Abstract
The self-assembling, mechanoenzymatic dynamin superfamily GTPase, dynamin-related protein 1 (Drp1), catalyzes mitochondrial and peroxisomal fission. Distinct intrinsically disordered regions (IDRs) in Drp1 substitute for the canonical pleckstrin homology (PH) domain and proline-rich domain (PRD) of prototypical dynamin, which cooperatively regulate endocytic vesicle scission. Whether the Drp1 IDRs function analogously to the corresponding dynamin domains however remains unknown. We show that an IDR unique to the Drp1 GTPase (G) domain, the ‘extended 80-loop’, albeit dissimilar in location, structure, and mechanism, functions akin to the dynamin PRD by enabling stable Drp1 mitochondrial recruitment and by suppressing Drp1 cooperative GTPase activity in the absence of specific partner-protein interactions. Correspondingly, we find that another IDR, the Drp1 variable domain (VD), in conjunction with the conserved stalk L1N loop, functions akin to the dynamin PH domain; first, in an ‘auto-inhibitory’ capacity that restricts Drp1 activity through a long-range steric inhibition of helical inter-rung G-domain dimerization, and second, as a ‘fulcrum’ for Drp1 self-assembly in the proper helical register. We show that the Drp1 VD is necessary and sufficient for specific Drp1-phospholipid interactions. We further demonstrate that the membrane-dependent VD conformational rearrangement essential for the alleviation of Drp1 auto-inhibition is contingent upon the basal GTP hydrolysis-dependent generation of Drp1 dimers from oligomers in solution. IDRs thus conformationally couple the enzymatic and membrane activities of Drp1 toward membrane fission.
Highlights
Dynamin superfamily proteins (DSPs) comprise a diverse collection of large, modular GTPases related by structure as well as by function[1,2,3]
Cooperative GTP hydrolysis in both dynamin and Drp[1] depends on nucleotide-dependent intermolecular G-domain dimerization, which increases with increasing protein concentration, and is facilitated conceivably by stalk-mediated higher-order self-assembly on target membranes[35,37,38]
Data shown are an average of three independent experiments ± SD. (E,F) size-exclusion chromatography (SEC)-multi-angle light scattering (MALS) profiles of dynamin-related protein 1 (Drp1)-short GG (D) and Drp1-long GG (E) (~40 kDa as monomers) each loaded at 25 μM onto a Superdex 75 10/300 GL column in the absence and presence of the transition-state analog, GDP.AlFx
Summary
Dynamin superfamily proteins (DSPs) comprise a diverse collection of large, modular GTPases related by structure as well as by function[1,2,3]. Grouped into ‘classical dynamins’ and ‘dynamin-related proteins’ (DRPs) based on distinctive variations in structure, it was until recently believed that the classical dynamins primarily function in the scission of small vesicles (≤100-nm diameter) from parent membranes, whereas the DRPs predominantly catalyze the fission (division) of significantly large organelles (>500-nm in diameter)[1,2]. How classical dynamins and DRPs, which are indispensable for numerous membrane fission events in the cell, remain functionally distinct during such disparate membrane remodeling processes is unclear. Recent data suggest that the maximally extended Drp1 80-loop participates in the targeting of Drp[1] to late endosomes, lysosomes, and the plasma membrane, indicating that this IDR may function as a recognition motif for specific binding partners located at the various membranes[20]
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