1.C.39 The Membrane Attack Complex/Perforin (MACPF) Family

Complement is part of the mammalian immune defense system against pathogenic microorganisms. More than 20 complement proteins control microbial invasion. One mechanism involves direct killing of the microbe, dependent on the C5b-8-9 or membrane attack complex (MAC) (Wang et al., 2000). This complex forms on the outer surface of a Gram-negative bacterial cell. The C5b-8 complex probably allows entry of C9 into the periplasm where the inactive protoxin is converted to the active toxin. This conversion process probably involves sulfhydryl oxidation to a disulfide linked protein. It has been shown that (C5b-8)1 (C9)1 which can lyse erythrocytes can not kill bacteria, but a (C5b-8)1 (C9)4 complex is bactericidal and also protozoan pathogens (Wang et al., 2000). Only the C-terminal portion of C9, residues 145-538 (the C9b fragment) is required to dissipate the membrane potential across respiring inner membrane vesicles, and only C9 is required if the protein is first targeted to the periplasm by any mechanism. While C9 therefore appears to form a pore in the inner membrane, C5b-8 appears to allow access of C9 to the periplasm. C6, C7, C8α, C8β and C9 all contain the pore forming MACPF domain (Rosado et al., 2008).

Native complement protein C9 is a soluble glycoprotein devoid of cellular toxicity, but its bactericidal activity is unmasked in the periplasm. This requires disulfide bond formation, a presumed conformational change, and insertion of a 'processed' toxin into the inner bacterial membrane, probably as an oligomer. Four disulfide bonds can potentially form in C9, three in the epidermal growth factor domain (residues 487-518) and one in the membrane attack/perforin region (Cys359 and Cys384). C9 has a 3-dimensional fold for its membrane interaction and pore forming domain as many other toxins of animal and bacterial origin (Anderluh and Lakey, 2008; Lukoyanova and Saibil, 2008). Kurschus et al., 2008 provided evidence that perforins and cholesterol-dependent cytolysins can deliver deadly proteins such as granzymes into the cytoplasm of target cells.

Proteins of the complement membrane attack complex (MAC) and the protein perforin (PF) share a common MACPF domain that is responsible for membrane insertion and pore formation (Cajnko et al. 2014). Hadders et al. 2007 determined the crystal structure of the MACPF domain of complement component C8α at 2.5 angstrom resolution and showed that it is structurally homologous to the bacterial, pore-forming, cholesterol-dependent cytolysins (TC #1.C.12). The structure displays two regions that (in the bacterial cytolysins) refold into transmembrane ß hairpins, forming the lining of a barrel pore. Local hydrophobicity explains why C8α is the first complement protein to insert into the membrane. The size of the MACPF domain is consistent with known C9 pore sizes. Thus, these mammalian and bacterial cytolytic proteins share a common mechanism of membrane insertion (Hadders et al., 2007). Insertion entails a dramatic refolding of the protein structure (Lukoyanova et al. 2016).

Proteins containing membrane attack complex/perforin (MACPF) domains play important roles in vertebrate immunity, embryonic development, and neural-cell migration among others (Anderluh and Lakey, 2008). In vertebrates, the ninth component of complement and perforin form oligomeric pores that lyse bacteria and kill virus-infected cells, respectively. Rosada et al. 2007 determined the crystal structure of a bacterial MACPF protein, Plu-MACPF from Photorhabdus luminescens, to 2.0 angstrom resolution. The MACPF domain revealed structural similarity with pore-forming cholesterol-dependent cytolysins (CDCs; 1.C.12) from Gram-positive bacteria. This suggests that lytic MACPF proteins may use a CDC-like mechanism to form pores and disrupt cell membranes. Sequence similarity between bacterial and vertebrate MACPF domains suggests that the fold of the CDCs, a family of proteins important for bacterial pathogenesis, is probably used by vertebrates for defense against infection.

The secretory granule-mediated cell death pathway is the key mechanism for elimination of virus-infected and transformed target cells by cytotoxic lymphocytes (Voskoboinik et al. 2010). The formation of the immunological synapse between an effector and a target cell leads to exocytic trafficking of the secretory granules and the release of their contents, which include pro-apoptotic serine proteases, granzymes, and pore-forming perforin into the synapse. There, perforin polymerizes and forms a transmembrane pore that allows the delivery of granzymes into the cytosol where they initiate various apoptotic death pathways. Unlike relatively redundant individual granzymes, functional perforin is absolutely essential for cytotoxic lymphocyte function and immune regulation in the host.  Perforin's structure and function as well as its role in immune-mediated diseases have been reviewed (Voskoboinik et al. 2010).  pH influsences perforin's permeabilizing activity but not its binding to membranes (Praper et al. 2010).

