Abstract
Topological knots are found in a considerable number of protein structures, but it is not clear how they knot and fold within the cellular environment. We investigated the behavior of knotted protein molecules as they are first synthesized by the ribosome using a cell-free translation system. We found that newly translated knotted proteins can spontaneously self-tie and do not require the assistance of molecular chaperones to fold correctly to their trefoil-knotted structures. This process is slow but efficient, and we found no evidence of misfolded species. A kinetic analysis indicates that the knotting process is rate limiting, occurs post-translationally, and is specifically and significantly (P < 0.001) accelerated by the GroEL–GroES chaperonin complex. This demonstrates a new active mechanism for this molecular chaperone and suggests that chaperonin-catalyzed knotting probably dominates in vivo. These results explain how knotted protein structures have withstood evolutionary pressures despite their topological complexity.
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References
Onuchic, J.N. & Wolynes, P.G. Theory of protein folding. Curr. Opin. Struct. Biol. 14, 70–75 (2004).
Dill, K.A. & Chan, H.S. From Levinthal to pathways to funnels. Nat. Struct. Biol. 4, 10–19 (1997).
Friel, C.T., Smith, D.A., Vendruscolo, M., Gsponer, J. & Radford, S.E. The mechanism of folding of Im7 reveals competition between functional and kinetic evolutionary contraints. Nat. Struct. Mol. Biol. 16, 318–324 (2009).
Watters, A.L. et al. The highly cooperative folding of small naturally occurring proteins is likely the result of natural selection. Cell 128, 613–624 (2007).
Baker, D. A surprising simplicity to protein folding. Nature 405, 39–42 (2000).
Bölinger, D. et al. A Stevedore's protein knot. PLOS Comput. Biol. 6, e1000731 (2010).
Taylor, W.R. Protein knots and fold complexity: some new twists. Comput. Biol. Chem. 31, 151–162 (2007).
Virnau, P., Mirny, L.A. & Kardar, M. Intricate knots in proteins: function and evolution. PLoS Comput. Biol. 2, e122 (2006).
Virnau, P., Mallam, A.L. & Jackson, S.E. Structures and folding pathways of topologically knotted proteins. J. Phys. Condens. Matter 23, 033101 (2011).
Lim, K. et al. Structure of the YibK methyltransferase from Haemophilus influenzae (HI0766): a cofactor bound at a site formed by a knot. Proteins 51, 56–67 (2003).
Mallam, A.L. & Jackson, S.E. Folding studies on a knotted protein. J. Mol. Biol. 346, 1409–1421 (2005).
Mallam, A.L. & Jackson, S.E. Probing Nature's knots: the folding pathway of a knotted homodimeric protein. J. Mol. Biol. 359, 1420–1436 (2006).
Mallam, A.L. & Jackson, S.E. A comparison of the folding of two knotted proteins: YbeA and YibK. J. Mol. Biol. 366, 650–665 (2007).
Mallam, A.L. & Jackson, S.E. The dimerization of an α/β-knotted protein is essential for structure and function. Structure 15, 111–122 (2007).
Mallam, A.L., Morris, E.R. & Jackson, S.E. Exploring knotting mechanisms in protein folding. Proc. Natl. Acad. Sci. USA 105, 18740–18745 (2008).
Mallam, A.L., Onuoha, S.C., Grossmann, J.G. & Jackson, S.E. Knotted fusion proteins reveal unexpected possibilities in protein folding. Mol. Cell 30, 642–648 (2008).
Sułkowska, J.I., Sulkowski, P. & Onuchic, J. Dodging the crisis of folding proteins with knots. Proc. Natl. Acad. Sci. USA 106, 3119–3124 (2009).
Wallin, S., Zeldovich, K.B. & Shakhnovich, E.I. The folding mechanics of a knotted protein. J. Mol. Biol. 368, 884–893 (2007).
Mallam, A.L., Rogers, J.M. & Jackson, S.E. Experimental detection of knotted conformations in denatured proteins. Proc. Natl. Acad. Sci. USA 107, 8189–8194 (2010).
Sułkowska, J.I., Sulkowska, P., Szymczak, P. & Cieplak, M. Untying Knots in Proteins. J. Am. Chem. Soc. 132, 13954–13956 (2010).
Mallam, A.L. How does a knotted protein fold? FEBS J. 276, 365–375 (2009).
Cabrita, L.D., Dobson, C.M. & Christodoulou, J. Protein folding on the ribosome. Curr. Opin. Struct. Biol. 20, 33–45 (2010).
Kim, M.S., Song, J. & Park, C. Determining protein stability in cell lysates by pulse proteolysis and western blotting. Protein Sci. 18, 1051–1059 (2009).
Park, C. & Marqusee, S. Pulse proteolysis: a simple method for quantitative determination of protein stability and ligand binding. Nat. Methods 2, 207–212 (2005).
