33 results found
Menny A, Lukassen M, Couves E, et al., 2021, Structural basis of soluble membrane attack complex packaging for clearance, Nature Communications, Vol: 12, ISSN: 2041-1723
Unregulated complement activation causes inflammatory and immunological pathologies with consequences for human disease. To prevent bystander damage during an immune response, extracellular chaperones (clusterin and vitronectin) capture and clear soluble precursors to the membrane attack complex (sMAC). However, how these chaperones block further polymerization of MAC and prevent the complex from binding target membranes remains unclear. Here, we address that question by combining cryo electron microscopy (cryoEM) and cross-linking mass spectrometry (XL-MS) to solve the structure of sMAC. Together our data reveal how clusterin recognizes and inhibits polymerizing complement proteins by binding a negatively charged surface of sMAC. Furthermore, we show that the pore-forming C9 protein is trapped in an intermediate conformation whereby only one of its two transmembrane β-hairpins has unfurled. This structure provides molecular details for immune pore formation and helps explain a complement control mechanism that has potential implications for how cell clearance pathways mediate immune homeostasis.
Mc Mahon O, Hallam TM, Patel S, et al., 2021, The rare C9 P167S risk variant for age-related macular degeneration increases polymerization of the terminal component of the complement cascade, Human Molecular Genetics, Vol: 30, Pages: 1188-1199, ISSN: 0964-6906
Age-related macular degeneration (AMD) is a complex neurodegenerative eye disease with behavioral and genetic etiology and is the leading cause of irreversible vision loss among elderly Caucasians. Functionally significant genetic variants in the alternative pathway of complement have been strongly linked to disease. More recently, a rare variant in the terminal pathway of complement has been associated with increased risk, Complement component 9 (C9) P167S. To assess the functional consequence of this variant, C9 levels were measured in two independent cohorts of AMD patients. In both cohorts, it was demonstrated that the P167S variant was associated with low C9 plasma levels. Further analysis showed that patients with advanced AMD had elevated sC5b-9 compared to those with non-advanced AMD, although this was not associated with the P167S polymorphism. Electron microscopy of membrane attack complexes (MACs) generated using recombinantly produced wild type or P167S C9 demonstrated identical MAC ring structures. In functional assays, the P167S variant displayed a higher propensity to polymerize and a small increase in its ability to induce hemolysis of sheep erythrocytes when added to C9-depleted serum. The demonstration that this C9 P167S AMD risk polymorphism displays increased polymerization and functional activity provides a rationale for the gene therapy trials of sCD59 to inhibit the terminal pathway of complement in AMD that are underway.
Bickel JK, Voisin TB, Tate EW, et al., 2021, How Structures of Complement Complexes Guide Therapeutic Design., Subcell Biochem, Vol: 96, Pages: 273-295, ISSN: 0306-0225
The complement system is essential for immune defence against infection and modulation of proinflammatory responses. Activation of the terminal pathway of complement triggers formation of the membrane attack complex (MAC), a multi-protein pore that punctures membranes. Recent advances in structural biology, specifically cryo-electron microscopy (cryoEM), have provided atomic resolution snapshots along the pore formation pathway. These structures have revealed dramatic conformational rearrangements that enable assembly and membrane rupture. Here we review the structural basis for MAC formation and show how soluble proteins transition into a giant β-barrel pore. We also discuss regulatory complexes of the terminal pathway and their impact on structure-guided drug discovery of complement therapeutics.
Shah NR, Voisin TB, Parsons ES, et al., 2020, Structural basis for tuning activity and membrane specificity of bacterial cytolysins, Nature Communications, Vol: 11, ISSN: 2041-1723
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.
