bioRxiv preprint doi: https://doi.org/10.1101/2020.06.16.154724; this version posted June 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 1 Structural basis for tuning activity and membrane specificity of bacterial 2 cytolysins 3 4 5 Authors: Nita R. Shah1, Tomas B. Voisin1, Edward S. Parsons2, Courtney M. Boyd1, Bart W. 6 Hoogenboom2,3, Doryen Bubeck1* 7 8 Affiliations: 9 1 Department of Life Sciences, Sir Ernst Chain Building, Imperial College London, London, SW7 10 2AZ, UK 11 2 London Centre for Nanotechnology, University College London, London, WC1H 0AH, UK 12 3 Department of Physics and Astronomy, University College London, Gower Street, London, 13 WC1E 6BT, UK 14 15 16 *Contact Information: [email protected] 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.16.154724; this version posted June 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 17 ABSTRACT 18 Cholesterol-dependent cytolysins (CDCs) form protein nanopores to lyse cells. They target 19 eukaryotic cells using different mechanisms, but all require the presence of cholesterol to pierce 20 lipid bilayers. How CDCs use cholesterol to selectively lyse cells is essential for understanding 21 virulence strategies of several pathogenic bacteria, and for repurposing CDCs to kill new cellular 22 targets. Here we address that question by trapping an early state of pore formation for the CDC 23 intermedilysin, bound to the human immune receptor CD59 in a nanodisc model membrane. 24 Our cryo-electron microscopy map reveals structural transitions required for oligomerization, 25 which include the lateral movement of a key amphipathic helix. We demonstrate that the charge 26 of this helix is crucial for tuning lytic activity of CDCs. Furthermore, we discover modifications 27 that overcome the requirement of cholesterol for membrane rupture, which will facilitate 28 engineering the target-cell specificity of pore-forming proteins. 29 30 31 32 33 34 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.16.154724; this version posted June 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 35 INTRODUCTION 36 Pore-forming proteins rupture lipid bilayers to kill target cells. They comprise the largest class of 37 virulence factors for pathogenic bacteria and are prevalent in all kingdoms of life1. Cholesterol- 38 dependent cytolysins (CDCs) are pore-forming proteins secreted by more than five genera of 39 Gram-positive bacteria, including human pathogens from Streptococcus, Clostridium, and 40 Listeria. Pore-forming proteins also play a crucial role in immune defence, killing Gram-negative 41 bacteria, cancer cells, and phagocytosed microbes2. Understanding how these proteins 42 discriminate between self-cells and target membranes will provide insight into fundamental 43 virulence strategies, as well as facilitate the application of engineered CDCs that lyse new 44 cellular targets. 45 46 Many pore-forming proteins depend on lipid specificity to select their targets. For example, the 47 immune protein perforin preferentially targets post-synaptic membranes through lipid disorder 48 phases and neutral charge headgroups3. Another immune protein, gasdermin, specifically binds 49 cardiolipin and phosphoinositide lipids to direct activity against mitochondria and the inner 50 leaflets of eukaryotic plasma membranes, triggering cell death4, 5. Lipid specificity is a common 51 theme for bacterial toxins in general6. For CDCs specifically, the requirement of cholesterol 52 directs lytic activity towards plasma membranes of eukaryotic hosts7. Since cholesterol is not 53 present in bacterial cells that secrete CDCs, this is one way the pathogen protects itself from 54 damage during infection and toxin production; it also limits the repurposing of CDCs to attack 55 bacterial pathogens. Many CDCs, such as pneumolysin (PLY), bind cholesterol directly through 56 a conserved cholesterol-recognition motif located on a membrane-binding loop8. Although 57 cholesterol is a well-known receptor for PLY, it may not be the only one. PLY also binds to a 58 mannose receptor in dendritic cells, downregulating inflammation and promoting bacterial 59 survival9. Other CDCs, such as intermedilysin (ILY), achieve species-specificity for their hosts 60 by hijacking the cell surface receptor CD59 to initiate membrane-binding10. For this subgroup of 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.16.154724; this version posted June 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 61 CDCs, the interaction with CD59 is sufficient for attachment and the role of cholesterol is 62 restricted to pore-formation11. While differences in membrane targeting may reflect diversity in 63 CDC virulence strategies, lipid dependency for membrane penetration remains highly 64 conserved. 