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 cell1,2,3,4,5,6. 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)7. 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) domain8,9 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, electron microscopy reveals that the orientation of the perforin MACPF domain in the pore is inside-out relative to the subunit arrangement in CDCs10,11. 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.

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Figure 1: Structure of perforin monomers.
Figure 2: Electron microscopy of perforin monomers.
Figure 3: Perforin pore structure.
Figure 4: Schematic comparison of pore formation in perforin and CDCs.

Accession codes

Accessions

Protein Data Bank

Data deposits

Structure factors and coordinates are deposited in the Protein Data Bank under accession number 3NSJ. Electron microscopy maps are deposited in the EM Databank (accession numbers EMD-1772 and EMD-1773 for the two conformations of perforin monomer and EMD-1769 for the pore).

References

  1. 1

    Tschopp, J., Masson, D. & Stanley, K. K. Structural/functional similarity between proteins involved in complement- and cytotoxic T-lymphocyte-mediated cytolysis. Nature 322, 831–834 (1986)

    CAS  ADS  Article  Google Scholar 

  2. 2

    Shinkai, Y., Takio, K. & Okumura, K. Homology of perforin to the ninth component of complement (C9). Nature 334, 525–527 (1988)

    CAS  ADS  Article  Google Scholar 

  3. 3

    Lichtenheld, M. G. et al. Structure and function of human perforin. Nature 335, 448–451 (1988)

    CAS  ADS  Article  Google Scholar 

  4. 4

    Lowin, B., Hahne, M., Mattmann, C. & Tschopp, J. Cytolytic T-cell cytotoxicity is mediated through perforin and Fas lytic pathways. Nature 370, 650–652 (1994)

    CAS  ADS  Article  Google Scholar 

  5. 5

    Kägi, D. et al. Cytotoxicity mediated by T cells and natural killer cells is greatly impaired in perforin-deficient mice. Nature 369, 31–37 (1994)

    ADS  Article  Google Scholar 

  6. 6

    Young, J. D., Cohn, Z. A. & Podack, E. R. The ninth component of complement and the pore-forming protein (perforin 1) from cytotoxic T cells: structural, immunological, and functional similarities. Science 233, 184–190 (1986)

    CAS  ADS  Article  Google Scholar 

  7. 7

    Voskoboinik, I., Smyth, M. J. & Trapani, J. A. Perforin-mediated target-cell death and immune homeostasis. Nature Rev. Immunol. 6, 940–952 (2006)

    CAS  Article  Google Scholar 

  8. 8

    Rosado, C. J. et al. A common fold mediates vertebrate defense and bacterial attack. Science 317, 1548–1551 (2007)

    CAS  ADS  Article  Google Scholar 

  9. 9

    Hadders, M. A., Beringer, D. X. & Gros, P. Structure of C8α-MACPF reveals mechanism of membrane attack in complement immune defense. Science 317, 1552–1554 (2007)

    CAS  ADS  Article  Google Scholar 

  10. 10

    Tilley, S. J., Orlova, E. V., Gilbert, R. J., Andrew, P. W. & Saibil, H. R. Structural basis of pore formation by the bacterial toxin pneumolysin. Cell 121, 247–256 (2005)

    CAS  Article  Google Scholar 

  11. 11

    Dang, T. X., Hotze, E. M., Rouiller, I., Tweten, R. K. & Wilson-Kubalek, E. M. Prepore to pore transition of a cholesterol-dependent cytolysin visualized by electron microscopy. J. Struct. Biol. 150, 100–108 (2005)

    CAS  Article  Google Scholar 

  12. 12

    Slade, D. J. et al. Crystal structure of the MACPF domain of human complement protein C8α in complex with the C8γ subunit. J. Mol. Biol. 379, 331–342 (2008)

    CAS  Article  Google Scholar 

  13. 13

    Rossjohn, J., Feil, S. C., McKinstry, W. J., Tweten, R. K. & Parker, M. W. Structure of a cholesterol-binding, thiol-activated cytolysin and a model of its membrane form. Cell 89, 685–692 (1997)

