The AAA+ protease ClpXP can easily degrade a 3₁ and a 5₂-knotted protein

Knots in proteins are hypothesized to make them resistant to enzymatic degradation by ATP-dependent proteases and recent studies have shown that whereas ClpXP can easily degrade a protein with a shallow 3₁ knot, it cannot degrade 5₂-knotted proteins if degradation is initiated at the C-terminus. Here, we present detailed studies of the degradation of both 3₁- and 5₂-knotted proteins by ClpXP using numerous constructs where proteins are tagged for degradation at both N- and C-termini. Our results confirm and extend earlier work and show that ClpXP can easily degrade a deeply 3₁-knotted protein. In contrast to recently published work on the degradation of 5₂-knotted proteins, our results show that the ClpXP machinery can also easily degrade these proteins. However, the degradation depends critically on the location of the degradation tag and the local stability near the tag. Our results are consistent with mechanisms in which either the knot simply slips along the polypeptide chain and falls off the free terminus, or one in which the tightened knot enters the translocation pore of ClpXP. Results of experiments on knotted protein fusions with a highly stable domain show partial degradation and the formation of degradation intermediates.