Crystal structure of a stable dimer reveals the molecular basis of serpin polymerization

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Abstract

Repeating intermolecular protein association by means of β-sheet expansion is the mechanism underlying a multitude of diseases including Alzheimer’s, Huntington’s and Parkinson’s and the prion encephalopathies1. A family of proteins, known as the serpins, also forms large stable multimers by ordered β-sheet linkages leading to intracellular accretion and disease2. These ‘serpinopathies’ include early-onset dementia caused by mutations in neuroserpin, liver cirrhosis and emphysema caused by mutations in α1-antitrypsin (α1AT), and thrombosis caused by mutations in antithrombin3. Serpin structure and function are quite well understood, and the family has therefore become a model system for understanding the β-sheet expansion disorders collectively known as the conformational diseases4. To develop strategies to prevent and reverse these disorders, it is necessary to determine the structural basis of the intermolecular linkage and of the pathogenic monomeric state. Here we report the crystallographic structure of a stable serpin dimer which reveals a domain swap of more than 50 residues, including two long antiparallel β-strands inserting in the centre of the principal β-sheet of the neighbouring monomer. This structure explains the extreme stability of serpin polymers, the molecular basis of their rapid propagation, and provides critical new insights into the structural changes which initiate irreversible β-sheet expansion.

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Figure 1: Crystallographic structures of active, latent and self-terminating dimer of the serpin antithrombin.
Figure 2: Biochemical properties of serpin polymers and the M* state.
Figure 3: Models of the serpin polymer and the M* state.

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Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors have been deposited in the Protein Data Bank under the accession code 2ZNH.

References

  1. 1

    Harrison, R. S., Sharpe, P. C., Singh, Y. & Fairlie, D. P. Amyloid peptides and proteins in review. Rev. Physiol. Biochem. Pharmacol. 159, 1–77 (2007)

  2. 2

    Lomas, D. A. & Carrell, R. W. Serpinopathies and the conformational dementias. Nature Rev. Genet. 3, 759–768 (2002)

  3. 3

    Lomas, D. A. et al. Molecular mousetraps and the serpinopathies. Biochem. Soc. Trans. 33, 321–330 (2005)

  4. 4

    Carrell, R. W. & Lomas, D. A. Conformational disease. Lancet 350, 134–138 (1997)

  5. 5

    Sharp, H. L., Bridges, R. A., Krivit, W. & Freier, E. F. Cirrhosis associated with α1-antitrypsin deficiency: a previously unrecognized inherited disorder. J. Lab. Clin. Med. 73, 934–939 (1969)

  6. 6

    Lomas, D. A., Evans, D. L., Finch, J. T. & Carrell, R. W. The mechanism of Z α1-antitrypsin accumulation in the liver. Nature 357, 605–607 (1992)

  7. 7

    Lomas, D. A. et al. Polymerisation underlies α1-antitrypsin deficiency, dementia and other serpinopathies. Front. Biosci. 9, 2873–2891 (2004)

  8. 8

    Huntington, J. A., Read, R. J. & Carrell, R. W. Structure of a serpin-protease complex shows inhibition by deformation. Nature 407, 923–926 (2000)

  9. 9

    Gettins, P. G. Serpin structure, mechanism, and function. Chem. Rev. 102, 4751–4804 (2002)

  10. 10

    Im, H., Ahn, H. Y. & Yu, M. H. Bypassing the kinetic trap of serpin protein folding by loop extension. Protein Sci. 9, 1497–1502 (2000)

  11. 11

    Corral, J. et al. Mutations in the shutter region of antithrombin result in formation of disulfide-linked dimers and severe venous thrombosis. J. Thromb. Haemost. 2, 931–939 (2004)

  12. 12

    Dafforn, T. R., Mahadeva, R., Elliott, P. R., Sivasothy, P. & Lomas, D. A. A kinetic mechanism for the polymerization of α1-antitrypsin. J. Biol. Chem. 274, 9548–9555 (1999)

  13. 13

    Kim, D. & Yu, M. H. Folding pathway of human α1-antitrypsin: characterization of an intermediate that is active but prone to aggregation. Biochem. Biophys. Res. Commun. 226, 378–384 (1996)

  14. 14

    Devlin, G. L., Chow, M. K., Howlett, G. J. & Bottomley, S. P. Acid denaturation of α1-antitrypsin: characterization of a novel mechanism of serpin polymerization. J. Mol. Biol. 324, 859–870 (2002)

  15. 15

    Tew, D. J. & Bottomley, S. P. Probing the equilibrium denaturation of the serpin α1-antitrypsin with single tryptophan mutants; evidence for structure in the urea unfolded state. J. Mol. Biol. 313, 1161–1169 (2001)

  16. 16

    Egelund, R. et al. A regulatory hydrophobic area in the flexible joint region of plasminogen activator inhibitor-1, defined with fluorescent activity-neutralizing ligands. Ligand-induced serpin polymerization. J. Biol. Chem. 276, 13077–13086 (2001)

