Conformational changes in solution multimers of dynamin-related protein 1 (Drp1) facilitates functional assembly

Dynamin superfamily proteins (DSPs) are present in all organisms, mediating critical membrane remodeling events throughout the cell. Despite the decades of structural and functional studies across the superfamily, no structure of a DSP in its native state has been determined. This gap in knowledge limits the field's understanding of the mechanisms required for functional assembly into membrane remodeling complexes. Dynamin-related protein 1 (Drp1) is the master regulator of membrane fission for mitochondria. Proper mitochondrial dynamics is essential for cellular health, and an imbalance in this cycle has been implicated in many diseases, from heart failure to prion-related neurodegeneration. Drp1 has been implicated as a potential therapeutic target; however, the underlying mechanisms governing its regulation are largely unclear. Drp1 exists predominantly as a mixture of dimers and tetramers in solution, but the specific interactions that stabilize these solution forms of Drp1 and prevent the assembly of larger complexes are not known. Using cryoEM, we have observed significant conformational rearrangements in native solution structures for both dimer and tetramer states when compared to existing DSP crystal structures. These changes provide insight into regulatory interactions that stabilize oligomer states or mediate the activation of assembly into a functional fission machinery. In the dimer, we have observed a locking of the GTPase domain against the distal end of the stalk, which stabilizes an inactive state. Additionally, we have observed a continuous interface between adjacent assembly domains (stalks), indicating that the solution dimer must undergo significant rearrangement before forming a helical oligomer. We hypothesize that the mutations used for crystallographic studies stabilize an “open”, assembly-poised state due to changes in critical regulatory regions. Our solution dimer and tetramer structures represent inactive states stabilized by cross talk between the stalk and GTPase domain.