Journal: Nature / Year: 2010 Title: The structural basis for membrane binding and pore formation by lymphocyte perforin. Authors: Ruby H P Law / Natalya Lukoyanova / Ilia Voskoboinik / Tom T Caradoc-Davies / Katherine Baran / Michelle A Dunstone / Michael E D'Angelo / Elena V Orlova / Fasséli Coulibaly / Sandra ...Authors: Ruby H P Law / Natalya Lukoyanova / Ilia Voskoboinik / Tom T Caradoc-Davies / Katherine Baran / Michelle A Dunstone / Michael E D'Angelo / Elena V Orlova / Fasséli Coulibaly / Sandra Verschoor / Kylie A Browne / Annette Ciccone / Michael J Kuiper / Phillip I Bird / Joseph A Trapani / Helen R Saibil / James C Whisstock / Abstract: Natural killer cells and cytotoxic T lymphocytes accomplish the critically important function of killing virus-infected and neoplastic cells. They do this by releasing the pore-forming protein ...Natural killer cells and cytotoxic T lymphocytes accomplish the critically important function of killing virus-infected and neoplastic cells. They do this by releasing the pore-forming protein perforin and granzyme proteases from cytoplasmic granules into the cleft formed between the abutting killer and target cell membranes. Perforin, a 67-kilodalton multidomain protein, oligomerizes to form pores that deliver the pro-apoptopic granzymes into the cytosol of the target cell. The importance of perforin is highlighted by the fatal consequences of congenital perforin deficiency, with more than 50 different perforin mutations linked to familial haemophagocytic lymphohistiocytosis (type 2 FHL). Here we elucidate the mechanism of perforin pore formation by determining the X-ray crystal structure of monomeric murine perforin, together with a cryo-electron microscopy reconstruction of the entire perforin pore. Perforin is a thin 'key-shaped' molecule, comprising an amino-terminal membrane attack complex perforin-like (MACPF)/cholesterol dependent cytolysin (CDC) domain followed by an epidermal growth factor (EGF) domain that, together with the extreme carboxy-terminal sequence, forms a central shelf-like structure. A C-terminal C2 domain mediates initial, Ca(2+)-dependent membrane binding. Most unexpectedly, however, electron microscopy reveals that the orientation of the perforin MACPF domain in the pore is inside-out relative to the subunit arrangement in CDCs. These data reveal remarkable flexibility in the mechanism of action of the conserved MACPF/CDC fold and provide new insights into how related immune defence molecules such as complement proteins assemble into pores.
History
Deposition
Jul 27, 2010
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Header (metadata) release
Sep 24, 2010
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Map release
Nov 3, 2010
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Update
Nov 16, 2010
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Current status
Nov 16, 2010
Processing site: PDBe / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Cryogen name: ETHANE / Chamber humidity: 100 % / Instrument: OTHER / Details: Vitrification instrument: Vitrobot Method: liposomes were applied to neg. glow discharged grids first, then protein solution was added. Grids were left inside Vitrobot equilibrated at 37C for 1-2 min for pore formaition. Blot for 2 seconds before plunging
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Electron microscopy
Microscope
FEI TECNAI F20
Electron beam
Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Specimen holder: Side entry LN2 cooled, single tilt / Specimen holder model: GATAN LIQUID NITROGEN
Alignment procedure
Legacy - Astigmatism: Objective lens astigmatism was corrected at 150,000x magnification
Date
Apr 23, 2007
Image recording
Category: CCD / Film or detector model: GENERIC GATAN (4k x 4k) / Digitization - Sampling interval: 15 µm / Number real images: 260 / Average electron dose: 20 e/Å2
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
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Image processing
CTF correction
Details: Estimated with CTFFIND3, then phases flipped for each particle
Final two d classification
Number classes: 10
Final reconstruction
Applied symmetry - Point group: C20 (20 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 28.5 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Imagic, Spider Details: 20-fold symmetrised reconstruction. Hand of the map has not been determined Number images used: 59
Details
Image regions containing the pores with small surrounding areas of membrane were selected manually using EMAN-Boxer software
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