New gadget in the membrane trafficking toolbox: A novel inhibitor of SNARE priming

Abstract

NSF (N-ethylmaleimide sensitive factor) and its yeast counterpart Sec18 are highly conserved homohexameric proteins that play vital roles in eukaryotic membrane trafficking. Sec18 functions by disrupting SNARE complexes formed in cis, on the same membrane. However, the molecular mechanisms of this process are poorly understood, in large part due to the lack of selective, reversible inhibitors. A new study by Sparks et al. now reports a small molecule that appears to selectively inhibit Sec18 action in an in vitro assay. Their finding now paves the way to elucidate further details of Sec18-mediated SNARE priming.

Introduction

Intracellular membrane–bound compartments are a hallmark of eukaryotic cells. These compartments are in flux, such that material is constantly entering and exiting each compartment; this movement underlies essential processes such as synaptic transmission, hormone secretion, cell motility, antigen presentation, and endocytosis. Seminal work by George Palade and colleagues in the 1950s and 60s showed that much of the flux of material between intracellular compartments was mediated by vesicles budding off of one compartment and fusing with another (1). These trafficking events were later shown to be mediated by SNAP receptor (SNARE)² protein complexes. SNAREs are integral membrane proteins that reside on apposed membranes about to fuse, and they interact in trans to catalyze selective membrane fusion events (2). After fusion, the SNARE complexes now reside on the same membrane and must be untangled to facilitate the next fusion cycle (Fig. 1a). NSF/Sec18 performs this function, pulling apart these SNARE complexes in a process called “priming,” but the mechanistic details of this process are still unclear (3, 4). A new paper by Sparks et al. (5), published in this issue of JBC, takes exciting new steps toward such additional mechanistic insight by describing the first specific small-molecule inhibitor of SNARE priming.

Figure 1.

Sec18 function and model for IPA action. a, scheme of SNARE-mediated fusion and Sec18-mediated recycling, known as priming. b, the functional form of Sec18 is a hexamer (middle). Phosphatidic acid binds a monomeric form of Sec18, sequestering Sec18 in a nonfunctional form (top). IPA binds to the same region in the Sec18 molecule but stabilizes the hexameric form while still preventing interaction of Sec18 with SNAREs (bottom).

In the 1980s, a groundbreaking in vitro assay developed in Jim Rothman's laboratory allowed the reconstitution of membrane fusion events in a cell-free system. This then allowed the purification of protein factors that are required for the process. One of the first observations made with regard to this assay was its sensitivity to N-ethylmaleimide (NEM), an alkylating agent that preferentially attacks thiol groups (6). It turned out that the sensitivity to NEM reflected a specific soluble protein factor that became alkylated, and the Rothman laboratory used this fact to purify the protein, which was named N-ethylmaleimide–sensitive factor, or NSF. Strikingly, it became obvious that NSF is a highly conserved homolog of a yeast protein, Sec18, whose gene was serendipitously identified in the Schekman laboratory and cloned in the Emr laboratory (7–9), establishing unexpected similarities between the yeast and mammalian systems. We now know that NSF and Sec18 are highly conserved ATPases that form homohexamers to interact with the SNARE proteins (10). Nonetheless, the molecular mechanism of NSF-mediated SNARE priming and reactivation has not been fully elucidated. Previous work in the Fratti laboratory showed that binding of individual Sec18 monomers to the phospholipid phosphatidic acid (PA) leads to a conformational change that is incompatible with Sec18 activity in membrane fusion assays (11). Thus, PA could theoretically serve as an inhibitor of the priming step, but it is not specific for Sec18 or this step in the process and is poorly soluble. Despite its important role in identifying NSF, NEM is similarly problematic as a selective inhibitor of priming, because it alkylates cysteines indiscriminately.

To identify novel Sec18 inhibitors, Sparks et al. used an in silico screen to look for molecules that would interact with the PA-binding site on NSF, as the structure of this complex is known. Initial docking pointed to 19 candidates that were further analyzed computationally, with two compounds receiving the best scores. One of these compounds, now named “inhibitor of priming activity” (IPA) was shown to bind NSF at low micromolar concentrations and to inhibit Sec18-mediated SNARE priming in vitro. The authors further demonstrated that IPA competes with PA for Sec18 binding, suggesting an overlapping binding site. Furthermore, IPA did not interfere with PA binding to another known target or with Sec18 binding to other kinds of lipids or with other steps in the in vitro fusion assay, indicating clear specificity.

The authors then characterized the effects of IPA on conformational switches in subdomains of the Sec18 molecule. Interestingly, whereas IPA was selected for its predicted ability to compete for the PA-binding site on Sec18 and initial data supported an overlapping point of contact, the authors convincingly demonstrate, using proteolytic cleavage, biophysical assays, and molecular docking simulations, that IPA binding to Sec18 stabilizes a conformation distinct from that stabilized by PA (Fig. 1b).

The discovery of a soluble small molecule that specifically inhibits Sec18 at micromolar concentrations could potentially be of huge importance, as it could supplant less specific and irreversible reagents, such as NEM. Using IPA, investigators may now be able to ask mechanistic questions that were previously inaccessible. First, it will be exciting to learn more about the potential utility of the compound with intact cells, experiments that the authors indicate are in progress, and to determine the structure of the Sec18-IPA complex. Additionally, it will be important to test whether IPA is also effective against mammalian NSF itself, to enable further studies of the human protein. Finally, titration of IPA on the SNARE priming reaction may reveal accumulations of novel intermediates, which can potentially shed light on the priming process itself. Historically, identification of selective inhibitors in other in vitro assays was followed by the experimental discovery of such novel intermediates. One therefore hopes that further work utilizing the IPA-Sec18 interaction will allow investigators to shed new light on the mechanism of Sec18- dependent SNARE priming.