Barnum SR, Bubeck D, Schein TN, 2020, Soluble Membrane Attack Complex: Biochemistry and Immunobiology, FRONTIERS IN IMMUNOLOGY, Vol: 11, ISSN: 1664-3224
McFarlane C, Shah N, Kabasakal B, et al., 2019, Structural basis of light-induced redox regulation in the Calvin-Benson cycle in cyanobacteria, Proceedings of the National Academy of Sciences of USA, Vol: 116, Pages: 20984-20990, ISSN: 0027-8424
Plants, algae, and cyanobacteria fix carbon dioxide to organic carbon with the Calvin-Benson (CB) cycle. Phosphoribulokinase (PRK) and glyceraldehyde 3 phosphate dehydrogenase (GAPDH) are essential Calvin-Benson cycle enzymes that control substrate availability for the carboxylation enzyme Rubisco. PRK consumes ATP to produce the Rubisco substrate ribulose bisphosphate (RuBP). GAPDH catalyses the reduction step of the CB cycle with NADPH to produce the sugar, glyceraldehyde 3-phosphate (GAP), which is used for regeneration of RuBP and is the main exit point of the cycle. GAPDH and PRK are co-regulated by the redox state of a conditionally disordered protein CP12, which forms a ternary complex with both enzymes. However, the structural basis of Calvin-Benson cycle regulation by CP12 is unknown. Here we show how CP12 modulates the activity of both GAPDH and PRK. Using thermophilic cyanobacterial homologues, we solve crystal structures of GAPDH with different cofactors and CP12 bound, and the ternary GAPDH-CP12-PRK complex by electron cryo-microscopy, we reveal that formation of the N-terminal disulfide pre-orders CP12 prior to binding the PRK active site, which is resolved in complex with CP12. We find that CP12 binding to GAPDH influences substrate accessibility of all GAPDH active sites in the binary and ternary inhibited complexes. Our structural and biochemical data explain how CP12 integrates responses from both redox state and nicotinamide dinucleotide availability to regulate carbon fixation.
Menny A, Serna M, Boyd C, et al., 2019, CRYOEM OF THE MAC, 17th European Meeting on Complement in Human Disease (EMCHD), Publisher: PERGAMON-ELSEVIER SCIENCE LTD, Pages: 493-494, ISSN: 0161-5890
Parsons E, Stanley G, Pyne A, et al., 2019, Single-molecule kinetics of pore assembly by the membrane attack complex, Nature Communications, Vol: 10, Pages: 1-10, ISSN: 2041-1723
The membrane attack complex (MAC) is a hetero-oligomeric protein assembly that kills pathogens by perforating their cell envelopes. The MAC is formed by sequential assembly of soluble complement proteins C5b, C6, C7, C8 and C9, but little is known about the rate-limiting steps in this process. Here, we use rapid atomic force microscopy (AFM) imaging to show that MAC proteins oligomerize within the membrane, unlike structurally homologous bacterial pore-forming toxins. C5b-7 interacts with the lipid bilayer prior to recruiting C8. We discover that incorporation of the first C9 is the kinetic bottleneck of MAC formation, after which rapid C9 oligomerization completes the pore. This defines the kinetic basis for MAC assembly and provides insight into how human cells are protected from bystander damage by the cell surface receptor CD59, which is offered a maximum temporal window to halt the assembly at the point of C9 insertion.
Menny A, Serna M, Boyd C, et al., 2018, CryoEM reveals how the complement membrane attack complex ruptures lipid bilayers, Nature Communications, Vol: 9, ISSN: 2041-1723
The membrane attack complex (MAC) is one of the immune system’s first responders. Complement proteins assemble on target membranes to form pores that lyse pathogens and impact tissue homeostasis of self-cells. How MAC disrupts the membrane barrier remains unclear. Here we use electron cryo-microscopy and flicker spectroscopy to show that MAC interacts with lipid bilayers in two distinct ways. Whereas C6 and C7 associate with the outer leaflet and reduce the energy for membrane bending, C8 and C9 traverse the bilayer increasing membrane rigidity. CryoEM reconstructions reveal plasticity of the MAC pore and demonstrate how C5b6 acts as a platform, directing assembly of a giant β-barrel whose structure is supported by a glycan scaffold. Our work provides a structural basis for understanding how β-pore forming proteins breach the membrane and reveals a mechanism for how MAC kills pathogens and regulates cell functions.