Law et al. (2010) elucidated 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, Ca²⁺-dependent membrane binding. Electron microscopy revealed 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 (Law et al., 2010). The 2.5Å structure of human C8 protein provided mechanistic insight into membrane pore formation by complement. C8-C9 and C9-C9 interactions facilitate refolding and insertion of putative MACPF transmembrane β-hairpins to form a circular pore (Lovelace et al., 2011). Perforin's helices function as TMSs after membrane insertion (Neely et al. 2016).

Perforin (PFN) is produced by cytotoxic lymphocytes and aids in the clearance of tumor or virus-infected cells by a pore forming mechanism. Praper et al. (2011) showed that perforin forms heterogeneous pores with a broad range of conductances, from 0.15 to 21 nanosiemens. In comparison with large pores that possessed low noise and remained stably open, small pores exhibited high noise and were unstable. Furthermore, the opening step and the pore size were dependent on the lipid composition of the membrane. The heterogeneity in pore sizes was confirmed with cryo-electron microscopy and showed a range of sizes matching that observed in the conductance measurements. Two different membrane-bound PFN conformations were  interpreted as pre-pore and pore states of the protein. PFN probably forms heterogeneous pores through a multistep mechanism.

The complement Membrane Attack Complex (MAC) forms transmembrane pores in pathogen membranes. The first step in MAC assembly is cleavage of C5 to generate metastable C5b, which forms a stable complex with C6, termed C5b-6. C5b-6 initiates pore formation via the sequential recruitment of homologous proteins: C7, C8, and 12-18 copies of C9, each of which comprise a central MACPF domain flanked by auxiliary domains. Aleshin et al. (2012) proposed a model of pore assembly, in which the auxiliary domains play key roles, both in stabilizing the closed conformation of the protomers, and in driving the sequential opening of the MACPF β-sheet of each new recruit to the growing pore. They also described an atomic model of C5b-6 at 4.2 Å resolution and showed that C5b provides 4 interfaces for the auxiliary domains of C6. The largest interface is created by the insertion of an interdomain linker from C6 into a hydrophobic groove created by a major reorganization of the α-helical domain of C5b. In combination with a rigid-body docking of N-terminal elements of both proteins, C5b becomes locked into a stable conformation. Both C6 auxiliary domains flanking the linker pack tightly against C5b. The net effect is to induce a clockwise rigid-body rotation of 4 auxiliary domains as well as an opening/twisting of the central β-sheet of C6, in the directions predicted to activate or prime C6 for the subsequent steps in MAC assembly. The complex also suggests novel small-molecule strategies for modulating pathological MAC assembly (Aleshin et al., 2012).

Cytotoxic lymphocytes use perforin to eliminate dangerous cells, while remaining refractory to lysis. At least two mechanisms jointly preserve the killer cell: the C-terminal residues of perforin dictate its rapid export from the ER, whose milieu otherwise favours pore formation. Perforin is then stored in secretory granules whose acidity prevent its oligomerisation. Following exocytosis, perforin delivers the proapoptotic protease, granzyme B, into the target cell by disrupting its plasma membrane. The defined crystal structure of the perforin monomer and cryo-electron microscopy (EM) of the entire pore suggest that passive transmembrane granzyme diffusion is the dominant proapoptotic mechanism (Lopez et al., 2012).

The membrane attack complex of complement (MAC), apart from its classical role of lysing cells, can also trigger a range of non-lethal effects on cells, acting to promote inflammation. Triantafilou et al. 2013 investigated these non-lethal effects on inflammasome activation and found that, following sublytic MAC attack, there is an increased cytosolic Ca²⁺ concentration, at least partly through Ca²⁺ release from the endoplasmic reticulum lumen via the inositol 1,4,5-triphosphate receptor (IP3R) and ryanodine receptor (RyR) channels (TC# 1.A.3). This increase in intracellular Ca²⁺ concentration leads to Ca²⁺ accumulation in the mitochondrial matrix via the 'mitochondrial calcium uniporter' (MCU; TC#1.A.77), and loss of mitochondrial transmembrane potential, triggering NLRP3 inflammasome activation and IL-1beta release. NLRP3 co-localises with the mitochondria, probably sensing the increase in calcium and the resultant mitochondrial dysfunction, leading to caspase activation and apoptosis (Triantafilou et al. 2013).

