将确定构象 Drp 1和其他发动蛋白超家族蛋白的取样。这些结果将通过识别 赋予功能弹性的特殊氨基酸。最后，该提议寻求解析Drp 1螺旋结构 在脂质纳米管上进行了实验。脂质酰基链的饱和状态已被确定为调节 Drp 1募集和自组装的因子。饱和和不饱和脂质模板上的多聚体形成 将识别由脂质膜扰动引起的螺旋结构和聚合物直径变化。的 二聚体为了形成螺旋组装体而必须经历的构象变化将提供宝贵的见解 Drp 1的生物物理特性有助于线粒体膜分裂。

PROJECT SUMMARY/ABSTRACT Mitochondria form dynamic networks in cells with a regulated cycle of fission and fusion to meet energy demands. An imbalance in the cycle has been implicated in a wide range of diseases—from heart failure to cancer. Dynamin-related protein 1 (Drp1) is the master regulator of fission, and its activity is controlled through several pathways, including self-assembly regulation, post-translational modifications, partner protein interactions, and mitochondria outer membrane contact sites enriched in unsaturated cardiolipin. This project seeks to understand the structure of the Drp1 dimer (the functional unit), and the conformational rearrangements required for helical assembly of these dimers on lipid templates. I propose to use cryo-EM single particle methods to resolve the structures of the WT dimer and an assembly defective mutant. These structures will identify dimer interfaces of the protein in solution and regions of the structure that contribute to the flexibility necessary to assume a wide range of geometries required for fission. While cryo-EM will identify distinct states of the Drp1 dimer, molecular dynamic simulations will be used to elucidate conformational motions through intermediate, or transition, states needed to build larger complexes on mitochondrial membranes. These in silico studies will identify conformation sampling of Drp1 and other dynamin superfamily proteins. The results will inform future studies by identifying specific amino acids that confer functional elasticity. Finally, this proposal seeks to resolve Drp1 helical structures on lipid nanotubes using cryo-EM. The saturation state of lipid acyl chains has been identified as a regulatory factor for Drp1 recruitment and self-assembly. Multimer formation on saturated and unsaturated lipid templates will identify helical structure and polymer diameter changes resulting from lipid membrane perturbations. The conformational changes the dimer must undergo in order to form helical assemblies will provide invaluable insight into the biophysical properties of Drp1 that contribute to mitochondrial membrane fission.