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Self-assembly of spider silk proteins is controlled by a pH-sensitive relay

Nature volume 465, pages 236–238 (2010)Cite this article

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Abstract

Nature’s high-performance polymer, spider silk, consists of specific proteins, spidroins, with repetitive segments flanked by conserved non-repetitive domains1,2. Spidroins are stored as a highly concentrated fluid dope. On silk formation, intermolecular interactions between repeat regions are established that provide strength and elasticity3,4. How spiders manage to avoid premature spidroin aggregation before self-assembly is not yet established. A pH drop to 6.3 along the spider’s spinning apparatus, altered salt composition and shear forces are believed to trigger the conversion to solid silk, but no molecular details are known. Miniature spidroins consisting of a few repetitive spidroin segments capped by the carboxy-terminal domain form metre-long silk-like fibres irrespective of pH5. We discovered that incorporation of the amino-terminal domain of major ampullate spidroin 1 from the dragline of the nursery web spider Euprosthenops australis (NT) into mini-spidroins enables immediate, charge-dependent self-assembly at pH values around 6.3, but delays aggregation above pH 7. The X-ray structure of NT, determined to 1.7 Å resolution, shows a homodimer of dipolar, antiparallel five-helix bundle subunits that lack homologues. The overall dimeric structure and observed charge distribution of NT is expected to be conserved through spider evolution and in all types of spidroins. Our results indicate a relay-like mechanism through which the N-terminal domain regulates spidroin assembly by inhibiting precocious aggregation during storage, and accelerating and directing self-assembly as the pH is lowered along the spider’s silk extrusion duct.

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Figure 1: pH-dependent assembly of NT and mini-spidroins.
Figure 2: Structural and conserved features of spidroin N-terminal domain.

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References

  1. Candelas, G. C. & Cintron, J. A spider fibroin and its synthesis. J. Exp. Zool. 216, 1–6 (1981)

    Article  CAS  Google Scholar 

  2. Ayoub, N. A., Garb, J. E., Tinghitella, R. M., Collin, M. A. & Hayashi, C. Y. Blueprint for a high-performance biomaterial: full-length spider dragline silk genes. PLoS ONE 2, e514

  3. Gosline, J. M., Guerette, P. A., Ortlepp, C. S. & Savage, K. N. The mechanical design of spider silks: From fibroin sequence to mechanical function. J. Exp. Biol. 202, 3295–3303 (1999)

    CAS  Google Scholar 

  4. Ittah, S., Cohen, S., Garty, S., Cohn, D. & Gat, U. An essential role for the C-terminal domain of a dragline spider silk protein in directing fiber formation. Biomacromolecules 7, 1790–1795 (2006)

    Article  CAS  Google Scholar 

  5. Stark, M. et al. Macroscopic fibers self-assembled from recombinant miniature spider silk proteins. Biomacromolecules 8, 1695–1701 (2007)

    Article  CAS  Google Scholar 

  6. Hedhammar, M. et al. Structural properties of recombinant nonrepetitive and repetitive parts of major ampullate spidroin 1 from Euprosthenops australis: implications for fiber formation. Biochemistry 47, 3407–3417 (2008)

    Article  CAS  Google Scholar 

  7. Holm, L. & Sander, C. Dali: a network tool for protein structure comparison. Trends Biochem. Sci. 20, 478–480 (1995)

    Article  CAS  Google Scholar 

  8. Lin, Z., Huang, W., Zhang, J., Fan, J. S. & Yang, D. Solution structure of eggcase silk protein and its implications for silk fiber formation. Proc. Natl Acad. Sci. USA 106, 8906–8911 (2009)

    Article  ADS  CAS  Google Scholar 

  9. Pace, C. N., Grimsley, G. R. & Scholtz, J. M. Protein ionizable groups: pK values and their contribution to protein stability and solubility. J. Biol. Chem. 284, 13285–13289 (2009)

    Article  CAS  Google Scholar 

  10. Flocco, M. M. & Mowbray, S. L. Strange bedfellows: interactions between acidic side-chains in proteins. J. Mol. Biol. 254, 96–105 (1995)

    Article  CAS  Google Scholar 

  11. Li, H., Robertson, A. D. & Jensen, J. H. Very fast empirical prediction and rationalization of protein pKa values. Proteins 61, 704–721 (2005)

    Article  CAS  Google Scholar 

  12. Gordon, J.C. et al. H++: a server for estimating pK as and adding missing hydrogens to macromolecules. Nucleic Acids Res. 33, (Web Server issue) W368–W371 (2005)

