Drp1 Structure: Regulatory Domains and Conformational Flexibility

项目摘要/摘要 线粒体在细胞中形成动态网络，通过分裂和融合的调节周期来满足能量需求。 从心力衰竭到癌症，这一周期的不平衡与一系列疾病有关。 动力蛋白相关蛋白1(Drp1)是核分裂的主要调节者，其活性通过多种途径控制。 途径，包括自组装调节，翻译后修饰，伙伴蛋白相互作用，以及 线粒体外膜接触部位富含不饱和心磷脂。这个项目试图理解 DRP1二聚体(功能单位)的结构，以及螺旋所需的构象重排 将这些二聚体组装在脂质模板上。我建议使用冷冻-EM单粒子方法来解决 WT二聚体和一个装配缺陷突变体的结构。这些结构将识别二聚体界面 溶液中的蛋白质和结构的区域，这些区域有助于承担广泛的 裂变所需的几何范围。而冷冻-EM将识别DRP1二聚体的不同状态，分子 动态模拟将用于阐明通过中间状态或过渡状态的构象运动 需要在线粒体膜上建立更大的复合体。这些在电子计算机研究中将确定构象 Drp1和其他Dynamin超家族蛋白的样本。这一结果将通过确定 赋予功能弹性的特定氨基酸。最后，这项提案试图解决Drp1螺旋结构 利用冷冻-EM对脂质纳米管进行了研究。脂酰链的饱和状态已被认为是一种调节 DRP1招募和自我组装的因素。饱和和不饱和脂肪模板上多聚体的形成 将确定脂膜扰动引起的螺旋结构和聚合物直径的变化。这个 为了形成螺旋组件，二聚体必须经历的构象变化将提供宝贵的洞察力 研究有助于线粒体膜分裂的Drp1的生物物理性质。

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.