    CAS  Article  Google Scholar 

  14. 14

    Hurley, J. H. & Misra, S. Signaling and subcellular targeting by membrane-binding domains. Annu. Rev. Biophys. Biomol. Struct. 29, 49–79 (2000)

    CAS  Article  Google Scholar 

  15. 15

    Baran, K. et al. The molecular basis for perforin oligomerization and transmembrane pore assembly. Immunity 30, 684–695 (2009)

    CAS  Article  Google Scholar 

  16. 16

    Shepard, L. A. et al. Identification of a membrane-spanning domain of the thiol-activated pore-forming toxin Clostridium perfringens perfringolysin O: an α-helical to β-sheet transition identified by fluorescence spectroscopy. Biochemistry 37, 14563–14574 (1998)

    CAS  Article  Google Scholar 

  17. 17

    Shatursky, O. et al. The mechanism of membrane insertion for a cholesterol-dependent cytolysin: a novel paradigm for pore-forming toxins. Cell 99, 293–299 (1999)

    CAS  Article  Google Scholar 

  18. 18

    Urrea Moreno, R. et al. Functional assessment of perforin C2 domain mutations illustrates the critical role for calcium-dependent lipid binding in perforin cytotoxic function. Blood 113, 338–346 (2009)

    Article  Google Scholar 

  19. 19

    Podack, E. R., Young, J. D. & Cohn, Z. A. Isolation and biochemical and functional characterization of perforin 1 from cytolytic T-cell granules. Proc. Natl Acad. Sci. USA 82, 8629–8633 (1985)

    CAS  ADS  Article  Google Scholar 

  20. 20

    Young, J. D., Nathan, C. F., Podack, E. R., Palladino, M. A. & Cohn, Z. A. Functional channel formation associated with cytotoxic T-cell granules. Proc. Natl Acad. Sci. USA 83, 150–154 (1986)

    CAS  ADS  Article  Google Scholar 

  21. 21

    Voskoboinik, I. et al. Calcium-dependent plasma membrane binding and cell lysis by perforin are mediated through its C2 domain: a critical role for aspartate residues 429, 435, 483, and 485 but not 491. J. Biol. Chem. 280, 8426–8434 (2005)

    CAS  Article  Google Scholar 

  22. 22

    Shin, O. H. et al. Munc13 C2B domain is an activity-dependent Ca2+ regulator of synaptic exocytosis. Nature Struct. Mol. Biol. 17, 280–288 (2010)

    CAS  Article  Google Scholar 

  23. 23

    Czajkowsky, D. M., Hotze, E. M., Shao, Z. & Tweten, R. K. Vertical collapse of a cytolysin prepore moves its transmembrane β-hairpins to the membrane. EMBO J. 23, 3206–3215 (2004)

    CAS  Article  Google Scholar 

  24. 24

    Grobler, J. A. & Hurley, J. H. Similarity between C2 domain jaws and immunoglobulin CDRs. Nature Struct. Biol. 4, 261–262 (1997)

    CAS  Article  Google Scholar 

  25. 25

    Ramachandran, R., Tweten, R. K. & Johnson, A. E. The domains of a cholesterol-dependent cytolysin undergo a major FRET-detected rearrangement during pore formation. Proc. Natl Acad. Sci. USA 102, 7139–7144 (2005)

    CAS  ADS  Article  Google Scholar 

  26. 26

    Dourmashkin, R. R., Deteix, P., Simone, C. B. & Henkart, P. Electron microscopic demonstration of lesions in target cell membranes associated with antibody-dependent cellular cytotoxicity. Clin. Exp. Immunol. 42, 554–560 (1980)

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27

    Thiery, J. et al. Perforin activates clathrin- and dynamin-dependent endocytosis, which is required for plasma membrane repair and delivery of granzyme B for granzyme-mediated apoptosis. Blood 115, 1582–1593 (2010)