    Article  CAS  Google Scholar 

  13. Kabsch, W. A solution for the best rotation to relate two sets of vectors. Acta Crystallogr. A 32, 922–923 (1976)

    Article  ADS  Google Scholar 

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

  15. Leslie, A. G. W. Recent changes to the MOSFLM package for processing film and image plate data. Joint CCP4 and ESF-EACBM Newsletter on Protein Crystallography no. 26, (1992)

  16. Evans, P. R. SCALA. Joint CCP4 ESF-EACBM Newsletter Protein Crystallography 33, 22–24 (1997)

    Google Scholar 

  17. Weeks, C. M. et al. Automatic solution of heavy-atom substructures. Methods Enzymol. 374, 37–83 (2003)

    Article  CAS  Google Scholar 

  18. Sheldrick, G. M. & Schneider, T. R. SHELXL: high-resolution refinement. Methods Enzymol. 277, 319–343 (1997)

    Article  CAS  Google Scholar 

  19. Adams, P. D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D 58, 1948–1954 (2002)

    Article  Google Scholar 

  20. Zwart, P. H. et al. Automated structure solution with the PHENIX suite. Methods Mol. Biol. 426, 419–435 (2008)

    Article  CAS  Google Scholar 

  21. 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)

    Article  Google Scholar 

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

    Article  Google Scholar 

  23. Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991)

    Article  Google Scholar 

  24. 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)

    Article  CAS  Google Scholar 

  25. Painter, J. & Merritt, E. A. Optimal description of a protein structure in terms of multiple groups undergoing TLS motion. Acta Crystallogr. D 62, 439–450 (2006)

    Article  Google Scholar 

  26. Painter, J. & Merritt, E. A. TLSMD web server for the generation of multi-group TLS models. J. Appl. Cryst. 39, 109–111 (2006)

    Article  CAS  Google Scholar 

  27. Rising, A., Hjalm, G., Engstrom, W. & Johansson, J. N-terminal nonrepetitive domain common to dragline, flagelliform, and cylindriform spider silk proteins. Biomacromolecules 7, 3120–3124 (2006)

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported by grants (to J.J., M.H. and S.D.K.) from the Swedish Research Council and FORMAS. We thank E. Andersson, K. Tars and D. Ericsson for collecting the high resolution wild-type X-ray diffraction data set, H. Bramfeldt and C. Aulin for help with collecting scanning electron microscopy images, L. Serpell, I. Andersson, A. Zavialov and T. Härd for comments on the manuscript, and a number of other colleagues for discussions. We thank the ESRF (Grenoble, France) beamline staff for help during data collection.

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Author notes

  1. Glareh Askarieh and My Hedhammar: These authors contributed equally to this work

Authors and Affiliations

  1. Department of Chemistry, Oslo University, 1033 Blindern, 0315 Oslo, Norway,

    Glareh Askarieh

  2. Department of Molecular Biology, Uppsala BioCenter, SLU, Biomedical Centre, P.O. Box 590, SE-751 24 Uppsala, Sweden,

    Glareh Askarieh & Stefan D. Knight

  3. Department of Anatomy, Physiology and Biochemistry, SLU, Biomedical Centre, P.O. Box 575, SE-751 23 Uppsala, Sweden,

    My Hedhammar, Kerstin Nordling, Anna Rising & Jan Johansson

  4. Department of Biochemistry and Molecular Biology I & CIBER Enfermedades Respiratorias, Complutense University of Madrid, 28040-Madrid, Spain

    Alejandra Saenz & Cristina Casals

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  1. Glareh Askarieh
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Contributions

G.A. and M.H. contributed equally to this work. G.A., M.H. performed experiments and wrote the paper, K.N., A.S. performed experiments, C.C., A.R., J.J., S.D.K. discussed experiments and wrote the paper.

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Correspondence to Jan Johansson or Stefan D. Knight.

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Competing interests

M.H., K.N., A.R. and J.J. own stocks and are funded by Spiber Technologies AB, a company that aims to commercialize recombinant spider silk for biomedical applications.

Additional information

X-ray crystallographic coordinates and structure factors have been deposited in the RCSB Protein Data Bank (PDB) with PDB ID codes 3LR2, 3LR6, 3LR8, 3LRD.

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This file contains Supplementary Figures 1-8 with legends and Supplementary Tables 1-2. (PDF 607 kb)

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Askarieh, G., Hedhammar, M., Nordling, K. et al. Self-assembly of spider silk proteins is controlled by a pH-sensitive relay. Nature 465, 236–238 (2010). https://doi.org/10.1038/nature08962

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