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. In 2023 we solved the structure of s-OPA1 by cryo-EM single particle analysis, (Nyenhuis et al, Nature 2023). Mitochondria are dynamic organelles that continuously undergo fission and fusion events, which are essential to maintain a healthy cell. OPA1 is directly involved in the fusion of the inner mitochondrial membrane as well as cristae remodeling. Mutations in OPA1 lead to Dominant Optic Atrophy, one of the leading causes of childhood blindness. OPA1 exists in two forms in the mitochondria, a long form (l-OPA1) that is tethered to the inner mitochondrial membrane through a transmembrane domain, and a short soluble form (s-OPA1) that is a proteolytic product of l-OPA1. We solved high-resolution structures of assembled s-OPA1 bound to lipid, in two states, apo and GDP-AlFx. Our cryo-EM maps reveal densely packed helical assemblies that exhibit nucleotide-dependent dimerization of the GTPase domains, a hallmark of DSPs that leads to stimulated GTPase activity. In addition, unlike other DSPs, OPA1 has a unique membrane-inserting region called the paddle domain that inserts into the membrane, likely enhancing membrane destabilization prior to fusion. And unlike the DSPs involved in fission, OPA1 does not constrict the underlying lipid bilayer upon GTP hydrolysis. Furthermore, OPA1 mutations that disrupt the assembly interfaces and membrane binding resulted in mitochondrial fragmentation in cell-based assays, providing evidence of the biological relevance of these interactions. In addition, we show for assembly and membrane inserting mutants, the cristae appear normal, limiting the mutant effects to inner mitochondrial fusion.