Biophysical Society Thematic Meeting| Padova 2019

Quantitative Aspects of Membrane Fusion and Fission

Poster Abstracts

3-POS Board 3 SNARE PRIMING INHIBITION VIA PHOSPHATIDIC ACID INDUCED SEC18 CONFORMATIONAL CHANGE Andres S. Arango 1,2 ; Robert P Sparks 3 ; Matthew L Starr 3 ; Zhiyu Zhao 1,2 ; Muyun Lihan 1,2 ; Rutilio Fratti 1,3 ; Emad Tajkhorshid 2,4 ; 1 University of Illinois at Urbana-Champaign, Center for Biophysics and Quantitative Biology, Champaign, Illinois, USA 2 University of Illinois at Urbana-Champaign, Beckman Institute, NIH Center for Macromolecular Modeling and Bioinformatics, Champaign, Illinois, USA 3 University of Illinois at Urbana-Champaign, Department of Biochemistry, Champaign, Illinois, USA 4 University of Illinois at Urbana-Champaign, Department of Biochemistry, Center for Biophysics and Quantitative Biology, Champaign, Illinois, USA Membrane fusion is mediated by SNARE proteins, and is vital for a multitude of cellular transport phenomena. Post fusion, cis-SNAREs are recycled for continued fusion, in an ATP dependent process called priming. The unique job of priming all cellular SNAREs is the responsibility of AAA+ ATPase NSF or Sec18. In a joint computational and experimental effort, we show that Phosphatidic Acid (PA) inhibits the priming of cis-SNARE complexes by inducing conformational changes in Sec18 protomer. We further studied priming inhibition via a new small molecule inhibitor of Sec18 called IPA, which inhibits fusion, priming, and competitively inhibits Sec18 binding to PA. We identify potential PA binding sites to Sec18 using computational flooding of short tailed PA, as well as using an HMMM PA rich membrane to capture unbiased spontaneous membrane insertion of Sec18 monomers. In addition, to further sample potential binding sites of both PA as well as IPA on Sec18 we employed ensemble molecular docking using AutoDock Vina, with consecutive molecular dynamics simulations performed for the top poses using NAMD for characterization of ligand stability. Finally, we performed Random Accelerated Molecular dynamics simulations on the most stable resultant poses to estimate dissociation constants, which were compared to results from liposome binding experiments providing insight into the mechanism of priming regulation via Sec18. Our experimental and computational results show that PA induces conformational change of Sec18, inhibiting priming.

96