Structural analysis of dynamins involved in mitochondrial morphology

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

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 and human dynamins involved in mitochondria fusion (Opa1 & Mfn1). A balance between mitochondrial membrane fusion and fission is required for normal mitochondrial morphology and function. Mitofusins have been shown to mediate mitochondrial outer membrane fusion in mammalian cells while Opa1 has been shown to play a role in the fusion of the inner mitochondrial membrane. Both mammalian mitofusins contain a large cytosolic GTPase domain at their N-terminus followed by two transmembrane domains and a short C-terminal domain. Like other dynamin family members, it is predicted that GTP binding and hydrolysis drive mitofusin conformational changes that mediate membrane fusion. However, the precise mechanism for mitofusin-mediated membrane fusion remains unclear. It is still unknown if mitochondrial outer membrane fusion proceeds through the canonical steps of tethering, docking, fusion, and disassembly. Similar to the SNARE protein complex, we expect that mitofusins will interact with each other on opposing membranes, undergo a conformational change that drives the membranes close enough to overcome the activation energy barrier for fusion, and after fusion disassemble to be available for the next round of fusion. To gain insight into the conformational changes that lead to membrane fusion we are examining the structure of mitofusins in a lipid bilayer. As with other transmembrane proteins, mitofusins are difficult to express and purify in their full-length state and are poor candidates for crystallography. Nevertheless, we have optimized a purification and liposome-incorporation strategy for full-length mitofusin 1, which can be visualized for the first time by cryo-EM. These electron micrographs show networks of tightly tethered proteoliposomes with electron dense seams. This suggests that mitofusin can tether proteoliposomes by forming oligomers that interact in trans between synthetic membranes. In addition, we are collaborating with Drs. John Hammer and Xufeng Wu to visualize mitofusins in cells by fluorescent microscopy and in the future we plan to determine the effects of mitofusin assembly-mutants on mitochondrial fusion. Previously, in collaboration with Dr. David Chan from Cal Tech, we examined the structure of OPA1 by negative stain and cryo electron microscopy. Mutations in OPA1 (autosomal dominant optic atrophy) can lead to an inherited neuropathy of the retinal ganglion cells. In the cell, OPA1 has been shown to be essential for the fusion of the inner mitochondrial membranes, but its mechanism of action remains poorly understood. Addition of OPA1 to liposomes containing cardiolipin results in enhanced GTP hydrolysis rate and promotes OPA1 to self-assemble into helical arrays around the lipid, forming protein-lipid tubes. This past year we calculated a high-resolution structure of OPA1 bound to lipid in a GTP transition state by cryoEM methods. The structure provides molecular details of how OPA assembles into a helical array on a lipid bilayer.