Macrophage-expressed gene 1 (MPEG1/Perforin-2) is a perforin-like protein that functions within the phagolysosome to damage engulfed microbes. MPEG1 is thought to form pores in target membranes, however, its mode of action remains unknown. We use cryo-Electron Microscopy (cryo-EM) to determine the 2.4 Å structure of a hexadecameric assembly of MPEG1 that displays the expected features of a soluble prepore complex. We further discover that MPEG1 prepore-like assemblies can be induced to perforate membranes through acidification, such as would occur within maturing phagolysosomes. We next solve the 3.6 Å cryo-EM structure of MPEG1 in complex with liposomes. These data reveal that a multi-vesicular body of 12 kDa (MVB12)-associated β-prism (MABP) domain binds membranes such that the pore-forming machinery of MPEG1 is oriented away from the bound membrane. This unexpected mechanism of membrane interaction suggests that MPEG1 remains bound to the phagolysosome membrane while simultaneously forming pores in engulfed bacterial targets.

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.This article is part of the themed issue ‘Membrane pores: from structure and assembly, to medicine and technology’.

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.

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The cholesterol-dependent cytolysin perfringolysin O (PFO) is secreted by Clostridium perfringens as a bacterial virulence factor able to form giant ring-shaped pores that perforate and ultimately lyse mammalian cell membranes. To resolve the kinetics of all steps in the assembly pathway, we have used single-molecule fluorescence imaging to follow the dynamics of PFO on dye-loaded liposomes that lead to opening of a pore and release of the encapsulated dye. Formation of a long-lived membrane-bound PFO dimer nucleates the growth of an irreversible oligomer. The growing oligomer can insert into the membrane and open a pore at stoichiometries ranging from tetramers to full rings (~35-mers), whereby the rate of insertion increases linearly with the number of subunits. Oligomers that insert before the ring is complete continue to grow by monomer addition post insertion. Overall, our observations suggest that PFO membrane insertion is kinetically controlled.

Macrophage-expressed gene 1 [MPEG1/Perforin-2 (PRF2)] is an ancient metazoan protein belonging to the Membrane Attack Complex/Perforin (MACPF) branch of the MACPF/Cholesterol Dependent Cytolysin (CDC) superfamily of pore-forming proteins (PFPs). MACPF/CDC proteins are a large and extremely diverse superfamily that forms large transmembrane aqueous channels in target membranes. In humans, MACPFs have known roles in immunity and development. Like perforin (PRF) and the membrane attack complex (MAC), MPEG1 is also postulated to perform a role in immunity. Indeed, bioinformatic studies suggest that gene duplications of MPEG1 likely gave rise to PRF and MAC components. Studies reveal partial or complete loss of MPEG1 causes an increased susceptibility to microbial infection in both cells and animals. To this end, MPEG1 expression is upregulated in response to proinflammatory signals such as tumor necrosis factor a (TNFa) and lipopolysaccharides (LPS). Furthermore, germline mutations in MPEG1 have been identified in connection with recurrent pulmonary mycobacterial infections in humans. Structural studies on MPEG1 revealed that it can form oligomeric pre-pores and pores. Strikingly, the unusual domain arrangement within the MPEG1 architecture suggests a novel mechanism of pore formation that may have evolved to guard against unwanted lysis of the host cell. Collectively, the available data suggest that MPEG1 likely functions as an intracellular pore-forming immune effector. Herein, we review the current understanding of MPEG1 evolution, regulation, and function. Furthermore, recent structural studies of MPEG1 are discussed, including the proposed mechanisms of action for MPEG1 bactericidal activity. Lastly limitations, outstanding questions, and implications of MPEG1 models are explored in the context of the broader literature and in light of newly available structural data.

The cholesterol-dependent cytolysin perfringolysin O (PFO) is secreted by Clostridium perfringens as a bacterial virulence factor able to form giant ring-shaped pores that perforate and ultimately lyse mammalian cell membranes. To resolve the kinetics of all steps in the assembly pathway, we have used single-molecule fluorescence imaging to follow the dynamics of PFO on dye-loaded liposomes that lead to opening of a pore and release of the encapsulated dye. Formation of a long-lived membrane-bound PFO dimer nucleates the growth of an irreversible oligomer. The growing oligomer can insert into the membrane and open a pore at stoichiometries ranging from tetramers to full rings (~35-mers), whereby the rate of insertion increases linearly with the number of subunits. Oligomers that insert before the ring is complete continue to grow by monomer addition post insertion. Overall, our observations suggest that PFO membrane insertion is kinetically controlled.

Pore-forming proteins (PFPs) are a broad class of molecules that comprise various families, structural folds, and assembly pathways. In nature, PFPs are most often deployed by their host organisms to defend against other organisms. In humans, this is apparent in the immune system, where several immune effectors possess pore-forming activity. Furthermore, applications of PFPs are found in next-generation low-cost DNA sequencing, agricultural crop protection, pest control, and biosensing. The advent of cryoEM has propelled the field forward. Nevertheless, significant challenges and knowledge-gaps remain. Overcoming these challenges is particularly important for the development of custom, purpose-engineered PFPs with novel or desired properties. Emerging single-molecule techniques and methods are helping to address these unanswered questions. Here we review the current challenges, problems, and approaches to studying PFPs.