Structural analysis of Dnm1, a dynamin involved in mitochondrial fission

Dynamin蛋白家族由独特的GTP酶组成，参与整个细胞的膜分裂和融合事件。我们的目标是了解这些蛋白质的动态结构特性，并将它们与不同的细胞功能联系起来。动力蛋白对细胞内吞和囊泡活动是必不可少的。更多的动力蛋白家族成员参与了多种基本的细胞过程，包括线粒体的分裂和融合、抗病毒活性、细胞板的形成和叶绿体的生物发生。在这些蛋白质中，自组装和齐聚成有序结构是一个共同的特征，对大多数蛋白质来说，是其功能所必需的。虽然有大量关于动力素的信息，但对动力素相关蛋白质的结构性质知之甚少。 为了确定动力蛋白家族成员之间是否存在共同的作用机制，我们研究了DNM1的结构和功能，DNM1是参与线粒体分裂的酵母动力蛋白家族成员。我们已经证明DNM1可以组装成直径100 nm的大螺旋，而Dynamin螺旋的直径为50 nm。值得注意的是，DNM1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。在没有或存在核苷酸的情况下，DNM1也会组装到脂质体上，形成装饰精美的管子。此外，DNM1的GTP水解率与其自组装状态和浓度具有高度的协同性，这与Dynamin的动力学特性是一致的。这些结果表明，尽管Dynamin家族成员有共同的特征，但他们的结构特性是独特的，以适应他们的功能。 虽然DNM1可以在体外组装到脂质体上，但它们在细胞内的组装受到严格的调控。另外两种线粒体蛋白Fis1和Mdv1是必需的，它们与DNM1一起在线粒体分裂中发挥作用。我们已经证明，只有当DNM1组装成GTP结合的环状或螺旋结构时，Mdv1才与DNM1相互作用。GTP酶结合缺陷，不能自组装成螺旋，不再定位于Mdv1。这些发现表明，MDv1通过稳定或促进DNM1形成螺旋状结构而在裂变中发挥作用。MDv1可能通过稳定DNM1的GTP结合形式或作为核因子，促进DNM1在膜收缩的位置形成螺旋，从而实现这一点。 我们用IHRSR方法解决了DNM1与脂质结合的结构，发现其螺旋参数与Dynamin有明显的不同。在DNM1的3D图谱中，每一轮螺旋有24个重复亚基，重复亚基由一个四聚体组成(每轮96个DNM1分子)。此外，我们还发现，加入GTP后，DNM1-脂质管的直径从120 nm缩小到70 nm。整体结构显示了与底层脂质双层的松散关联，这支持了高度灵活的螺旋模型，该螺旋能够经历大的构象变化。

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.