Biophysical Society Conference | Estes Park 2023

Membrane Budding and Fusion

Poster Abstracts

3-POS Board 1 SNAPSHOTS OF DYNAMIN-MEDIATED FISSION BY CRYOEM

Nidhi Kundu 1 ; Jonathan T Harrison 1 ; Jenny E Hinshaw 1 ; 1 NIH, NIDDK, Bethesda, MD, USA

Dynamin drives the process of endocytosis by pinching off the invaginating vesicles from the plasma membrane via its GTPase activity. Dynamin mediates endocytosis in three steps; 1) binding and assembling as a helical polymer on the neck of invaginating vesicles, 2) constricting the membrane neck upon GTP binding and, 3) further constriction and fission upon GTP hydrolysis, which ultimately leads to release of vesicles. By a time-resolved cryoEM method, we showed that dynamin constricts the membranes within 150 milliseconds. Furthermore, by cryo-electron tomography, we provided evidence that dynamin is involved in early stages of endocytosis by wrapping around large diameter vesicles (200-300 nm). Dynamin is being targeted using many drugs like dyngo-4a, an inhibitor of dynamin GTPase activity, to block endocytosis. This is a potential target for antiviral, anti-cancer, and anti-epilepsy treatment as dynamin-mediated endocytosis facilitates entry of viruses, including HIV and SARS-CoV-2, and dysregulation of dynamin is associated with many cancers and epilepsy. While dynamin inhibitors have been identified, the structural basis and mechanism of inhibition of these dynamin inhibitors is unknown. Using Pull-down, GTPase assays, and mass spectrometry analysis, we determined that dyngo-4a binds to dynamin 1 and inhibits it’s functional GTPase activity. In order to determine the binding site and mechanism of action of dyngo-4a inhibition of dynamin, we are solving a high-resolution structure of dynamin 1 with dyngo-4a, using cryo electron microscopy. To improve resolution for dyngo-4a, we are optimizing conditions to improve homogeneity of the tubes and acquiring a larger data set. Our goal is to provide the first available structure of dynamin with its inhibitor dyngo-4a and identify the drug binding site, which potentially will open new avenue of designing drugs against dynamin for blocking endocytosis.

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