A small-molecule competitive inhibitor of phosphatidic acid binding by the AAA+ protein NSF/Sec18 blocks the SNARE-priming stage of vacuole fusion

J Biol Chem. 2019 Nov 15;294(46):17168-17185. doi: 10.1074/jbc.RA119.008865. Epub 2019 Sep 12.

Abstract

The homeostasis of most organelles requires membrane fusion mediated by soluble N -ethylmaleimide-sensitive factor (NSF) attachment protein receptors (SNAREs). SNAREs undergo cycles of activation and deactivation as membranes move through the fusion cycle. At the top of the cycle, inactive cis-SNARE complexes on a single membrane are activated, or primed, by the hexameric ATPase associated with the diverse cellular activities (AAA+) protein, N-ethylmaleimide-sensitive factor (NSF/Sec18), and its co-chaperone α-SNAP/Sec17. Sec18-mediated ATP hydrolysis drives the mechanical disassembly of SNAREs into individual coils, permitting a new cycle of fusion. Previously, we found that Sec18 monomers are sequestered away from SNAREs by binding phosphatidic acid (PA). Sec18 is released from the membrane when PA is hydrolyzed to diacylglycerol by the PA phosphatase Pah1. Although PA can inhibit SNARE priming, it binds other proteins and thus cannot be used as a specific tool to further probe Sec18 activity. Here, we report the discovery of a small-molecule compound, we call IPA (inhibitor of priming activity), that binds Sec18 with high affinity and blocks SNARE activation. We observed that IPA blocks SNARE priming and competes for PA binding to Sec18. Molecular dynamics simulations revealed that IPA induces a more rigid NSF/Sec18 conformation, which potentially disables the flexibility required for Sec18 to bind to PA or to activate SNAREs. We also show that IPA more potently and specifically inhibits NSF/Sec18 activity than does N-ethylmaleimide, requiring the administration of only low micromolar concentrations of IPA, demonstrating that this compound could help to further elucidate SNARE-priming dynamics.

Keywords: NSF; SNARE proteins; Sec17; Sec18; membrane fusion; membrane lipid; membrane trafficking; phosphatidic acid; α-SNAP.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • ATPases Associated with Diverse Cellular Activities / chemistry
  • ATPases Associated with Diverse Cellular Activities / genetics
  • Adenosine Triphosphatases / chemistry
  • Adenosine Triphosphatases / genetics*
  • Ethylmaleimide / metabolism*
  • Membrane Fusion / drug effects
  • Membrane Fusion / genetics
  • Membrane Lipids / chemistry
  • Membrane Lipids / genetics
  • Membrane Transport Proteins / chemistry
  • Membrane Transport Proteins / genetics
  • Molecular Dynamics Simulation
  • N-Ethylmaleimide-Sensitive Proteins / chemistry
  • N-Ethylmaleimide-Sensitive Proteins / genetics
  • Phosphatidic Acids / antagonists & inhibitors
  • Phosphatidic Acids / chemistry*
  • SNARE Proteins / chemistry
  • SNARE Proteins / genetics
  • Saccharomyces cerevisiae / chemistry
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae Proteins / chemistry
  • Saccharomyces cerevisiae Proteins / genetics*
  • Small Molecule Libraries / chemistry*
  • Small Molecule Libraries / pharmacology
  • Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins / chemistry
  • Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins / genetics
  • Vacuoles / genetics
  • Vesicular Transport Proteins / chemistry
  • Vesicular Transport Proteins / genetics*

Substances

  • Membrane Lipids
  • Membrane Transport Proteins
  • Phosphatidic Acids
  • SEC17 protein, S cerevisiae
  • SNARE Proteins
  • Saccharomyces cerevisiae Proteins
  • Small Molecule Libraries
  • Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins
  • Vesicular Transport Proteins
  • Adenosine Triphosphatases
  • SEC18 protein, S cerevisiae
  • ATPases Associated with Diverse Cellular Activities
  • N-Ethylmaleimide-Sensitive Proteins
  • Ethylmaleimide

Associated data

  • PDB/3J94