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

Robert P Sparks,Andres S Arango,Matthew L Starr,Zachary L Aboff,Logan R Hurst,David A Rivera-Kohr,Chi Zhang,Kevin A Harnden,Jermaine L Jenkins,Wayne C Guida,Emad Tajkhorshid,Rutilio A Fratti

doi:10.1074/jbc.ra119.008865

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.

Highlights

The homeostasis of most organelles requires membrane fusion mediated by soluble N-ethylmaleimide–sensitive factor (NSF) attachment protein receptors (SNAREs)
Because phosphatidic acid (PA) acts a potent inhibitor of Sec18 function, we used computational modeling to search for small molecules that docked at the previously identified PA-binding regions of Sec18 [12]
We found that inhibitor of priming activity (IPA) and epirubicin had the highest gscores of the candidate compounds (Fig. 2E)

Summary

Introduction

The homeostasis of most organelles requires membrane fusion mediated by soluble N-ethylmaleimide–sensitive factor (NSF) attachment protein receptors (SNAREs). 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 ␣-SNAP/ Sec. PA can inhibit SNARE priming, it binds other proteins and cannot be used as a specific tool to further probe Sec activity. SNAREs remain as inactive cisSNARE complexes on this newly formed single bilayer These complexes need to be disassembled so that a new cycle of fusion may occur. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Even though the dosage of NEM required to inhibit Sec18/NSF is in the millimolar range [5, 7], its promiscuity may have even been advantageous [8] to its use in adopting early models identifying the interaction of Sec18/NSF with Sec17/␣-SNAP as being crucial to the continual cycle of fusion by membranes within the cell [9]

Methods

Results

Conclusion