Biophysical Society Conference | Estes Park 2023

Membrane Budding and Fusion

Poster Abstracts

5-POS Board 2 OPA1 HELICAL STRUCTURES SHED LIGHT ON THE MECHANISM OF MITOCHONDRIAL FUSION

Sarah B Nyenhuis 1 ; Xufeng Wu 2 ; Marie-Paule Strub 2 ; Yang-In Yim 2 ; Abigail E Stanton 1 ; Bertram Canagarajah 1 ; John A Hammer 2 ; Jenny E Hinshaw 1 ;

1 National Institutes of Health, NIDDK, Bethesda , MD, USA 2 National Institutes of Health, NHLBI, Bethesda, MD, USA

Dominant Optic Atrophy is the leading cause of childhood blindness, with 60-80% of cases caused by mutation of the gene encoding the protein Optic Atrophy 1, OPA1. These mutations putatively dysregulate GTPase-mediated mitochondrial inner-membrane (MIM) fusion, leading to mitochondrial network disruption and the DOA pathology. OPA1 is a dynamin superfamily GTPase which exists in two forms in vivo: the long-form which is tethered to the inner mitochondrial membrane through a single transmembrane domain, and the short-form that results from proteolytic cleavage in the inner membrane space as a response to mitochondrial membrane depolarization. A balance of these forms is thought to drive MIM fusion. The mechanics of this process, however, remain unknown due to a lack of OPA1 structural information. Using cryo electron microscopy, we solved helical structures of the short form of human OPA1 isoform-1 (s-OPA1) in the presence and absence of nucleotide, revealing both the domain organization of s-OPA1 and its mode of higher order assembly. These helical assemblies formed around a lipid core, with densely packed protein rungs marked by minimal inter-rung connectivity. Notably, we observed nucleotide-dependent dimerization of the GTPase domains, a hallmark of the mechano chemical, membrane-remodeling dynamin superfamily proteins. The OPA1 assemblies contained several unique secondary structures which strengthen membrane association, through monotopic membrane-inserting helices; pointing to the importance of OPA1 membrane interaction to its function during MIM fusion. These novel structural features and interfaces also shed light on the effects of pathogenic point mutations on protein folding, inter-protein assembly, and membrane binding. Further, the biological relevance of the OPA1 helical assembly and membrane binding was supported by single-cell based assays, where disruption of these regions in a balance of long- and short-OPA1 caused fragmentation of the mitochondrial network in a dominant-negative manner.

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