Schlebach, J.P., Kim, M.S., Joh, N.H., Bowie, J.U. & Park, C. Probing membrane protein unfolding with pulse proteolysis. J. Mol. Biol. 406, 545–551 (2011).
Shimizu, Y. et al. Cell-free translation reconstituted with purified components. Nat. Biotechnol. 19, 751–755 (2001).
Shimizu, Y., Kanamori, T. & Ueda, T. Protein synthesis by pure translation systems. Methods 36, 299–304 (2005).
Ying, B.W., Taguchi, H., Ueda, H. & Ueda, T. Chaperone-assisted folding of a single-chain antibody in a reconstituted translation system. Biochem. Biophys. Res. Commun. 320, 1359–1364 (2004).
Purta, E., Kaminska, K.H., Kasprzak, J.M., Bujnicki, J.M. & Douthwaite, S. YbeA is the m3Psi methyltransferase RlmH that targets nucleotide 1915 in 23S rRNA. RNA 14, 2234–2244 (2008).
Hartl, F.U. & Hayer-Hartl, M. Converging concepts of protein folding in vitro and in vivo. Nat. Struct. Mol. Biol. 16, 574–581 (2009).
Hartl, F.U. & Hayer-Hartl, M. Molecular chaperones in the cytosol: from nascent chain to folded protein. Science 295, 1852–1858 (2002).
Noel, J.K., Sulkowska, J.I. & Onuchic, J.N. Slipknotting upon native-like loop formation in a trefoil knot protein. Proc. Natl. Acad. Sci. USA 107, 15403–15408 (2010).
Jackson, S.E. How do small single-domain proteins fold? Fold. Des. 3, R81–R91 (1998).
Prentiss, M.C., Wales, D.J. & Wolynes, P.G. The energy landscape, folding pathways and the kinetics of a knotted protein. PLOS Comput. Biol. 6, e1000835 (2010).
King, N.P., Jacobitz, A.W., Sawaya, M.R., Goldschmidt, L. & Yeates, T.O. Structure and folding of a designed knotted protein. Proc. Natl. Acad. Sci. USA 107, 20732–20737 (2010).
Hsu, S.T., Blaser, G. & Jackson, S.E. The folding, stability and conformational dynamics of β-barrel fluorescent proteins. Chem. Soc. Rev. 38, 2951–2965 (2009).
Plaxco, K.W., Simons, K.T. & Baker, D. Contact order, transition state placement and the refolding rates of single domain proteins. J. Mol. Biol. 277, 985–994 (1998).
Kerner, M.J. et al. Proteome-wide analysis of chaperonin-dependent protein folding in Escherichia coli. Cell 122, 209–220 (2005).
Fujiwara, K., Ishihama, Y., Nakahigashi, K., Soga, T. & Taguchi, H. A systematic survey of in vivo obligate chaperonon-dependent substrates. EMBO J. 29, 1552–1564 (2010).
Maisnier-Patin, S. et al. Genomic buffering mitigates the effects of deleterious mutations in bacteria. Nat. Genet. 37, 1376–1379 (2005).
Chakraborty, K. et al. Chaperonin-catalyzed rescue of kinetically trapped states in protein folding. Cell 142, 112–122 (2010).
Cheung, M.S. & Thirumalai, D. Nanopore-protein interactions dramatically alter stability and yield of the native state in restricted spaces. J. Mol. Biol. 357, 632–643 (2006).
Baumketner, A., Jewett, A. & Shea, J.E. Effects of confinement in chaperonin assisted protein folding: Rate enhancement by decreasing the roughness of the folding energy landscape. J. Mol. Biol. 332, 701–713 (2003).
Virnau, P., Kantor, Y. & Kardar, M. Knots in globule and coil phases of a model polyethylene. J. Am. Chem. Soc. 127, 15102–15106 (2005).
Micheletti, C., Marenduzzo, D., Orlandini, E. & Sumners, D.W. Knotting of random ring polymers in confined spaces. J. Chem. Phys. 124, 64903 (2006); erratum 7, 219903 (2006).
Johnson, K.A., Simpson, Z.B. & Blom, T. Global kinetic explorer: a new computer program for dynamic simulation and fitting of kinetic data. Anal. Biochem. 387, 20–29 (2009).
Acknowledgements
We thank E. O'Brien, D. Hsu, F. Andersson, G. Blaser and E. Werrell for helpful discussions. This research was supported by a fellowship and a grant from St. John's College, University of Cambridge, UK (to A.L.M.).
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A.L.M. and S.E.J. designed research; A.L.M. carried out research; A.L.M. and S.E.J. analyzed data, and A.L.M. and S.E.J. wrote the paper.
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Mallam, A., Jackson, S. Knot formation in newly translated proteins is spontaneous and accelerated by chaperonins. Nat Chem Biol 8, 147–153 (2012). https://doi.org/10.1038/nchembio.742
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DOI: https://doi.org/10.1038/nchembio.742
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