ATP-dependent proteases translocate proteins through a narrow pore for their controlled destruction. However, how a protein substrate containing a knotted topology affects this process remains unknown. Here, we characterized the effects of the trefoil-knotted protein MJ0366 from Methanocaldococcus jannaschii on the operation of the ClpXP protease from Escherichia coli. ClpXP completely degrades MJ0366 when pulling from the C-terminal ssrA-tag. However, when a GFP moiety is appended to the N terminus of MJ0366, ClpXP releases intact GFP with a 47-residue tail. The extended length of this tail suggests that ClpXP tightens the trefoil knot against GFP, which prevents GFP unfolding. Interestingly, if the linker between the knot core of MJ0366 and GFP is longer than 36 residues, ClpXP tightens and translocates the knot before it reaches GFP, enabling the complete unfolding and degradation of the substrate. These observations suggest that a knot-induced stall during degradation of multidomain proteins by AAA proteases may constitute a novel mechanism to produce partially degraded products with potentially new functions.

Knots are natural topologies of chains. Yet, little is known about spontaneous knot formation in a polypeptide chain—an event that can potentially impair its folding—and about the effect of a knot on the stability and folding kinetics of a protein. Here we used optical tweezers to show that the free energy cost to form a trefoil knot in the denatured state of a polypeptide chain of 120 residues is 5.8 ± 1 kcal mol⁻¹. Monte Carlo dynamics of random chains predict this value, indicating that the free energy cost of knot formation is of entropic origin. This cost is predicted to remain above 3 kcal mol⁻¹ for denatured proteins as large as 900 residues. Therefore, we conclude that naturally knotted proteins cannot attain their knot randomly in the unfolded state but must pay the cost of knotting through contacts along their folding landscape.

Knots are remarkable topological features in nature. The presence of knots in crystallographic structures of proteins have stimulated considerable research to determine the kinetic and thermodynamic consequences of threading a polypeptide chain. By mechanically manipulating MJ0366, a small single domain protein harboring a shallow trefoil knot, we allow the protein to refold from either the knotted or the unknotted denatured state to characterize the free energy profile associated to both folding pathways. By comparing the stability of the native state with reference to the knotted and unknotted denatured state we find that knotting the polypeptide chain of MJ0366 increase the folding energy barrier in a magnitude close to the energy cost of forming a knot randomly in the denatured state. These results support that a protein knot can be formed during a single cooperative step of folding but occurs at the expenses of a large increment on the free energy barrier.