Conformational Changes during Pore Formation by the Perforin-Related Protein Pleurotolysin

Abstract

Membrane attack complex/perforin-like (MACPF) proteins comprise the largest superfamily of pore-forming proteins, playing crucial roles in immunity and pathogenesis. Soluble monomers assemble into large transmembrane pores via conformational transitions that remain to be structurally and mechanistically characterised. Here we present an 11 Å resolution cryo-electron microscopy (cryo-EM) structure of the two-part, fungal toxin Pleurotolysin (Ply), together with crystal structures of both components (the lipid binding PlyA protein and the pore-forming MACPF component PlyB). These data reveal a 13-fold pore 80 Å in diameter and 100 Å in height, with each subunit comprised of a PlyB molecule atop a membrane bound dimer of PlyA. The resolution of the EM map, together with biophysical and computational experiments, allowed confident assignment of subdomains in a MACPF pore assembly. The major conformational changes in PlyB are a ∼70° opening of the bent and distorted central β-sheet of the MACPF domain, accompanied by extrusion and refolding of two α-helical regions into transmembrane β-hairpins (TMH1 and TMH2). We determined the structures of three different disulphide bond-trapped prepore intermediates. Analysis of these data by molecular modelling and flexible fitting allows us to generate a potential trajectory of β-sheet unbending. The results suggest that MACPF conformational change is triggered through disruption of the interface between a conserved helix-turn-helix motif and the top of TMH2. Following their release we propose that the transmembrane regions assemble into β-hairpins via top down zippering of backbone hydrogen bonds to form the membrane-inserted β-barrel. The intermediate structures of the MACPF domain during refolding into the β-barrel pore establish a structural paradigm for the transition from soluble monomer to pore, which may be conserved across the whole superfamily. The TMH2 region is critical for the release of both TMH clusters, suggesting why this region is targeted by endogenous inhibitors of MACPF function.

Stonefish toxin defines an ancient branch of the perforin-like superfamily

“…We suggest that the structure of the SNTX heterodimer represents a soluble and stable snapshot of this event. Consistent with this idea, the orientation of the two MACPF/CDC domains in the SNTX heterodimer resembles the arrangement of MACPF/CDC domains seen in low-resolution EM structures (>15 Å) of MACPF/CDC prepore assemblies (22,23,31). Furthermore, our phylogenetic data suggest that, after duplication, SNTX-α and -β initially functioned as monomers but then, coevolved to function as a stable dimer.…”

“…Crucially and consistent with this idea, mutagenesis of the equivalent β4-α6 loop structure in CDCs (the β5-region) showed that mobility in this region is essential for initial stable dimer formation as well as subsequent oligomerization events (30,32). Similarly, EM data and mutagenesis data suggest that the equivalent region is also important for assembly of the fungal MACPF protein pleurotolysin (23). Taken together, our data on SNTX suggest that the region equivalent to the β4-α6 loop structure in MACPF and CDCs represents a key and conserved feature that is critical for initial prepore assembly.…”

“…This structure can, thus, be considered analogous to a partly closed zipper. Previously, for MACPF and CDCs, it has been suggested that polar interactions between the β4-and β1-strands in the prepore function to pull the strands together during formation of the full β-barrel pore (23). Finally, analysis of the general surface charge at the SNTX dimer interface reveals that the charge distribution of each face is largely complementary, with the α-subunit interface positively charged and the β-subunit interface negatively charged (Fig.…”

“…Subsequently, the central β-sheet substantially untwists, and both TMH1 and TMH2 unwind to form the β-strands of the final β-barrel pore ( Fig. S4) (19)(20)(21)(22)(23). However, our current molecular understanding of pore assembly is severely limited by a lack of any high-resolution structural examples of MACPF/CDC pores or assembly intermediates in a pore-compatible conformation.…”

The membrane attack complex, perforin and cholesterol-dependent cytolysin superfamily of pore-forming proteins

“…Both CDC and MACPF prepores can be trapped by disulphide bonds that have been engineered to limit either TMH1 or TMH2 release (Hotze et al, 2001;Ramachandran et al, 2005;Leung et al, 2014;Lukoyanova et al, 2015). This will be discussed in greater detail below.…”