  17. 17

    Zhou, A. & Carrell, R. W. Dimers initiate and propagate serine protease inhibitor polymerisation. J. Mol. Biol. 375, 36–42 (2008)

  18. 18

    Yu, M. H., Lee, K. N. & Kim, J. The Z type variation of human α1-antitrypsin causes a protein folding defect. Nature Struct. Biol. 2, 363–367 (1995)

  19. 19

    James, E. L., Whisstock, J. C., Gore, M. G. & Bottomley, S. P. Probing the unfolding pathway of α1-antitrypsin. J. Biol. Chem. 274, 9482–9488 (1999)

  20. 20

    Kjoller, L. et al. Conformational changes of the reactive-centre loop and β-strand 5A accompany temperature-dependent inhibitor-substrate transition of plasminogen-activator inhibitor 1. Eur. J. Biochem. 241, 38–46 (1996)

  21. 21

    Jung, C. H., Na, Y. R. & Im, H. Retarded protein folding of deficient human α1-antitrypsin D256V and L41P variants. Protein Sci. 13, 694–702 (2004)

  22. 22

    Bennett, M. J., Sawaya, M. R. & Eisenberg, D. Deposition diseases and 3D domain swapping. Structure 14, 811–824 (2006)

  23. 23

    Liu, Y. & Eisenberg, D. 3D domain swapping: as domains continue to swap. Protein Sci. 11, 1285–1299 (2002)

  24. 24

    James, E. L. & Bottomley, S. P. The mechanism of α1-antitrypsin polymerization probed by fluorescence spectroscopy. Arch. Biochem. Biophys. 356, 296–300 (1998)

  25. 25

    Pearce, M. C., Cabrita, L. D., Ellisdon, A. M. & Bottomley, S. P. The loss of tryptophan 194 in antichymotrypsin lowers the kinetic barrier to misfolding. FEBS J. 274, 3622–3632 (2007)

  26. 26

    Pedersen, K. E. et al. Plasminogen activator inhibitor-1 polymers, induced by inactivating amphipathic organochemical ligands. Biochem. J. 372, 747–755 (2003)

  27. 27

    Crowther, D. C., Serpell, L. C., Dafforn, T. R., Gooptu, B. & Lomas, D. A. Nucleation of α1-antichymotrypsin polymerization. Biochemistry 42, 2355–2363 (2003)

  28. 28

    Davis, R. L. et al. Familial dementia caused by polymerization of mutant neuroserpin. Nature 401, 376–379 (1999)

  29. 29

    Soto, C. et al. β-sheet breaker peptides inhibit fibrillogenesis in a rat brain model of amyloidosis: implications for Alzheimer’s therapy. Nature Med. 4, 822–826 (1998)

  30. 30

    Olson, S. T., Bjork, I. & Shore, J. D. Kinetic characterization of heparin-catalyzed and uncatalyzed inhibition of blood coagulation proteinases by antithrombin. Methods Enzymol. 222, 525–559 (1993)

  31. 31

    Stock, D., Perisic, O. & Lowe, J. Robotic nanolitre protein crystallisation at the MRC Laboratory of Molecular Biology. Prog. Biophys. Mol. Biol. 88, 311–327 (2005)

  32. 32

    Leslie, A. W. G. in Joint CCP4 and ESF-EACMB Newsletter on Protein Crystallography (Daresbury Laboratory, 1992)

  33. 33

    McCoy, A. J., Grosse-Kunstleve, R. W., Storoni, L. C. & Read, R. J. Likelihood-enhanced fast translation functions. Acta Crystallogr. D 61, 458–464 (2005)

  34. 34

    Brunger, A. T. et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D. 54, 905–921 (1998)

  35. 35

    McRee, D. E. A visual protein crystallographic software system for X11/XView. J. Mol. Graph. 10, 44–46 (1992)

  36. 36

    Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

  37. 37

    Lovell, S. C. et al. Structure validation by Cα geometry: φ, ψ and Cβ deviation. Proteins 50, 437–450 (2003)

  38. 38

    DeLano, W. L. The PyMOL Molecular Graphics System. <http://www.pymol.org> (2002)

  39. 39

    Zhou, A., Carrell, R. W. & Huntington, J. A. The serpin inhibitory mechanism is critically dependent on the length of the reactive center loop. J. Biol. Chem. 276, 27541–27547 (2001)

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Acknowledgements

We thank R. Carrell, D. Crowther and D. Lomas for their comments on the manuscript. We are also grateful to J. Löwe for access to the robotic crystallisation facility in the MRC-LMB, and to M. Weldon for N-terminal sequencing. This work was supported by the National Institutes of Health (USA) and the Uehara Memorial Foundation (Japan, to M.Y.), and J.A.H. is a senior MRC non-clinical fellow. Data were collected at beamlines I02 and I04 at the Diamond Light Source and we acknowledge the support of L. Duke, G. Evans, R. Flaig, J. Sandy and T. Sorensen.

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Correspondence to James A. Huntington.

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