title: 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.
authors: Department of Crystallography/Biological Sciences, Institute of Structural and Molecular Biology, Birkbeck College, London, United Kingdom.
authors: Natalya
authors: N
authors: Lukoyanova
authors: Department of Biochemistry and Molecular Biology, Monash University, Clayton, Melbourne, Australia; The ARC Centre of Excellence in Advanced Molecular Imaging, Monash University, Melbourne, Australia.
authors: Stephanie C
authors: SC
authors: Kondos
authors: Department of Crystallography/Biological Sciences, Institute of Structural and Molecular Biology, Birkbeck College, London, United Kingdom.
authors: Irene
authors: I
authors: Farabella
authors: Department of Biochemistry and Molecular Biology, Monash University, Clayton, Melbourne, Australia; The ARC Centre of Excellence in Advanced Molecular Imaging, Monash University, Melbourne, Australia.
authors: Ruby H P
authors: RH
authors: Law
authors: Department of Biochemistry and Molecular Biology, Monash University, Clayton, Melbourne, Australia; The ARC Centre of Excellence in Advanced Molecular Imaging, Monash University, Melbourne, Australia.
authors: Cyril F
authors: CF
authors: Reboul
authors: The ARC Centre of Excellence in Advanced Molecular Imaging, Monash University, Melbourne, Australia; Australian Synchrotron, Clayton, Melbourne, Australia.
authors: Tom T
authors: TT
authors: Caradoc-Davies
authors: Department of Biochemistry and Molecular Biology, Monash University, Clayton, Melbourne, Australia.
authors: Bradley A
authors: BA
authors: Spicer
authors: Department of Biochemistry and Molecular Biology, Monash University, Clayton, Melbourne, Australia.
authors: Oded
authors: O
authors: Kleifeld
authors: Department of Biochemistry and Molecular Biology, Monash University, Clayton, Melbourne, Australia.
authors: Daouda A K
authors: DA
authors: Traore
authors: Department of Biochemistry and Molecular Biology, Monash University, Clayton, Melbourne, Australia.
authors: Susan M
authors: SM
authors: Ekkel
authors: Peter MacCallum Cancer Centre, East Melbourne, Australia.
authors: Ilia
authors: I
authors: Voskoboinik
authors: Peter MacCallum Cancer Centre, East Melbourne, Australia.
authors: Joseph A
authors: JA
authors: Trapani
authors: Department of Biochemistry and Molecular Biology, Monash University, Clayton, Melbourne, Australia.
authors: Tamas
authors: T
authors: Hatfaludi
authors: Institute of Structural and Molecular Biology, University College London, London, United Kingdom.
authors: Katherine
authors: K
authors: Oliver
authors: Department of Microbiology and Immunology, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States of America.
authors: Eileen M
authors: EM
authors: Hotze
authors: Department of Microbiology and Immunology, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States of America.
authors: Rodney K
authors: RK
authors: Tweten
authors: Department of Biochemistry and Molecular Biology, Monash University, Clayton, Melbourne, Australia; The ARC Centre of Excellence in Advanced Molecular Imaging, Monash University, Melbourne, Australia.
authors: James C
authors: JC
authors: Whisstock
authors: Department of Crystallography/Biological Sciences, Institute of Structural and Molecular Biology, Birkbeck College, London, United Kingdom.
authors: Maya
authors: M
authors: Topf
authors: Department of Crystallography/Biological Sciences, Institute of Structural and Molecular Biology, Birkbeck College, London, United Kingdom.
authors: Helen R
authors: HR
authors: Saibil
authors: Department of Biochemistry and Molecular Biology, Monash University, Clayton, Melbourne, Australia; The ARC Centre of Excellence in Advanced Molecular Imaging, Monash University, Melbourne, Australia; Department of Microbiology, Monash University, Melbourne, Australia.
authors: Michelle A
authors: MA
authors: Dunstone
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000737:chemistry*; D015938:Complement Membrane Attack Complex / Q000737:chemistry* / Q000378:metabolism; D020285:Cryoelectron Microscopy; D018360:Crystallography, X-Ray; D004912:Erythrocytes / Q000737:chemistry / Q000166:cytology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D020076:Pleurotus / Q000737:chemistry*; D011485:Protein Binding; D017510:Protein Folding; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012756:Sheep
chemical_list: D015938:Complement Membrane Attack Complex; D005656:Fungal Proteins; D006460:Hemolysin Proteins; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; C586632:pleurotolysin B, oyster mushroom
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