Abstract:
A host of dynamin superfamily proteins undertake diverse membrane-active processes in cells. Classical dynamins are paradigmatic membrane fission catalysts that release vesicles by acting on the neck of nascent endocytic buds during clathrin-mediated endocytosis (CME). Notably, among fission dynamins, endocytic dynamins have evolved a specialized membrane-binding pleckstrin-homology domain (PHD). PHD facilitates dynamin-mediated membrane fission catalytically, but the underlying mechanism is unclear. PHD-membrane interaction occurs via hydrophobic and basic residues enriched in the highly conserved unstructured ‘variable loops’ (VLs). Literature identifies dynamin PHD as a hotspot for pathological mutations associated with congenital neuropathy and myopathy. Molecular dynamics (MD) simulations and structural modeling recently reported a novel loop viz. VL4 as a preferred membrane anchor after the previously known VL1, whose relevance in dynamin function remains unexplored. To this end, we perform a suite of biochemical and cellular assays and mechanistically link in silico data to physiology. In minimal lipid-based recruitment assays, we find that mutations perturbing VL4 hydrophobic character manifest partial defects in membrane binding and fission. Fine real-time analysis establishes discrete and separable effects of VL1 and VL4 in dynamin-mediated membrane fission. Remarkably, more native-like reconstitution involving lipid and protein interactions reveals a complete loss in fission despite dynamin being effectively captured on the membrane, signifying an active contribution of the PHD in membrane fission. Importantly, expression of VL4 mutants in cells inhibit CME, consistent with the autosomal dominant phenotype associated with VL4-linked Charcot-Marie-Tooth neuropathy. These results bring out the basis of catalytic contribution of PHD in dynamin function and emphasize the significance of finely tuned lipid and protein interactions for efficient vesicle release by dynamin.
