Structural analysis of Dnm1, a dynamin involved in mitochondrial fission

Dnm1（一种参与线粒体裂变的动力）的结构分析

• 批准号: 7967689
• 负责人: Jenny E Hinshaw
• 金额: $ 50.54万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7967689
• 关键词: Binding; Biogenesis; Biological Process; Caliber; Cell physiology; Cells; Characteristics; Chloroplasts; Collaborations; Dynamin; Endocytosis; Event; Family member; GTP Binding; Goals; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Hydrolysis; In Vitro; Kinetics; Lipid Bilayers; Lipids; Liposomes; Maps; Membrane; Methods; Mitochondria; Mitochondrial Proteins; Modeling; Nucleotides; Property; Protein Family; Proteins; Site; Structure; Testing; Tube; Viral Physiology; Work; Yeasts; constriction; flexibility; mutant; self assembly

The dynamin family of proteins consists of unique GTPases involved in membrane fission and fusion events throughout the cell. Our goal is to understand the dynamic structural properties of these proteins and correlate them with their diverse cellular functions. Dynamin is essential for endocytosis and vesiculation events in the cell. Additional dynamin family members have been implicated in a variety of fundamental cellular processes, including mitochondrial fission and fusion, anti-viral activity, cell plate formation and chloroplast biogenesis. Among these proteins, self-assembly and oligomerization into ordered structures is a common characteristic and, for the majority, is essential for their function. Although there is a wealth of information regarding dynamin, little is known about the structural properties of dynamin-related proteins. To determine if a common mechanism of action exists among the dynamin family members, we examined the structure and function of Dnm1, a yeast dynamin family member involved in mitochondria fission. We have shown that Dnm1 assembles into large spirals, 100 nm in diameter compared to the 50 nm for dynamin spirals. Remarkably, the diameter of Dnm1 spirals is the same as that of mitochondrial constriction sites observed in cells. Dnm1 also assembles onto liposomes in the absence or presence of nucleotides, forming well-decorated tubes. In addition, the GTP hydrolysis rate of Dnm1 is highly cooperative with respect to its self-assembly state and concentration, which is consistent with the kinetic properties of dynamin. These results suggest that although dynamin family members share common characteristics, their structural properties are uniquely tailored to fit their function. Though Dnm1 can assemble onto liposomes in vitro, their assembly in cells is tightly regulated. Two additional mitochondrial proteins, Fis1 and Mdv1, are required and function together with Dnm1 in mitochondrial division. We have shown that Mdv1 interacts with Dnm1 only when Dnm1 is assembled into GTP-bound ring or spiral structures. GTPase mutants defective in binding GTP, which failed to self-assemble into spirals, no longer localized with Mdv1. These findings suggest Mdv1 functions in fission by stabilizing or promoting the formation of Dnm1 into spiral-like structures. Mdv1 may accomplish this by stabilizing the GTP bound form of Dnm1 or by acting as a nucleator, promoting Dnm1 to form spirals at sites of membrane constriction. We have solved the structure of Dnm1 bound to lipid using the IHRSR method and found the helical parameters significantly different than dynamin. In the 3D map of Dnm1 there are 24 repeating subunits per turn of the helix and the repeating subunit consists of a tetramer (96 Dnm1 molecules per turn). In addition, we have shown that upon GTP addition the Dnm1-lipid tubes constrict in diameter from 120 nm to 70 nm. The overall structure reveals a loose association with the underlying lipid bilayer, which supports the model of a highly flexible helix that is capable of undergoing a large conformational change.

发动蛋白家族的蛋白质由参与整个细胞的膜分裂和融合事件的独特GTP酶组成。我们的目标是了解这些蛋白质的动态结构特性，并将其与其不同的细胞功能相关联。发动蛋白是细胞内吞和囊泡形成所必需的。另外的动力蛋白家族成员涉及多种基本细胞过程，包括线粒体分裂和融合、抗病毒活性、细胞板形成和叶绿体生物发生。在这些蛋白质中，自组装和寡聚成有序结构是一个共同的特征，对于大多数蛋白质来说，这对它们的功能至关重要。虽然有丰富的信息关于发动蛋白，很少有人知道发动蛋白相关的蛋白质的结构特性。 为了确定发动蛋白家族成员之间是否存在共同的作用机制，我们研究了Dnm 1的结构和功能，Dnm 1是参与线粒体分裂的酵母发动蛋白家族成员。我们已经表明，Dnm 1组装成大的螺旋，直径为100 nm，而发动蛋白螺旋的直径为50 nm。值得注意的是，Dnm 1螺旋的直径与细胞中观察到的线粒体收缩部位的直径相同。Dnm 1也组装到脂质体在核苷酸的存在或不存在下，形成良好的装饰管。此外，GTP水解速率的Dnm 1是高度合作的自组装状态和浓度，这是一致的动力学特性。这些结果表明，虽然动力蛋白家族成员具有共同的特征，但它们的结构特性是独特的，以适应其功能。 虽然Dnm 1可以在体外组装到脂质体上，但它们在细胞中的组装受到严格调控。另外两个线粒体蛋白，Fis 1和Mdv 1，需要和功能与Dnm 1在线粒体分裂。我们已经表明，Mdv 1与Dnm 1只有当Dnm 1组装成GTP结合的环或螺旋结构。GTP结合缺陷突变体，未能自组装成螺旋，不再与Mdv 1本地化。这些发现表明Mdv 1通过稳定或促进Dnm 1形成螺旋状结构而在裂变中起作用。Mdv 1可以通过稳定GTP结合形式的Dnm 1或通过充当成核剂，促进Dnm 1在膜收缩部位形成螺旋来实现这一点。 我们已经解决了结构的Dnm 1结合到脂质使用IHRSR方法，并发现螺旋参数显着不同的发动蛋白。 在Dnm 1的3D图谱中，螺旋的每圈有24个重复亚基，重复亚基由四聚体组成（每圈96个Dnm 1分子）。 此外，我们已经表明，在GTP除了Dnm 1-脂质管收缩直径从120 nm到70 nm。 整体结构揭示了一个松散的协会与底层脂质双层，这支持了一个高度灵活的螺旋，能够经历一个大的构象变化的模型。