    CAS  Article  Google Scholar 

  28. 28

    Bird, C. H. et al. Cationic sites on granzyme B contribute to cytotoxicity by promoting its uptake into target cells. Mol. Cell. Biol. 25, 7854–7867 (2005)

    CAS  Article  Google Scholar 

  29. 29

    Brickner, A. & Sodetz, J. M. Functional domains of the α subunit of the eighth component of human complement: identification and characterization of a distinct binding site for the γ chain. Biochemistry 24, 4603–4607 (1985)

    CAS  Article  Google Scholar 

  30. 30

    Strong, M. et al. Toward the structural genomics of complexes: crystal structure of a PE/PPE protein complex from Mycobacterium tuberculosis. Proc. Natl Acad. Sci. USA 103, 8060–8065 (2006)

    CAS  ADS  Article  Google Scholar 

  31. 31

    Kabsch, W. Xds. Acta Crystallogr. D 66, 125–132 (2010)

    CAS  Article  Google Scholar 

  32. 32

    Evans, P. Scaling and assessment of data quality. Acta Crystallogr. D 62, 72–82 (2006)

    Article  Google Scholar 

  33. 33

    Vonrhein, C., Blanc, E., Roversi, P. & Bricogne, G. Automated structure solution with autoSHARP. Methods Mol. Biol. 364, 215–230 (2007)

    CAS  Google Scholar 

  34. 34

    Sheldrick, G. M. Experimental phasing with SHELXC/D/E: combining chain tracing with density modification. Acta Crystallogr. D 66, 479–485 (2010)

    CAS  Article  Google Scholar 

  35. 35

    de la Fortelle, E. & Bricogne, G. Maximum-likelihood heavy-atom parameter refinement for multiple isomorphous replacement and multiwavelength anomalous diffraction methods. Methods Enzymol. 276, 472–494 (1997)

    CAS  Article  Google Scholar 

  36. 36

    Abrahams, J. P. & Leslie, A. G. Methods used in the structure determination of bovine mitochondrial F1 ATPase. Acta Crystallogr. D 52, 30–42 (1996)

    CAS  Article  Google Scholar 

  37. 37

    Cowtan, K. D. & Zhang, K. Y. Density modification for macromolecular phase improvement. Prog. Biophys. Mol. Biol. 72, 245–270 (1999)

    CAS  Article  Google Scholar 

  38. 38

    Cowtan, K. The Buccaneer software for automated model building. 1. Tracing protein chains. Acta Crystallogr. D 62, 1002–1011 (2006)

    Article  Google Scholar 

  39. 39

    Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010)

    CAS  Article  Google Scholar 

  40. 40

    Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010)

    CAS  Article  Google Scholar 

  41. 41

    Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997)

    CAS  Article  Google Scholar 

  42. 42

    Bricogne, G. et al. BUSTER, version 2.8.0 (Global Phasing Ltd, Cambridge, UK, 2009)

  43. 43

    Collaborative Computational Project, 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994)

    Article  Google Scholar 

  44. 44

    Vriend, G. WHAT IF: a molecular modeling and drug design program. J. Mol. Graph. 8, 52–56 (1990)

    CAS  Article  Google Scholar 

  45. 45

    Konagurthu, A. S., Whisstock, J. C., Stuckey, P. J. & Lesk, A. M. MUSTANG: a multiple structural alignment algorithm. Proteins 64, 559–574 (2006)

    CAS  Article  Google Scholar 

  46. 46

    DeLano, W. L. The PyMOL molecular graphics system. (DeLano Scientific, Palo Alto, 2008); 〈http://www.pymol.org〉.

