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

Abstract

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...

Phosphatidic acid in membrane rearrangements

“…Phosphatidic acid is known to play an important role in membrane fusion mediated by SNARE proteins ( and references therein). Interactions of PA with various SNARE proteins, such as sporulation‐specific yeast homolog of SNAP‐25 (Spo20p) , Sso1p and Sso2p (yeast homologs of syntaxin1) , syntaxin1A , stimulates membrane fusion.…”

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

“…Thus, the temporal regulation of balancing PA and DAG concentrations has a direct effect on progression through the fusion pathway. Subsequent studies showed that PA binding by monomeric Sec18/NSF triggers large conformational changes that appear to be incompatible with the assembly of the active homohexamer needed to bind and prime SNAREs (12). The major site of conformational change, as shown by molecular dynamics, is the predominant PA-binding site between the D1 and D2 domains of NSF.…”

“…Because 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). To accomplish this, we used the cryo-EM-guided resolution of the hexameric structure of NSF bound to SNAREs (17).…”

Non-Canonical Binding of a Small Molecule to Sortilin Alters Cellular Trafficking of ApoB and PCSK9 in Liver Derived Cells

“…Thermophoresis measurements were performed using a Monolith NT.115 labeled thermophoresis instrument 72,73 . Sortilin-His6 was labeled with Ni-NTA Atto-488 according to the manufacturer's protocol as previously described 73,74 curves were fit using a specific binding equation with hill slope, whereas all other SPR saturation curves were fit using a 1:1 specific binding model.…”