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

Scientific Reports
Elin M SivertssonLaura S Itzhaki

Abstract

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 intermed...Continue Reading

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Citations

Jul 29, 2020·Nature Communications·Antonio SumaCristian Micheletti
Jan 10, 2021·Trends in Biochemical Sciences·Jennifer Michelle Simien, Ellinor Haglund
Nov 23, 2019·Biochimica Et Biophysica Acta. Proteins and Proteomics·Manoj Kumar SriramojuShang-Te Danny Hsu
Feb 18, 2020·Current Opinion in Structural Biology·Joanna Ida Sulkowska

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Methods Mentioned

BETA
protein folding
optical tweezers
gel filtration
size-exclusion chromatography
circular dichroism
AFM
optical tweezer
PCR
ion exchange chromatography
size exclusion chromatography

Software Mentioned

GraphPad Prism
ImageJ
ProtParam
FoldX
ClpXP

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