65 66 CDCs bind target membranes through interactions located in their domain 4 (D4). Both the 67 cholesterol recognition loops of PLY8 and CD59-binding site of ILY lie within this domain10. A 68 long extended domain 2 (D2) flexibly links D4 to the membrane attack complex perforin 69 (MACPF)/CDC (also referred to as D1 and D3) domain responsible for pore formation12. 70 Membrane-binding of CDC monomers triggers a series of conformational re-arrangements that 71 are required for oligomerization into an assembly referred to as a prepore. A dramatic vertical 72 collapse of the oligomeric prepore brings two helical bundles (HB1 and HB2) within the 73 MACPF/CDC domain close to the target membrane. HB1 and HB2 residues undergo 74 rearrangement in secondary structure to form adjacent transmembrane β-strands in the pore13- 75 16. These rearrangements are facilitated by stabilizing interactions between a number of amino 76 acids at the MACPF/CDC domain interface17, but structural details of intermediate 77 conformations remain unresolved. 78 79 Here we report the cryo-electron microscopy (cryoEM) structure of an early prepore-trapped 80 conformation of the CDC intermedilysin (ILY). By binding to the human immune receptor CD59, 81 ILY oligomerizes and triggers movement of a highly conserved amphipathic helix that encodes 82 activity and lipid specificity of CDC pore formation. This helix can be modified to create 83 cholesterol-independent CDCs or “super-CDCs” with significantly enhanced activity. Hence, our 84 results provide a blueprint for engineering broad-purpose pore-forming proteins with tunable 85 lytic activity. 86 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.16.154724; this version posted June 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 87 RESULTS 88 CryoEM structure of an ILY early prepore 89 ILY from Streptococcus intermedius targets human cells by binding the GPI-anchored 90 complement regulator CD5910 through an extended β-hairpin of D418. To understand how this 91 interaction initiates oligomerization on a target membrane, we trapped ILY in an early prepore 92 state using a disulfide lock that restricts movement between D2 and the MACPF/CDC domain11. 93 This disulfide-locked ILY variant binds CD59 and forms SDS-sensitive loosely-associated 94 oligomers11, analogous to previously characterized CDC early prepore states19. We then used 95 cryo-electron microscopy (cryoEM) to visualize this ILY variant in complex with CD59 anchored 96 to cholesterol-containing lipid nanodiscs. We collected data on a Titan Krios microscope 97 equipped with a direct electron detector and processed images using RELION20. 2D 98 classification revealed heterogeneity in the number of subunits within a single nanodisc 99 (Supplementary Fig. 1). Extensive 2D and 3D classification resulted in a final reconstruction 100 comprised of 51,041 particles. The data were further refined with local symmetry, resulting in a 101 map with an average resolution of 4.6 Å. (Supplementary Fig. 2). The final local resolution- 102 sharpened density map illustrates an arc of five ILY-CD59 complexes, with the central subunit 103 best resolved (Fig. 1a, Supplementary Fig. 1). We therefore built a model into the density of the 104 central subunit and applied this fit as a rigid-body into the neighboring densities to model a 105 three-subunit oligomer (Fig. 1a, Supplementary Fig. 3). 106 107 Our cryoEM structure reveals an oligomeric complex whereby a single CD59 can interact with 108 two neighboring ILY monomers (ILY and ILY’). Similar to the soluble monomeric ILY-CD59 109 crystal structures18, 21, a β-hairpin of one ILY D4 extends the central β-sheet of CD59. The same 110 β-hairpin of an adjacent monomer (ILY’), together with the tip of D2, sandwiches an α-helix of 111 CD59 to form the second interface (Fig. 1b). This interface is dominated by two CD59 residues 5 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.16.154724; this version posted June 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.