  47. 47

    Davis, I. W. et al. MolProbity: all-atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res. 35, W375–383 (2007)

    ADS  Article  Google Scholar 

  48. 48

    Crowther, R. A., Henderson, R. & Smith, J. M. MRC image processing programs. J. Struct. Biol. 116, 9–16 (1996)

    CAS  Article  Google Scholar 

  49. 49

    Ludtke, S. J., Baldwin, P. R. & Chiu, W. EMAN: semiautomated software for high-resolution single-particle reconstructions. J. Struct. Biol. 128, 82–97 (1999)

    CAS  Article  Google Scholar 

  50. 50

    van Heel, M., Harauz, G., Orlova, E. V., Schmidt, R. & Schatz, M. A new generation of the IMAGIC image processing system. J. Struct. Biol. 116, 17–24 (1996)

    CAS  Article  Google Scholar 

  51. 51

    Frank, J. et al. SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields. J. Struct. Biol. 116, 190–199 (1996)

    CAS  Article  Google Scholar 

  52. 52

    White, H. E., Saibil, H. R., Ignatiou, A. & Orlova, E. V. Recognition and separation of single particles with size variation by statistical analysis of their images. J. Mol. Biol. 336, 453–460 (2004)

    CAS  Article  Google Scholar 

  53. 53

    Pettersen, E. F. et al. UCSF Chimera — a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004)

    CAS  Article  Google Scholar 

  54. 54

    Phillips, J. C. et al. Scalable molecular dynamics with NAMD. J. Comput. Chem. 26, 1781–1802 (2005)

    CAS  Article  Google Scholar 

  55. 55

    Kawasaki, A. et al. Perforin, a pore-forming protein detectable by monoclonal antibodies, is a functional marker for killer cells. Int. Immunol. 2, 677–684 (1990)

    CAS  Article  Google Scholar 

  56. 56

    Sutton, V. R. et al. Measuring cell death mediated by cytotoxic lymphocytes or their granule effector molecules. Methods 44, 241–249 (2008)

    CAS  Article  Google Scholar 

  57. 57

    Sun, J. et al. Expression and purification of recombinant human granzyme B from Pichia pastoris. Biochem. Biophys. Res. Commun. 261, 251–255 (1999)

    CAS  Article  Google Scholar 

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Acknowledgements

J.C.W. is an Australian Research Council Federation Fellow and Honorary National Health and Medical Research Council of Australia Principal Research Fellow. I.V., F.C. and M.A.D. are NHMRC Career Development Fellows. K.B. is an NHMRC C.J. Martin overseas training fellow. J.A.T. acknowledges the support of an NHMRC Senior Principal Research Fellowship during the course of the work. The authors thank the NHMRC, the ARC, the UK BBSRC and the Wellcome Trust for grant support. We thank the Australian synchrotron beamline scientists for technical support and access to the MX-2 Microfocus Beamline; we thank D. Clare and L. Wang for electron microscopy support, and D. Houldershaw, R. Westlake and K. Mahmood for computing support. We thank D. Steer and the Monash University Proteomics Unit for technical support.

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R.H.P.L., N.L. and I.V. are joint first authors; J.A.T., H.R.S. and J.C.W. contributed equally to this work. R.H.P.L. crystallized perforin, performed the soaks, collected diffraction data, determined the structure and co-wrote the paper. N.L. performed electron microscopy structural analysis, and co-wrote the paper. I.V. developed the perforin expression system, designed and developed the oligomerization defective variants, produced the perforin variant, co-led the research and co-wrote the paper. T.T.C. collected data and determined the structure, and co-wrote the paper. K.B. developed perforin variants with defective oligomerization. M.A.D. analysed the structure, and co-wrote the paper. M.E.D. performed the bioinformatic research. E.V.O. developed procedures for image processing and analysis. F.C. assisted with determining the structure. S.V., K.A.B. and A.C. produced perforin. M.J.K. performed the modelling experiments. P.I.B. performed bioinformatic experiments, interpreted the data and co-wrote the paper. J.A.T., H.R.S. and J.C.W. analysed the data, led the research and co-wrote the paper.

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Correspondence to Joseph A. Trapani or Helen R. Saibil or James C. Whisstock.

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The authors declare no competing financial interests.

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Law, R., Lukoyanova, N., Voskoboinik, I. et al. The structural basis for membrane binding and pore formation by lymphocyte perforin. Nature 468, 447–451 (2010). https://doi.org/10.1038/nature09518

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