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.
We have a new 11 Å resolution cryo-Electron Microscopy (cryo-EM) structure of the two-part fungal toxin Pleurotolysin (Ply), together with crystal structures of both components. These data revealed 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 allowed unambiguous assignment of subdomains in a MACPF pore assembly. The major conformational changes in PlyB are a 70º opening of the bent and distorted central bsheet of the MACPF domain, accompanied by extrusion and refolding of two a-helical regions (TMH1 and TMH2) into transmembrane b-hairpins.
Moreover, three different disulphide bond-trapped prepore intermediates have been determined. Analysis of these data by molecular modelling and flexible fitting allow us to generate a molecular trajectory of b-sheet unbending. These data 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 b-hairpins via top down zippering of backbone hydrogen bonds to form the membrane-inserted b-barrel.
The intermediate structures of the MACPF/CDC domain during its refolding into the β-barrel pore establish a structural paradigm for the transition from soluble monomer to membrane embedded oligomers (pores). Our data further reveal that the TMH2 region is critical for the release of both TMH regions, suggesting why this region is targeted by endogenous inhibitors of MACPF function.