data_sheet_1.PDF

<p>The ability of cytotoxic lymphocytes (CL) to eliminate virus-infected or cancerous target cells through the granule exocytosis death pathway is critical to immune homeostasis. Congenital loss of CL function due to bi-allelic mutations in PRF1, UNC13D, STX11, or STXBP2 leads to a potentially fatal immune dysregulation, familial haemophagocytic lymphohistiocytosis (FHL). This occurs due to the failure of CLs to release functional pore-forming protein perforin and, therefore, inability to kill the target cell. Bi-allelic mutations in partner proteins STXBP2 or STX11 impair CL cytotoxicity due to failed docking/fusion of cytotoxic secretory granules with the plasma membrane. One unique feature of STXBP2- and STX11-deficient patient CLs is that their short-term in vitro treatment with a low concentration of IL-2 partially or completely restores natural killer (NK) cell degranulation and cytotoxicity, suggesting the existence of a secondary, yet unknown, pathway for secretory granule exocytosis. In the current report, we studied NK and T-cell function in an individual with late presentation of FHL due to hypomorphic bi-allelic mutations in STXBP2. Intriguingly, in addition to the expected alterations in the STXBP2 and STX11 proteins, we also observed a concomitant significant reduction in the expression of homologous STXBP1 protein and its partner STX1, which had never been implicated in CL function. Further analysis of human NK and T cells demonstrated a functional role for the STXBP1/STX1 axis in NK and CD8+ T-cell cytotoxicity, where it appears to be responsible for as much as 50% of their cytotoxic activity. This discovery suggests a unique and previously unappreciated interplay between STXBP/Munc proteins regulating the same essential granule exocytosis pathway.</p

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

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, Ca2+-dependent membrane binding. Most unexpectedly, however, cryo-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

Exploration of a Series of 5‑Arylidene-2-thioxoimidazolidin-4-ones as Inhibitors of the Cytolytic Protein Perforin

A series of novel 5-arylidene-2-thioxoimidazolidin-4-ones were investigated as inhibitors of the lymphocyte-expressed pore-forming protein perforin. Structure–activity relationships were explored through variation of an isoindolinone or 3,4-dihydroisoquinolinone subunit on a fixed 2-thioxoimidazolidin-4-one/thiophene core. The ability of the resulting compounds to inhibit the lytic activity of both isolated perforin protein and perforin delivered in situ by natural killer cells was determined. A number of compounds showed excellent activity at concentrations that were nontoxic to the killer cells, and several were a significant improvement on previous classes of inhibitors, being substantially more potent and soluble. Representative examples showed rapid and reversible binding to immobilized mouse perforin at low concentrations (≤2.5 μM) by surface plasmon resonance and prevented formation of perforin pores in target cells despite effective target cell engagement, as determined by calcium influx studies. Mouse PK studies of two analogues showed <i>T</i>_1/2 values of 1.1–1.2 h (dose of 5 mg/kg iv) and MTDs of 60–80 mg/kg (ip)

Electron microscopy of pleurotolysin pores.

<p>Representative views of negatively stained (A) and vitrified (B) Ply pores on liposomes. (C) Averaged views of 12-fold and 13-fold symmetric pores on lipid monolayers (negative stain). (D) Averaged side view of Ply pores on liposomes (cryo-EM). Scale bar, 20 nm.</p

Three dimensional (3-D) reconstructions of disulphide locked pleurotolysin prepores.

<p>(A) PlyB crystal structure with positions of TMH1 disulphide lock indicated by magenta spheres and corresponding side view average of the liposome-bound prepore (cryo-EM). Scale bar, 20 nm. Main panel, cut-away view of the prepore cryo-EM map with the model obtained by flexible fitting. No TMH density is seen in the TMH1 lock prepore map. (B) The equivalent panels are shown for the TMH2 helix lock map and model. Partial density is seen for the TMH1 region. (C) Equivalent views of the TMH2 strand lock map and model. No density is seen for the TMH regions. These regions must therefore be disordered and they were omitted from the fits. The disordered regions are shown schematically as yellow dashed lines; the long TMH1 helix is illustrated in (B) but was not part of the fitting. Mutated residues are shown: TMH1 lock; F138C (located in the TMH1 region, yellow), H221C. TMH2 helix lock; Y166C, G266C (located in the TMH2 helix region, yellow). TMH2 strand lock; V277C (located in the fifth β-strand, TMH2 region, yellow), K291C, all on the C487A background.</p

Structure of the pleurotolysin pore.

<p>(A) Cut away side and (B) tilted surface views of the cryo-EM reconstruction of a pleurotolysin pore with the fitted atomic structures. (C) Segment of the pore map corresponding to a single subunit with pore model fitted into the density. The PlyB crystal structure is superposed to show a 70° opening of the MACPF β-sheet (red) and movement of the HTH motif (cyan). TMH regions (yellow) are refolded into transmembrane β-hairpins. The PlyB C-terminal trefoil (green) sits on top of the PlyA dimer (pink). (D) Interface between TMH2, the HTH region, and the underlying sheet in the PlyB crystal structure. The position of the TMH2 helix lock (pink spheres) and TMH2 strand lock (grey spheres) are shown. The highly conserved “GG” motif (296–297) in the HTH region is represented as yellow spheres.</p

Schematic diagram of sheet opening in pleurotolysin pore formation.

<p>(A) PlyA and B monomer structures with coloured shapes indicating the major elements used in flexible fitting. (B) and (C) show the two types of prepore structures found, which can be considered as early and later stages of sheet opening. The top 20 models (pink for the MACPF β-sheet and grey for HTH region) and best scoring model (red) in the pleurotolysin prepore maps are shown. (B) TMH2 helix lock, in which the best scoring model has a rotation angle (β-sheet opening) of 37° relative to the monomer structure. (C) TMH2 strand lock, in which the best scoring model has a rotation angle of 49° relative to the monomer. (D) Pore model with 70° open sheet and membrane inserted TMH regions.</p

Validation of the orientation of PlyA.

<p>(A) Proposed orientation of PlyA dimer (pink) and interface with PlyB C-terminal trefoil (green). Trp 6 is shown as purple spheres. (B) Western blot showing PlyA binding to red blood cells when untagged or C-terminally tagged but not when N-terminally tagged.</p