Biophysical and Molecular-Dynamics Studies of Phosphatidic Acid Binding by the Dvl-2 DEP Domain

Capelluto, Daniel G. S.; Zhao, Xiaolin; Lucas, Andrew T.; Lemkul, Justin A.; Xiao, S. Y.; Fu, Xiangping; Sun, Fanping; Bevan, David R.; Finkielstein, Carla V.

doi:10.1016/j.bpj.2014.01.032

Cited by 23 publications

(28 citation statements)

References 53 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…These data were consistent with previous SPR competition experiments using PA containing nanodiscs (24). To examine whether IPA blocked other PA-binding proteins, we used the DEP domain from the murine protein Dvl2 (25). Previously, we used DEP to bind PA liposomes and vacuolar PA to displace Sec18 from membranes (11).…”

Section: Sec18 Monomer D1 and D2 Domains Bind Ipa With High Affinitysupporting

confidence: 85%

See 1 more Smart Citation

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

Sparks

Arango

Starr

et al. 2019

Journal of Biological Chemistry

View full text Add to dashboard Cite

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.

show abstract

Section: Sec18 Monomer D1 and D2 Domains Bind Ipa With High Affinitysupporting

confidence: 85%

“…Each pool was collected and concentrated before use. The DEP PA-binding domain from murine Dvl2 was purified as a GST fusion as described previously (25). Membrane scaffold protein 1D1 (MSP1D1-His) was prepared as described previously (39).…”

Section: Recombinant Proteinsmentioning

confidence: 99%

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

Sparks

Arango

Starr

et al. 2019

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…The analysis of DEP domain mutants performed in this study provided evidence that none of the previously described mutants with mutations connected with the deregulation of the PCP pathway, such as K438M ( 9 , 12 ), membrane binding residue mutations RRRKA (R464A/R465A/R468A/K469A) and H482A/K486A ( 19 , 28 ), or Y494F ( 20 ), is defective in the capacity to rescue the Wnt3a response and activation of Wnt/β-catenin signaling. This is largely in agreement with the situation in Drosophila .…”

Section: Discussionmentioning

confidence: 66%

The N-Terminal Part of the Dishevelled DEP Domain Is Required for Wnt/β-Catenin Signaling in Mammalian Cells

Paclíková

Bernatík

Radaszkiewicz

et al. 2017

Molecular and Cellular Biology

View full text Add to dashboard Cite

Dishevelled (DVL) proteins are key mediators of the Wnt/β-catenin signaling pathway. All DVL proteins contain three conserved domains: DIX, PDZ, and DEP. There is a consensus in the field that the DIX domain is critical for Wnt/β-catenin signaling, but contradictory evidence regarding the function of the DEP domain exists. It has been difficult, until recently, to test the importance of the DEP domain rigorously because of the interference with endogenous DVL, expressed in all Wnt-responsive cell lines. In this study, we took advantage of DVL knockout (DVL1/DVL2/DVL3 triple knockout) cells fully deficient in Wnt3a-induced signaling events and performed a series of rescue experiments. Using these complementation assays, we analyzed the role of individual DVL isoforms. Further domain mapping of DVL1 showed that both the DVL1 DEP domain and especially its N-terminal region are required and sufficient for Wnt3a-induced phosphorylation of LRP6 and TopFlash reporter activation. On the contrary, multiple DEP domain mutants deficient in the planar cell polarity (PCP) pathway could fully rescue the Wnt3a response. This study provides conclusive evidence that the DVL DEP domain is essential for Wnt/β-catenin signaling in mammalian cells and establishes an experimental system suitable for further functional testing of DVL.

show abstract

“…The ND were linked to the Ni-NTA chip through the His tags of the membrane scaffold proteins. First we tested the known PA-binding domain DEP of the murine protein Dvl2 (34). GST-DEP bound to PA-ND with a K D of 18.9 Ϯ 2 M ( Fig.…”

