The structural basis for membrane binding and pore formation by lymphocyte perforin

Abstract

Natural killer cells and cytotoxic T lymphocytes accomplish the critically important function of killing virus-infected and neoplastic cells. They do this by releasing the pore-forming protein perforin and granzyme proteases from cytoplasmic granules into the cleft formed between the abutting killer and target cell membranes. Perforin, a 67-kilodalton multidomain protein, oligomerizes to form pores that deliver the pro-apoptopic granzymes into the cytosol of the target cell. The importance of perforin is highlighted by the fatal consequences of congenital perforin deficiency, with more than 50 different perforin mutations linked to familial haemophagocytic lymphohistiocytosis (type 2 FHL). Here we elucidate 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, Ca(2+)-dependent membrane binding. Most unexpectedly, however, electron microscopy reveals 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 and provide new insights into how related immune defence molecules such as complement proteins assemble into pores.

Perforin oligomers form arcs in cellular membranes: a locus for intracellular delivery of granzymes

“…2 This structure has seemed the obvious candidate for providing granzyme passage into a target cell cytosol. Perplexingly, however, PFN efficiently induces translocation of GzmB in targets that exclude even low molecular weight cationic fluorophores, 5,6,17,18 suggesting that cylindrical pores are somehow dispensable for granzyme delivery leaving another form of PFN responsible.…”

“…Examples include: (1) Melittin, the principal component of bee venom; 23 (2) Magainin, an antimicrobial peptide; 24 (3) sticholysin II, 25 equinatoxin II, 26 pore forming toxins from sea anemone and pneumolysin; 27 (4) Colicin E1, a bacteriocin; 28 and (5) Bax. 29 Whether cytolytic proteins generate these structures is uncertain.…”

“…1 Ring-shaped transmembrane PFN pores hereafter called 'cylindrical pores', are presumed to act as the gateway for cytosolic entry, either at the plasma membrane or after endocytosis. [2][3][4] In either case the highly cationic Gzms are thought to diffuse through these cylindrical pores formed by poly-PFN. Nevertheless, a mechanistic understanding of the phenomenon (how the cationic globular protein exchanges from its carrier proteoglycan, serglycin, to the pore and crosses the plasma and/or vesicular membranes) has been lacking due to limitations in imaging technology and in our detailed understanding of the molecular forms that PFN may adopt following interaction with a target cell plasma membrane.…”

FADD cleavage by NK cell granzyme M enhances its self-association to facilitate procaspase-8 recruitment for auto-processing leading to caspase cascade

“…1 Perforin assists the entry of Gzms into the target cell cytosol, whose structural basis was just defined. 2 Gzms are a number of serine proteases that induce distinct cell death pathways through proteolysis of different target cell substrates. [3][4][5][6][7][8][9][10] Granzyme M (GzmM) is a chymotrypsin-like serine protease that preferentially degrades its substrates after Met or Leu.…”

Granzyme B enters the mitochondria in a Sam50-, Tim22- and mtHsp70-dependent manner to induce apoptosis

“…[1][2][3][4] Among the five human granzymes (A, B, H, K and M), granzyme B (GB) triggers cell death in both a caspasedependent and caspase-independent manner, although human and mouse GB differ in their requirement for caspase activation. [5][6][7][8][9][10] GB-induced cell death also involves Bid/Bax/ Bak-mediated mitochondrial outer membrane permeabilization for the release of the apoptogenic factors cytochrome c, Smac/Diablo, Endo G, HtrA2 and AIF.…”