Exploring knotting mechanisms in protein folding

Mallam, Anna L.; Morris, Elizabeth R.; Jackson, Sophie

doi:10.1073/pnas.0806697105

Cited by 68 publications

(72 citation statements)

References 40 publications

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“…The question is whether the topology forces native-like monomers to fold before dimerization or whether the dimerization step could be coupled to the folding process as in so-called obligatory dimers (29). YibK, a 3 1 knotted protein, has been shown experimentally to first fold to a nativelike monomeric state before a slow dimerization step (10). MJ0366 has a homodimeric interface similar to that of YibK: Both interfaces include the C-terminal helix directly involved in the knotted structure.…”

Section: Resultsmentioning

confidence: 99%

Slipknotting upon native-like loop formation in a trefoil knot protein

Noel

Sułkowska

Onuchic

2010

Proc. Natl. Acad. Sci. U.S.A.

109

163

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Protein knots and slipknots, mostly regarded as intriguing oddities, are gradually being recognized as significant structural motifs. Recent experimental results show that knotting, starting from a fully extended polypeptide, has not yet been observed. Understanding the nucleation process of folding knots is thus a natural challenge for both experimental and theoretical investigation. In this study, we employ energy landscape theory and molecular dynamics to elucidate the entire folding mechanism. The full free energy landscape of a knotted protein is mapped using an all-atom structure-based protein model. Results show that, due to the topological constraint, the protein folds through a three-state mechanism that contains (i) a precise nucleation site that creates a correctly twisted native loop (first barrier) and (ii) a rate-limiting free energy barrier that is traversed by two parallel knot-forming routes. The main route corresponds to a slipknot conformation, a collapsed configuration where the C-terminal helix adopts a hairpin-like configuration while threading, and the minor route to an entropically limited plug motion, where the extended terminus is threaded as through a needle. Knot formation is a late transition state process and results show that random (nonspecific) knots are a very rare and unstable set of configurations both at and below folding temperature. Our study shows that a native-biased landscape is sufficient to fold complex topologies and presents a folding mechanism generalizable to all known knotted protein topologies: knotting via threading a native-like loop in a preordered intermediate.free energy landscape | knotted protein kinetics | nontrivial protein topology | protein folding | structure-based model P rotein structures have been observed with several complex folding motifs including knots and slipknots. These include nontrivial topologies containing 3 1 , 4 1 , 5 2 , and 6 1 knots (1-5). While the mechanism by which these proteins manage to reliably fold from a disordered linear polypeptide into complicated topologies is still largely a mystery, energy landscape theory is starting to provide us the key to resolve this challenge. In a minimally frustrated, funnel-like energy landscape, one expects that native contacts are on average favorable and dominate over nonfavorable nonnative ones (6-8). Topological constraints imposed by the existence of a native knot radically alters the funneled landscape. Many routes are barred from reaching the native state due to the obstacle imposed by the knot. Forming a knot requires intricate crossings of the polypeptide; any one made incorrectly leads to an unknotted protein or a wrong chirality. Therefore at first sight the problem of folding knots appears perplexing, but there is no reason to doubt that clues will be found in the native structure itself. Here it is shown how an all-atom structure-based model, which is dominated by native attractive interactions, is sufficient to uncover the energy landscape and folding routes of the smallest kn...

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Section: Resultsmentioning

confidence: 99%

Slipknotting upon native-like loop formation in a trefoil knot protein

Noel

Sułkowska

Onuchic

2010

Proc. Natl. Acad. Sci. U.S.A.

109

163

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“…Previous investigations on the folding mechanisms of a few knotted proteins established that these proteins have complex multistep folding pathways with slow kinetics rather than the simple two-state mechanisms that are commonly seen for small, fast-folding, single-domain proteins (6)(7)(8)(9)(10)(11). Despite experimental and computational work over the last 10 years (12)(13)(14)(15)(16)(17), the folding mechanisms of knotted proteins remain poorly understood compared with those described for small model systems that have been studied in great detail (18)(19)(20)(21)(22)(23).…”

Section: Introductionmentioning

confidence: 99%

Characterization of the Folding of a 5 2 -Knotted Protein Using Engineered Single-Tryptophan Variants

