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 made mitofusin 1 and 2 CRISPER knock-in cells. 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 two high-resolution structures of OPA1 bound to lipid in an apo and GTP transition state by cryoEM methods. The structures provide novel molecular details of how OPA1 assembles into a helical array on a lipid bilayer and the importance of OPA1 higher order assembles to mitochondrial morphology and dominant optic atrophy (DOA) pathologies. In collaboration with Drs. Xufeng Wu and Marie-Paule Strub (NHLBI), we examined DOA mutants that correlated to our higher order assembly interfaces in cells and in each case observed mitochondrial fragmentation. Recently we started exploring the structure neurolastin, a brain-specific dynamin-related protein associated with the mitochondria and also plays a role in synaptic transmission. In the evolutionary phylogenetic tree, neurolastin is most similar to atlastin, a dynamin-related protein involved in ER morphology. Previously, in collaboration with Dr. Tina Lee (Carnegie Melon U), we revealed atlastins ability to tether liposomes by cryoEM, a crucial step prior to membrane fusion (Crosby et al, 2021). In the future we plan to examine neurolastins ability to tether and fuse membranes in vitro and within cells by cryoEM and cryo-electron tomography methods.