Proteins with MACPF domains have a variety of biological roles, including defence and attack, organism development, and cell adhesion and signalling (Ota et al. 2014). The distribution of these proteins in fungi appears to be restricted to some Pezizomycotina and Basidiomycota species only, in correlation with another group of proteins with unknown biological function, known as aegerolysins. These two protein groups coincide in only a few species, and they might operate in concert as cytolytic bi-component pore- forming agents. Representative proteins here include pleurotolysin B, which has a MACPF domain, and the aegerolysin-like protein pleurotolysin A, and the very similar ostreolysin A, which have been purified from oyster mushroom (Pleurotus ostreatus). These act in concert to perforate natural and artificial lipid membranes with high cholesterol and sphingomyelin content. The aegerolysin-like proteins provide the membrane cholesterol/sphingomyelin selectivity and recruit oligomerized pleurotolysin B molecules, to create a membrane-inserted pore complex. The resulting protein structure has been imaged with electron microscopy, and it has a 13-meric rosette-like structure, with a central lumen that is ~4-5 nm in diameter. The opened transmembrane pore is non-selectively permeable for ions and smaller neutral solutes and is a cause of cytolysis of a colloid-osmotic type.

D'Angelo et al. (2012) (PMID 22551122) identified orthologs and homologs of human perforin in all but one species analysed from Euteleostomi, and presented evidence for an earlier ortholog in Gnathostomata but not in more primitive chordates. In placental mammals, perforin is a single copy gene, but there are multiple perforin genes in all lineages predating marsupials, except birds. Comparisons of these many-to-one homologs of human perforin showed that they mainly arose from lineage-specific gene duplications in multiple taxa, suggesting acquisition of new roles or different modes of regulation. Perforins probably arose by duplication of the ancient MPEG1 gene (TC# 3.C.39.14), and that they share a common ancestor with the functionally related complement proteins.  The evolution of perforin in vertebrates involved a complex pattern of gene/intron, gain and loss. The primordial perforin gene arose at least 500 million years ago, at around the time that the major histocompatibility complex-T cell receptor antigen recognition system was established. As it is absent from primitive chordates and invertebrates, cytotoxic cells from these lineages must possess a different effector molecule or cytotoxic mechanism. 

CDC/MACPF proteins contain a characteristic four-stranded beta-sheet that is flanked by two alpha-helical bundles, which unfold to form two transmembrane beta-hairpins. Apicomplexan parasites express MACPFs termed perforin-like proteins (PLPs). Wade and Tweten 2015 present insights into the assembly and regulation of the Apicomplexan PLP molecular pore-forming mechanisms, necessary for osmotically driven rupture of the parasitophorous vacuole and host cell membrane, and cell traversal by these parasites. 

Membrane Attack Complex (MAC) pores are assembled when surface-bound convertase enzymes convert C5 into C5b, which together with C6, C7, C8 and multiple copies of C9 forms transmembrane pores that damage the bacterial cell envelope. Bacterial killing requires local conversion of C5 by surface-bound convertases, and Doorduijn et al. 2020 showed that rapid interaction of C7 with C5b6 is required to form bactericidal MAC pores. This rapid interaction is crucial to efficiently anchor C5b-7 to the bacterial cell envelope. Bacterial pathogens can prevent complement-dependent killing by interfering with the anchoring of C5b-7 and thus prevented stable insertion of MAC pores into the bacterial cell envelope.

Unregulated complement activation causes inflammatory and immunological pathologies. To prevent damage during an immune response, extracellular chaperones (clusterin and vitronectin) capture and clear soluble precursors to the membrane attack complex (sMAC). Menny et al. 2021 combined cryoEM and cross-linking mass spectrometry (XL-MS) to solve the structure of sMAC. Clusterin recognizes and inhibits polymerizing complement proteins by binding a negatively charged surface of sMAC. The pore-forming C9 protein is trapped in an intermediate conformation whereby only one of its two transmembrane beta-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 (Menny et al. 2021).

Perforin is a granular effector pore-forming protein formed in NK cells and Cytotoxic T lymphocytes. These cytotoxic proteins are part of the first-line immune defense in the human body. They ensure apoptosis of pathogen-infected cells or tumor cells in the human body. Activation of receptors on NK cells or T cells triggers secondary proteins in these cells. Further, it leads to Ca²⁺-dependent perforin egress towards the target cell, ensued by a PI3K signaling pathway. Perforin undergoes oligomerization over the target cell membrane and forms transmembrane pores with the membrane-spanning domain, the MACPF domain. Granzymes, proapoptotic serine proteases, are released through these pores and initiate the target cell apoptotic pathway leading to cell death. Although perforin is a savior for humans from tumor and viral infections, uncontrolled expression of the perforins leads to autoimmune conditions, including Familial Hemophagocytic lymphohistiocytosis, insulin-dependent diabetes, and cerebral myocarditis. The review by Sankar et al. 2022 is a concerted effort to highlight the mechanistic pathways concerning perforin secretion, NK cell and T cell-mediated cytotoxicity towards virus-infected and transformed cells. This is followed by a discussion on synthetic derivatives tested so far to inhibit the perforin in pre and clinical arena for certain unusual conditions (Sankar et al. 2022).

The generalized transport reaction catalyzed by MACPF family members is:

Small and large molecules (in) ⇌ small and large molecules (out)

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