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Knot formation in newly translated proteins is spontaneous and accelerated by chaperonins

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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Figure 1: Structures of the trefoil-knotted methyltransferases YibK and YbeA.
Figure 2: Characterization of the oligomeric state of in vitro–translated knotted proteins.
Figure 3: Analysis of stability of knotted proteins produced by cell-free translation.
Figure 4: Kinetics of translation and folding for in vitro–synthesized knotted proteins.
Figure 5: A model for the knotting and folding of newly translated topologically knotted proteins in bacteria.

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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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Correspondence to Sophie E Jackson.

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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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