Journal: Nat Commun / Year: 2020 Title: Structural basis for tuning activity and membrane specificity of bacterial cytolysins. Authors: Nita R Shah / Tomas B Voisin / Edward S Parsons / Courtney M Boyd / Bart W Hoogenboom / Doryen Bubeck / Abstract: Cholesterol-dependent cytolysins (CDCs) are pore-forming proteins that serve as major virulence factors for pathogenic bacteria. They target eukaryotic cells using different mechanisms, but all ...Cholesterol-dependent cytolysins (CDCs) are pore-forming proteins that serve as major virulence factors for pathogenic bacteria. They target eukaryotic cells using different mechanisms, but all require the presence of cholesterol to pierce lipid bilayers. How CDCs use cholesterol to selectively lyse cells is essential for understanding virulence strategies of several pathogenic bacteria, and for repurposing CDCs to kill new cellular targets. Here we address that question by trapping an early state of pore formation for the CDC intermedilysin, bound to the human immune receptor CD59 in a nanodisc model membrane. Our cryo electron microscopy map reveals structural transitions required for oligomerization, which include the lateral movement of a key amphipathic helix. We demonstrate that the charge of this helix is crucial for tuning lytic activity of CDCs. Furthermore, we discover modifications that overcome the requirement of cholesterol for membrane rupture, which may facilitate engineering the target-cell specificity of pore-forming proteins.
History
Deposition
Jun 13, 2020
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Header (metadata) release
Nov 18, 2020
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Map release
Nov 18, 2020
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Update
May 1, 2024
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Current status
May 1, 2024
Processing site: PDBe / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Supramolecule #1: Early prepore of intermedilysin-CD59
Supramolecule
Name: Early prepore of intermedilysin-CD59 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: Prepore is formed on a lipid bilayer on a nanodisc
Model: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: GRAPHENE OXIDE / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR Details: Grids were glow discharged before application of graphene oxide, then left to dry for 1 hour before use.
Vitrification
Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 294 K / Instrument: FEI VITROBOT MARK III / Details: Wait time of 60 s, blot time 2.5 s, blot force 3.
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Electron microscopy
Microscope
FEI TITAN KRIOS
Details
Pixel size 1.4 A
Image recording
Film or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Average exposure time: 1.0 sec. / Average electron dose: 66.0 e/Å2 Details: Some images were collected at a 30 degree tilt. Images were collected in movie mode at 39 frames per second.
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Chain - Source name: PDB / Chain - Initial model type: experimental model
Details
Model of ILY-CD59 was fit and refined into the central subunit density. A combination of rigid body fitting and global minimization with secondary structure restraints was used to fit and refine the central subunit model. Then the central subunit model was rigid body fit as one body into the neighbouring subunits to generate a 3 subunit oligomer model.
Refinement
Space: REAL / Protocol: RIGID BODY FIT
Output model
PDB-6zd0: Disulfide-locked early prepore intermedilysin-CD59
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