Mechanistic analysis of a novel membrane-interacting variable loop in the pleckstrin-homology domain critical for dynamin function

Abstract

Classical dynamins are best understood for their ability to generate vesicles by membrane fission. During clathrin-mediated endocytosis (CME), dynamin is recruited to the membrane through multivalent protein and lipid interactions between its Proline Rich Domain (PRD) with SRC Homology 3 (SH3) domains in endocytic proteins and its Pleckstrin Homology Domain (PHD) with membrane lipids. Variable loops (VL) in the PHD bind lipids and partially insert into the membrane thereby anchoring the PHD to the membrane. Recent molecular dynamics (MD) simulations reveal a novel VL4 that interacts with the membrane. Importantly, a missense mutation that reduces VL4 hydrophobicity is linked to an autosomal dominant form of Charcot Marie Tooth (CMT) neuropathy. To mechanistically link data from simulations with the CMT neuropathy, we analyzed functions of a VL4 mutant with reduced hydrophobicity. In assays that rely solely on lipid-based membrane recruitment, this mutant showed an acute membrane curvature-dependent binding and fission. Remarkably, in assays that mimic a physiological multivalent lipid- and protein-based recruitment, this mutant was completely defective in fission across a range of membrane curvatures. Importantly, expression of this mutant in cells inhibited CME, consistent with the autosomal dominant phenotype associated with the CMT neuropathy. Together, our results emphasize the significance of finely tuned lipid and protein interactions for efficient dynamin function.