Mechanistic Insights into the Folding of Knotted Proteins In Vitro and In Vivo

JOURNAL OF MOLECULAR BIOLOGY

卷:427

Article

Lim, Nicole C. H.^1,2  Jackson, Sophie E.¹ 

[1] Univ Cambridge, Dept Chem, Cambridge CB2 1EW, England

[2] Univ Brunei Darussalam, Fac Sci, BE-1410 Gadong, Brunei

关键词: protein folding;    knotted;    chaperonins;    GroEL-GroES;    nascent chain;   

DOI  :  10.1016/j.jmb.2014.09.007

来源: Elsevier

【 摘 要 】

The importance of knots and entanglements in biological systems is increasingly being realized and the number of proteins with topologically complex knotted structures has risen. However, the mechanism as to how these proteins knot and fold efficiently remains unclear. Using a cell-free expression system and pulse-proteolysis experiments, we have investigated the mechanism of knotting and folding for two bacterial trefoil-knotted methyltransferases. This study provides the first experimental evidence for a knotting mechanism. Results on fusions of stable protein domains to N-terminus, C-terminus or both termini of the knotted proteins clearly demonstrate that threading of the nascent chain through a knotting loop occurs via the C-terminus. Our results strongly suggest that this mechanism occurs even when the C-terminus is severely hindered by the addition of a large stable structure, in contrast to some simulations indicating that even the folding pathways of knotted proteins have some plasticity. The same strategy was employed to probe the effects of GroEL-GroES. In this case, results suggest active mechanisms for the molecular chaperonin. We demonstrate that a simple model in which GroEL-GroES sterically confines the unknotted polypeptide chain thereby promoting knotting is unlikely, and we propose two alternatives: (a) the chaperonin facilitates unfolding of kinetically and topologically trapped intermediates or (b) the chaperonin stabilizes interactions that promote knotting. These findings provide mechanistic insights into the folding of knotted proteins both in vitro and in vivo, thus elucidating how they have withstood evolutionary pressures regardless of their complex topologies and intrinsically slow folding rates. Crown Copyright (C) 2014 Published by Elsevier Ltd. All rights reserved.

【 授权许可】

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