Boyd CM, Bubeck DA, 2018, Advances in cryoEM and its impact on beta-pore forming proteins, Current Opinion in Structural Biology, Vol: 52, Pages: 41-49, ISSN: 0959-440X
Deployed by both hosts and pathogens, β-pore-forming proteins (β-PFPs) rupture membranes and lyse target cells. Soluble protein monomers oligomerize on the lipid bilayer where they undergo dramatic structural rearrangements, resulting in a transmembrane β-barrel pore. Advances in electron cryo-microscopy (cryoEM) sample preparation, image detection, and computational algorithms have led to a number of recent structures that reveal a molecular mechanism of pore formation in atomic detail.
McFarlane C, Shah N, Kabasakal B, et al., 2018, Structural basis of light-induced redox regulation in the Calvin cycle, biorxiv
Abstract In plants, carbon dioxide is fixed via the Calvin cycle in a tightly regulated process. Key to this regulation is the conditionally disordered protein CP12. CP12 forms a complex with two Calvin cycle enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK), inhibiting their activities. The mode of CP12 action was unknown. By solving crystal structures of CP12 bound to GAPDH, and the ternary GAPDH-CP12-PRK complex by electron cryo-microscopy, we reveal that formation of the N-terminal disulfide pre-orders CP12 prior to binding the PRK active site. We find that CP12 binding to GAPDH influences substrate accessibility of all GAPDH active sites in the binary and ternary inhibited complexes. Our model explains how CP12 integrates responses from both redox state and nicotinamide dinucleotide availability to regulate carbon fixation. One Sentence Summary How plants turn off carbon fixation in the dark.
Morgan BP, Boyd C, Bubeck DA, 2017, Molecular cell biology of complement membrane attack, Seminars in Cell and Developmental Biology, Vol: 72, Pages: 124-132, ISSN: 1084-9521
The membrane attack complex (MAC) is the pore-forming toxin of the complement system, a relatively early evolutionary acquisition that confers upon complement the capacity to directly kill pathogens. The MAC is more than just a bactericidal missile, having the capacity when formed on self-cells to initiate a host of cell activation events that can have profound consequences for tissue homeostasis in the face of infection or injury. Although the capacity of complement to directly kill pathogens has been recognised for over a century, and the pore-forming killing mechanism for at least 50 years, there remains considerable uncertainty regarding precisely how MAC mediates its killing and cell activation activities. A recent burst of new information on MAC structure provides context and opportunity to re-assess the ways in which MAC kills bacteria and modulates cell functions. In this brief review we will describe key aspects of MAC evolution, function and structure and seek to use the new structural information to better explain how the MAC works.
Bayly-Jones C, Bubeck DA, Dunstone M, 2017, The mystery behind membrane insertion: a review of the complement membrane attack complex, Philosophical Transactions of the Royal Society B: Biological Sciences, Vol: 372, ISSN: 1471-2970
The membrane attack complex (MAC) is an important innate immune effector of the complement terminal pathway that forms cytotoxic pores on the surface of microbes. Despite many years of research, MAC structure and mechanism of action have remained elusive, relying heavily on modelling and inference from biochemical experiments. Recent advances in structural biology, specifically cryo electron microscopy, have provided new insights into the molecular mechanism of MAC assembly. Its unique “split washer” shape, coupled with an irregular giant β barrel architecture, enable an atypical mechanism of hole punching and represent a novel system for which to study pore formation. This review will introduce the complement terminal pathway that leads to formation of the MAC. Moreover, it will discuss how structures of the pore and component proteins underpin a mechanism for MAC function, modulation and inhibition.
