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

Research output: Contribution to journalReview article

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.

Original languageEnglish (US)
Pages (from-to)17168-17185
Number of pages18
JournalJournal of Biological Chemistry
Volume294
Issue number46
DOIs
StatePublished - Jan 1 2019

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N-Ethylmaleimide-Sensitive Proteins
SNARE Proteins
Phosphatidic Acids
Vacuoles
Carrier Proteins
Fusion reactions
Proteins
Molecules
Membranes
Chemical activation
Phosphatidate Phosphatase
Membrane Fusion
Ethylmaleimide
Diglycerides
Molecular Dynamics Simulation
Organelles
Conformations
Molecular dynamics

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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A small-molecule competitive inhibitor of phosphatidic acid binding by the AAA+ protein NSF/Sec18 blocks the SNARE-priming stage of vacuole fusion. / Sparks, Robert P.; Arango, Andres S.; Starr, Matthew L.; Aboff, Zachary L.; Hurst, Logan R.; Rivera-Kohr, David A.; Zhang, Chi; Harnden, Kevin A.; Jenkins, Jermaine L.; Guida, Wayne C.; Tajkhorshid, Emad; Fratti, Rutilio A.

In: Journal of Biological Chemistry, Vol. 294, No. 46, 01.01.2019, p. 17168-17185.

Research output: Contribution to journalReview article

Sparks, RP, Arango, AS, Starr, ML, Aboff, ZL, Hurst, LR, Rivera-Kohr, DA, Zhang, C, Harnden, KA, Jenkins, JL, Guida, WC, Tajkhorshid, E & Fratti, RA 2019, 'A small-molecule competitive inhibitor of phosphatidic acid binding by the AAA+ protein NSF/Sec18 blocks the SNARE-priming stage of vacuole fusion', Journal of Biological Chemistry, vol. 294, no. 46, pp. 17168-17185. https://doi.org/10.1074/jbc.RA119.008865
Sparks, Robert P. ; Arango, Andres S. ; Starr, Matthew L. ; Aboff, Zachary L. ; Hurst, Logan R. ; Rivera-Kohr, David A. ; Zhang, Chi ; Harnden, Kevin A. ; Jenkins, Jermaine L. ; Guida, Wayne C. ; Tajkhorshid, Emad ; Fratti, Rutilio A. / A small-molecule competitive inhibitor of phosphatidic acid binding by the AAA+ protein NSF/Sec18 blocks the SNARE-priming stage of vacuole fusion. In: Journal of Biological Chemistry. 2019 ; Vol. 294, No. 46. pp. 17168-17185.
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T1 - A small-molecule competitive inhibitor of phosphatidic acid binding by the AAA+ protein NSF/Sec18 blocks the SNARE-priming stage of vacuole fusion

AU - Sparks, Robert P.

AU - Arango, Andres S.

AU - Starr, Matthew L.

AU - Aboff, Zachary L.

AU - Hurst, Logan R.

AU - Rivera-Kohr, David A.

AU - Zhang, Chi

AU - Harnden, Kevin A.

AU - Jenkins, Jermaine L.

AU - Guida, Wayne C.

AU - Tajkhorshid, Emad

AU - Fratti, Rutilio A.

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N2 - 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.

AB - 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.

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