Section: Both D1 and D2 Domains Bind Sec18mentioning

confidence: 99%

“…GST-N was purified in the same way using cells transformed with pGST-N. The DEP PA-binding domain from murine Dvl2 was purified as a GST fusion as described (34). Membrane scaffold protein 1D1 (MSP1D1-His) was prepared as described (62).…”

Section: Protein Purificationmentioning

confidence: 99%

Phosphatidic acid induces conformational changes in Sec18 protomers that prevent SNARE priming

Starr

Sparks

Arango

et al. 2019

Journal of Biological Chemistry

View full text Add to dashboard Cite

Eukaryotic cell homeostasis requires transfer of cellular components among organelles and relies on membrane fusion catalyzed by SNARE proteins. Inactive SNARE bundles are reactivated by hexameric N-ethylmaleimide-sensitive factor, vesicle-fusing ATPase (Sec18/NSF)-driven disassembly that enables a new round of membrane fusion. We previously found that phosphatidic acid (PA) binds Sec18 and thereby sequesters it from SNAREs and that PA dephosphorylation dissociates Sec18 from the membrane, allowing it to engage SNARE complexes. We now report that PA also induces conformational changes in Sec18 protomers and that hexameric Sec18 cannot bind PA membranes. Molecular dynamics (MD) analyses revealed that the D1 and D2 domains of Sec18 contain PA-binding sites and that the residues needed for PA binding are masked in hexameric Sec18. Importantly, these simulations also disclosed that a major conformational change occurs in the linker region between the D1 and D2 domains, which is distinct from the conformational changes that occur in hexameric Sec18 during SNARE priming. Together, these findings indicate that PA regulates Sec18 function by altering its architecture and stabilizing membrane-bound Sec18 protomers.Membrane fusion is necessary for all eukaryotes to effectively transport cellular components between organelles. The trafficking and fusion of vesicles is carried out through a series of events that are highly conserved across eukarya (1). Although many proteins that drive the process may differ between eukaryotic species, they all perform similar roles allowing compartment contact, bilayer fusion, and luminal content mixing (2). The final stage of membrane fusion, and luminal content mixing, is catalyzed by SNARE 3 proteins. Each participating membrane contributes either an R-SNARE or three Q-SNARE coils that wrap around each other to form a parallel four-helical trans-SNARE complex that brings membranes into close apposition. The formation of such complexes releases free energy that is transmitted to the membranes to trigger fusion. Once fusion occurs and membranes are merged, the four-helical SNARE bundle, now a cis-SNARE complex, is inactive and requires disassembly to undergo a new round of fusion.The disassembly of cis-SNAREs, also known as Priming, is carried out by the AAA ϩ protein Sec18/NSF and its adaptor protein Sec17/␣-SNAP (3) (Fig. 1A). Current models suggest that NSF primes cis-SNAREs through a "loaded spring" mechanism triggered by cis-SNARE recognition and ATP hydrolysis (4). NSF binds to cis-SNAREs with the help of ␣-SNAP to form what is known as the 20S complex (5-8). Although NSF was originally isolated as a trimer or tetramer, it can only prime SNAREs as a homohexamer that surrounds the cis-SNAREs and ␣-SNAP proteins to form the 20S particle (9 -11). Association with cis-SNARE-␣-SNAP complexes triggers ATP hydrolysis, which leads to a large conformational change in the protein, with the major change occurring at the N terminus where it folds back over the D1-D2 rings (8). This generates eno...

show abstract

Biophysical and Molecular-Dynamics Studies of Phosphatidic Acid Binding by the Dvl-2 DEP Domain

Cited by 23 publications

References 53 publications

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

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

The N-Terminal Part of the Dishevelled DEP Domain Is Required for Wnt/β-Catenin Signaling in Mammalian Cells

Phosphatidic acid induces conformational changes in Sec18 protomers that prevent SNARE priming

Contact Info

Product

Resources

About