Boyd C, Parsons ES, Smith RAG, et al., 2016, Disentangling the roles of cholesterol and CD59 in intermedilysin pore formation, Scientific Reports, Vol: 6, ISSN: 2045-2322
The plasma membrane provides an essential barrier, shielding a cell from the pressures of its external environment. Pore-forming proteins, deployed by both hosts and pathogens alike, breach this barrier to lyse target cells. Intermedilysin is a cholesterol-dependent cytolysin that requires the human immune receptor CD59, in addition to cholesterol, to form giant β-barrel pores in host membranes. Here we integrate biochemical assays with electron microscopy and atomic force microscopy to distinguish the roles of these two receptors in mediating structural transitions of pore formation. CD59 is required for the specific coordination of intermedilysin (ILY) monomers and for triggering collapse of an oligomeric prepore. Movement of Domain 2 with respect to Domain 3 of ILY is essential for forming a late prepore intermediate that releases CD59, while the role of cholesterol may be limited to insertion of the transmembrane segments. Together these data define a structural timeline for ILY pore formation and suggest a mechanism that is relevant to understanding other pore-forming toxins that also require CD59.
Morgan BP, Walters D, Serna M, et al., 2016, Terminal complexes of the complement system: new structural insights and their relevance to function, Immunological Reviews, Vol: 274, Pages: 141-151, ISSN: 1600-065X
Complement is a key component of innate immunity in health and a powerful driver of inflammation and tissue injury in disease. The biological and pathological effects of complement activation are mediated by activation products. These come in two flavors: (i) proteolytic fragments of complement proteins (C3, C4, C5) generated during activation that bind specific receptors on target cells to mediate effects; (ii) the multimolecular membrane attack complex generated from the five terminal complement proteins that directly binds to and penetrates target cell membranes. Several recent publications have described structural insights that have changed perceptions of the nature of this membrane attack complex. This review will describe these recent advances in understanding of the structure of the membrane attack complex and its by-product the fluid-phase terminal complement complex and relate these new structural insights to functional consequences and cell responses to complement membrane attack.
Taylor JD, Hawthorne WJ, Lo J, et al., 2016, Electrostatically-guided inhibition of Curli amyloid nucleation by the CsgC-like family of chaperones, Scientific Reports, Vol: 6, ISSN: 2045-2322
Polypeptide aggregation into amyloid is linked with several debilitating human diseases.Despite the inherent risk of aggregation-induced cytotoxicity, bacteria control the export ofamyloid-prone subunits and assemble adhesive amyloid fibres during biofilm formation. AnEscherichia protein, CsgC potently inhibits amyloid formation of curli amyloid proteins.Here we unlock its mechanism of action, and show that CsgC strongly inhibits primarynucleation via electrostatically-guided molecular encounters, which expands theconformational distribution of disordered curli subunits. This delays the formation of higherorder intermediates and maintains amyloidogenic subunits in a secretion-competent form.New structural insight also reveal that CsgC is part of diverse family of bacterial amyloidinhibitors. Curli assembly is therefore not only arrested in the periplasm, but the preservationof conformational flexibility also enables efficient secretion to the cellsurface. Understanding how bacteria safely handle amyloidogenic polypeptides contributetowards efforts to control aggregation in disease-causing amyloids and amyloid-based biotechnological applications.
Serna Gil M, Bubeck D, Giles JL, et al., 2016, Structural basis of complement membrane attack complex formation, Nature Communications, Vol: 7, Pages: 1-7, ISSN: 2041-1723
In response to complement activation, the membrane attack complex (MAC) assembles from fluid-phase proteins to form pores in lipid bilayers. MAC directly lyses pathogens by a ‘multi-hit’ mechanism; however, sublytic MAC pores on host cells activate signalling pathways. Previous studies have described the structures of individual MAC components and subcomplexes; however, the molecular details of its assembly and mechanism of action remain unresolved. Here we report the electron cryo-microscopy structure of human MAC at subnanometre resolution. Structural analyses define the stoichiometry of the complete pore and identify a network of interaction interfaces that determine its assembly mechanism. MAC adopts a ‘split-washer’ configuration, in contrast to the predicted closed ring observed for perforin and cholesterol-dependent cytolysins. Assembly precursors partially penetrate the lipid bilayer, resulting in an irregular β-barrel pore. Our results demonstrate how differences in symmetric and asymmetric components of the MAC underpin a molecular basis for pore formation and suggest a mechanism of action that extends beyond membrane penetration.
