Membrane pore formation by human complement: functional importance of the transmembrane ?-hairpin (TMH) segments of C8? and C9.
ABSTRACT: Human C8 and C9 have a key role in forming the pore-like "membrane attack complex" (MAC) of complement on bacterial cells. A possible mechanism for membrane insertion of these proteins was suggested when studies revealed a structural similarity between the MACPF domains of the C8? and C8? subunits and the pore-forming bacterial cholesterol-dependent cytolysins (CDCs). This similarity includes a pair of ?-helical bundles that in the CDCs refold during pore formation to produce two transmembrane ?-hairpins (TMH1 and TMH2). C9 is the major pore-forming component of the MAC and is also likely to contain two TMH segments because of its homology to C8? and C8?. To determine their potential for membrane insertion, the TMH sequences in C8? and those predicted to be in C9 were substituted for the TMH sequences in perfringolysin O (PFO), a well-characterized CDC. Only chimeric proteins containing TMH2 from C8? (PFO/?T2) or C9 (PFO/C9T2) could be expressed in soluble, active form. The PFO/?T2 and PFO/C9T2 chimeras retained significant hemolytic activity, formed pore-like structures on membranes, and could combine with PFO to form hemolytically active mixed complexes that were functionally similar to PFO alone. These results provide experimental evidence in support of the hypothesis that TMH segments in C8? and those predicted to be in C9 have a direct role in MAC membrane penetration and pore formation.
Project description:C8 is one of five complement proteins that assemble on bacterial membranes to form the lethal pore-like "membrane attack complex" (MAC) of complement. The MAC consists of one C5b, C6, C7, and C8 and 12-18 molecules of C9. C8 is composed of three genetically distinct subunits, C8?, C8?, and C8?. The C6, C7, C8?, C8?, and C9 proteins are homologous and together comprise the MAC family of proteins. All contain N- and C-terminal modules and a central 40-kDa membrane attack complex perforin (MACPF) domain that has a key role in forming the MAC pore. Here, we report the 2.5 Å resolution crystal structure of human C8 purified from blood. This is the first structure of a MAC family member and of a human MACPF-containing protein. The structure shows the modules in C8? and C8? are located on the periphery of C8 and not likely to interact with the target membrane. The C8? subunit, a member of the lipocalin family of proteins that bind and transport small lipophilic molecules, shows no occupancy of its putative ligand-binding site. C8? and C8? are related by a rotation of ?22° with only a small translational component along the rotation axis. Evolutionary arguments suggest the geometry of binding between these two subunits is similar to the arrangement of C9 molecules within the MAC pore. This leads to a model of the MAC that explains how C8-C9 and C9-C9 interactions could facilitate refolding and insertion of putative MACPF transmembrane ?-hairpins to form a circular pore.
Project description: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.
Project description:Human C8 is one of five complement components (C5b, C6, C7, C8, and C9) that assemble on bacterial membranes to form a porelike structure referred to as the "membrane attack complex" (MAC). C8 contains three genetically distinct subunits (C8 alpha, C8 beta, C8 gamma) arranged as a disulfide-linked C8 alpha-gamma dimer that is noncovalently associated with C8 beta. C6, C7 C8 alpha, C8 beta, and C9 are homologous. All contain N- and C-terminal modules and an intervening 40-kDa segment referred to as the membrane attack complex/perforin (MACPF) domain. The C8 gamma subunit is unrelated and belongs to the lipocalin family of proteins that display a beta-barrel fold and generally bind small, hydrophobic ligands. Several hundred proteins with MACPF domains have been identified based on sequence similarity; however, the structure and function of most are unknown. Crystal structures of the secreted bacterial protein Plu-MACPF and the human C8 alpha MACPF domain were recently reported and both display a fold similar to those of the bacterial pore-forming cholesterol-dependent cytolysins (CDCs). In the present study, we determined the crystal structure of the human C8 alpha MACPF domain disulfide-linked to C8 gamma (alphaMACPF-gamma) at 2.15 A resolution. The alphaMACPF portion has the predicted CDC-like fold and shows two regions of interaction with C8 gamma. One is in a previously characterized 19-residue insertion (indel) in C8 alpha and fills the entrance to the putative C8 gamma ligand-binding site. The second is a hydrophobic pocket that makes contact with residues on the side of the C8 gamma beta-barrel. The latter interaction induces conformational changes in alphaMACPF that are likely important for C8 function. Also observed is structural conservation of the MACPF signature motif Y/W-G-T/S-H-F/Y-X(6)-G-G in alphaMACPF and Plu-MACPF, and conservation of several key glycine residues known to be important for refolding and pore formation by CDCs.
