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

Nature volume 468, pages 447–451 (2010)Cite this article

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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).

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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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Author notes

  1. Ruby H. P. Law, Natalya Lukoyanova, Ilia Voskoboinik, Joseph A. Trapani, Helen R. Saibil and James C. Whisstock: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Monash University, Clayton, Melbourne, Victoria 3800, Australia,

    Ruby H. P. Law, Michelle A. Dunstone, Michael E. D’Angelo, Fasséli Coulibaly, Phillip I. Bird & James C. Whisstock

  2. The ARC Centre of Excellence in Structural and Functional Microbial Genomics, Monash University, Melbourne, Victoria 3800, Australia,

    Ruby H. P. Law & James C. Whisstock

  3. Crystallography, Institute of Structural and Molecular Biology, Birkbeck College, Malet Street, London WC1E 7HX, UK,

    Natalya Lukoyanova, Elena V. Orlova & Helen R. Saibil

  4. Cancer Immunology Program, Peter MacCallum Cancer Centre, St Andrew's Place, East Melbourne, Victoria 3002, Australia,

    Ilia Voskoboinik, Katherine Baran, Sandra Verschoor, Kylie A. Browne, Annette Ciccone & Joseph A. Trapani

  5. Department of Genetics, The University of Melbourne, Parkville, Victoria 3010, Australia,

    Ilia Voskoboinik

  6. Australian Synchrotron, 800 Blackburn Road, Clayton, Melbourne, Victoria 3168, Australia,

    Tom T. Caradoc-Davies

  7. Department of Microbiology, Monash University, Melbourne, Victoria 3800, Australia,

    Michelle A. Dunstone

  8. Life Sciences Department, Victorian Partnership of Advanced Computing, Carlton South, Victoria 3053, Australia,

    Michael J. Kuiper

  9. Department of Microbiology and Immunology, The University of Melbourne, Parkville, Victoria 3010, Australia,

    Joseph A. Trapani

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Contributions

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.

Corresponding authors

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

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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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