Bubeck D, 2015, Unraveling Structural Polymorphism of Amyloid Fibers, Structure, Vol: 23, Pages: 10-11, ISSN: 0969-2126
Guenther C, Kind B, Reijns MAM, et al., 2015, Defective removal of ribonucleotides from DNA promotes systemic autoimmunity, JOURNAL OF CLINICAL INVESTIGATION, Vol: 125, Pages: 413-424, ISSN: 0021-9738
Bubeck D, 2014, The Making of a Macromolecular Machine: Assembly of the Membrane Attack Complex, BIOCHEMISTRY, Vol: 53, Pages: 1908-1915, ISSN: 0006-2960
Johnson S, Brooks NJ, Smith RAG, et al., 2013, Structural basis for recognition of the pore-forming toxin intermedilysin by human complement receptor CD59, Cell Reports, Vol: 3
Pore-forming proteins containing the structurally conserved membrane attack complex/perforin fold play an important role in immunity and host-pathogen interactions. Intermedilysin (ILY) is an archetypal member of a cholesterol-dependent cytolysin subclass that hijacks the complement receptor CD59 to make cytotoxic pores in human cells. ILY directly competes for the membrane attack complex binding-site on CD59, rendering cells susceptible to complement lysis. To understand how these bacterial pores form in lipid bilayers and the role CD59 plays in complement regulation, we determined the crystal structure of human CD59 bound to ILY. Here we show the ILY-CD59 complex at 3.5 Å resolution and identify two interfaces mediating this hostpathogen interaction. An ILY-derived peptide based on the binding-site inhibits pore formation in a CD59-containing liposome model system. These data provide insight into how CD59 coordinates ILY monomers, nucleating an early prepore state, and suggest a potential mechanism of inhibition for the complement terminal pathway.
Hadders MA, Bubeck D, Roversi P, et al., 2012, Assembly and Regulation of the Membrane Attack Complex Based on Structures of C5b6 and sC5b9, CELL REPORTS, Vol: 1, Pages: 200-207, ISSN: 2211-1247
Chen S, Bubeck D, MacDonald BT, et al., 2011, Structural and functional studies of LRP6 ectodomain reveal a platform for Wnt signaling, Vol: 21, Pages: 848-861
Bubeck D, Reijns MAM, Graham SC, et al., 2011, PCNA directs type 2 RNase H activity on DNA replication and repair substrates, Nucleic Acids Research, Vol: 39, Pages: 3652-3666
Reijns MAM, Bubeck D, Gibson LCD, et al., 2011, The structure of the human RNase H2 complex defines key interaction interfaces relevant to enzyme function and human disease, Journal of Biological Chemistry, Vol: 286, Pages: 10530-10539
Bubeck D, Roversi P, Donev R, et al., 2011, Structure of human complement C8, a precursor to membrane attack, Journal of Molecular Biology, Vol: 405, Pages: 325-330
Bubeck D, Filman DJ, Kuzmin M, et al., 2008, Post-imaging fiducial markers aid in the orientation determination of complexes with mixed or unknown symmetry, Journal of Structural Biology, Vol: 162, Pages: 480-490
Bostina M, Bubeck D, Schwartz C, et al., 2007, Single particle cryoelectron tomography characterization of the structure and structural variability of poliovirus-receptor-membrane complex at 30 angstrom resolution, JOURNAL OF STRUCTURAL BIOLOGY, Vol: 160, Pages: 200-210, ISSN: 1047-8477
Huiskonen JT, de Haas F, Bubeck D, et al., 2006, Structure of the bacteriophage phi 6 nucleocapsid suggests a mechanism for sequential RNA packaging, STRUCTURE, Vol: 14, Pages: 1039-1048, ISSN: 0969-2126
Tuthill TJ, Bubeck D, Rowlands DJ, et al., 2006, Characterization of early steps in the poliovirus infection process: Receptor-decorated liposomes induce conversion of the virus to membrane-anchored entry-intermediate particles, JOURNAL OF VIROLOGY, Vol: 80, Pages: 172-180, ISSN: 0022-538X
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