Project description:The membrane attack complex (MAC)/perforin-like protein complement component 9 (C9) is the major component of the MAC, a multi-protein complex that forms pores in the membrane of target pathogens. In contrast to homologous proteins such as perforin and the cholesterol-dependent cytolysins (CDCs), all of which require the membrane for oligomerisation, C9 assembles directly onto the nascent MAC from solution. However, the molecular mechanism of MAC assembly remains to be understood. Here we present the 8 Å cryo-EM structure of a soluble form of the poly-C9 component of the MAC. These data reveal a 22-fold symmetrical arrangement of C9 molecules that yield an 88-strand pore-forming β-barrel. The N-terminal thrombospondin-1 (TSP1) domain forms an unexpectedly extensive part of the oligomerisation interface, thus likely facilitating solution-based assembly. These TSP1 interactions may also explain how additional C9 subunits can be recruited to the growing MAC subsequent to membrane insertion.
Project description: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 ?-barrel whose structure is supported by a glycan scaffold. Our work provides a structural basis for understanding how ?-pore forming proteins breach the membrane and reveals a mechanism for how MAC kills pathogens and regulates cell functions.
Project description:The complement membrane attack complex (MAC) is formed by the sequential assembly of C5b with four homologous proteins as follows: one copy each of C6, C7, and C8 and 12-14 copies of C9. Together these form a lytic pore in bacterial membranes. C6 through C9 comprise a MAC-perforin domain flanked by 4-9 "auxiliary" domains. Here, we report the crystal structure of C6, the first and longest of the pore proteins to be recruited by C5b. Comparisons with the structures of the C8??? heterodimer and perforin show that the central domain of C6 adopts a "closed" (perforin-like) state that is distinct from the "open" conformations in C8. We further show that C6, C8?, and C8? contain three homologous subdomains ("upper," "lower," and "regulatory") related by rotations about two hinge points. In C6, the regulatory segment includes four auxiliary domains that stabilize the closed conformation, inhibiting release of membrane-inserting elements. In C8?, rotation of the regulatory segment is linked to an opening of the central ?-sheet of its clockwise partner, C8?. Based on these observations, we propose a model for initiation and unidirectional propagation of the MAC in which the auxiliary domains play key roles: in the assembly of the C5b-8 initiation complex; in driving and regulating the opening of the ?-sheet of the MAC-performin domain of each new recruit as it adds to the growing pore; and in stabilizing the final pore. Our model of the assembled pore resembles those of the cholesterol-dependent cytolysins but is distinct from that recently proposed for perforin.
Project description:CD59 is a glycosylphosphatidylinositol-anchored protein that inhibits the assembly of the terminal complement membrane attack complex (MAC) pore, whereas Streptococcus intermedius intermedilysin (ILY), a pore forming cholesterol-dependent cytolysin (CDC), specifically binds to human CD59 (hCD59) to initiate the formation of its pore. The identification of the residues of ILY and hCD59 that form their binding interface revealed a remarkably deep correspondence between the hCD59 binding site for ILY and that for the MAC proteins C8α and C9. ILY disengages from hCD59 during the prepore to pore transition, suggesting that loss of this interaction is necessary to accommodate specific structural changes associated with this transition. Consistent with this scenario, mutants of hCD59 or ILY that increased the affinity of this interaction decreased the cytolytic activity by slowing the transition of the prepore to pore but not the assembly of the prepore oligomer. A signature motif was also identified in the hCD59 binding CDCs that revealed a new hCD59-binding member of the CDC family. Although the binding site on hCD59 for ILY, C8α, and C9 exhibits significant homology, no similarity exists in their binding sites for hCD59. Hence, ILY and the MAC proteins interact with common amino acids of hCD59 but lack detectable conservation in their binding sites for hCD59.
Project description:Complement component 9 (C9) functions as the pore-forming component of the Membrane Attack Complex (MAC). During MAC assembly, multiple copies of C9 are sequentially recruited to membrane associated C5b8 to form a pore. Here we determined the 2.2?Å crystal structure of monomeric murine C9 and the 3.9?Å resolution cryo EM structure of C9 in a polymeric assembly. Comparison with other MAC proteins reveals that the first transmembrane region (TMH1) in monomeric C9 is uniquely positioned and functions to inhibit its self-assembly in the absence of C5b8. We further show that following C9 recruitment to C5b8, a conformational change in TMH1 permits unidirectional and sequential binding of additional C9 monomers to the growing MAC. This mechanism of pore formation contrasts with related proteins, such as perforin and the cholesterol dependent cytolysins, where it is believed that pre-pore assembly occurs prior to the simultaneous release of the transmembrane regions.
Project description: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 ?-sheet of the MACPF domain, accompanied by extrusion and refolding of two ?-helical regions into transmembrane ?-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 ?-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 ?-hairpins via top down zippering of backbone hydrogen bonds to form the membrane-inserted ?-barrel. The intermediate structures of the MACPF domain during refolding into the ?-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.
Project description:The effect of nine monoclonal antibodies to complement component C8 on the interaction of C9 with preformed cell-surface C5b-8 complexes and on the functional insertion of C8 into the membrane-attack complex (MAC) was investigated. None of the antibodies prevented C9 insertion into a preformed C5b-8 complex. One antibody (F1) directed to the C8 alpha subunit clearly inhibited formation of a functional MAC. It is proposed that this antibody prevents the C8 alpha subunit unfolding and distorting the bilayer to allow C9 insertion.