Zhang

Jackson

2016

Biophysical Journal

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An increasing number of proteins that contain topological knots have been identified over the past two decades; however, their folding mechanisms are still not well understood. UCH-L1 has a 5 2 -knotted topology. Here, we constructed a series of variants that contain a single tryptophan at different locations along the polypeptide chain. A study of the thermodynamic properties of the variants shows that the structure of UCH-L1 is remarkably tolerant to incorporation of bulky tryptophan side chains. Comprehensive kinetic studies of the variants reveal that they fold via parallel pathways on which there are two intermediate states very similar to wild-type UCH-L1. The structures of the intermediate states do not change significantly with mutation and therefore occupy local minima on the energy landscape that have relatively narrow basins. The kinetic studies also establish that there are considerably more tertiary interactions in the intermediate states than results from previous NMR studies suggested. The two intermediates differ from each other in the extent to which tertiary interactions between the highly stable central b-sheet and flanking a-helices and loop regions are formed. There is strong evidence that these states are aggregation prone. The transition states from both I 1 and I 2 to the native state are plastic and change with mutation and denaturant concentration. Previous studies indicated that the threading event that creates the 5 2 knot occurs during these steps, suggesting that there is a broad energy barrier consistent with the chain undergoing some searching of conformational space as the threading takes place.

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“…It has been shown experimentally that both these proteins unfold spontaneously and reversibly on addition of chemical denaturant (8)(9)(10)(11) and they are able to fold even when additional domains are attached to one or both termini (12). In very recent experimental work (13), based on analysis of the effect of mutations in the knotted region of the protein, a folding model for YibK was also proposed. In this model the threading of the polypeptide chain and formation of the native structure in the knotted region can occur independently as successive events.…”

mentioning

confidence: 99%

Dodging the crisis of folding proteins with knots

Sułkowska

Sułkowski

Onuchic

2009

Proc. Natl. Acad. Sci. U.S.A.

167

217

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Proteins with nontrivial topology, containing knots and slipknots, have the ability to fold to their native states without any additional external forces invoked. A mechanism is suggested for folding of these proteins, such as YibK and YbeA, that involves an intermediate configuration with a slipknot. It elucidates the role of topological barriers and backtracking during the folding event. It also illustrates that native contacts are sufficient to guarantee folding in Ϸ1-2% of the simulations, and how slipknot intermediates are needed to reduce the topological bottlenecks. As expected, simulations of proteins with similar structure but with knot removed fold much more efficiently, clearly demonstrating the origin of these topological barriers. Although these studies are based on a simple coarse-grained model, they are already able to extract some of the underlying principles governing folding in such complex topologies. molecular dynamics ͉ slipknots ͉ backtracking ͉ topological barriers D uring the past 2 decades, a joint theoretical and experimental effort has largely advanced the quantitative understanding of the protein folding mechanism. Most small-and intermediate-size proteins live on a minimally frustrated funnel-like energy landscape, which allows fast and robust folding (1-3). Because proteins have been able to solve the energy problem, the final challenge is the structural complexity of the protein folding motifs. Most proteins avoid complex topologies, but recent discoveries have shown that some proteins are actually able to fold into nontrivial topologies where the main chain folds into a knotted conformation (4-6). Although these ''knotted'' folding motifs have been observed, we still have to face the challenging question of how the protein overcomes the kinetic barrier associated with the search of the knotted conformation. We suggest a possible mechanism where the knot formation is preceded by a conformation called a ''slipknot.'' A slipknot is topologically similar to a knot, except that an internal knot is effectively undone as the pathway of the backbone folds back on itself. The fact that such slipknots have already been observed in some protein final structures (7) adds support to this suggestion.This folding mechanism is explored in the context of the two most experimentally investigated knotted families of proteins, Haemophilus influenzae YibK and Escherichia coli YbeA, which are homodimeric ␣/␤-knot methyltransferases (MTases). A schematic representation of these proteins is shown in Fig. 1, [see also supporting information (SI) Fig. S1]. It has been shown experimentally that both these proteins unfold spontaneously and reversibly on addition of chemical denaturant (8-11) and they are able to fold even when additional domains are attached to one or both termini (12). In very recent experimental work (13), based on analysis of the effect of mutations in the knotted region of the protein, a folding model for YibK was also proposed. In this model the threading of the polypeptide chain and fo...

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Exploring knotting mechanisms in protein folding

Cited by 68 publications

References 40 publications

Slipknotting upon native-like loop formation in a trefoil knot protein

Slipknotting upon native-like loop formation in a trefoil knot protein

Characterization of the Folding of a 5 2 -Knotted Protein Using Engineered Single-Tryptophan Variants

Dodging the crisis of folding proteins with knots

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