ClpAP proteolysis does not require rotation of the ClpA unfoldase relative to ClpP

Elife. 2020 Dec 1:9:e61451. doi: 10.7554/eLife.61451.

Abstract

AAA+ proteases perform regulated protein degradation in all kingdoms of life and consist of a hexameric AAA+ unfoldase/translocase in complex with a self-compartmentalized peptidase. Based on asymmetric features of cryo-EM structures and a sequential hand-over-hand model of substrate translocation, recent publications have proposed that the AAA+ unfoldases ClpA and ClpX rotate with respect to their partner peptidase ClpP to allow function. Here, we test this model by covalently crosslinking ClpA to ClpP to prevent rotation. We find that crosslinked ClpAP complexes unfold, translocate, and degrade protein substrates in vitro, albeit modestly slower than uncrosslinked enzyme controls. Rotation of ClpA with respect to ClpP is therefore not required for ClpAP protease activity, although some flexibility in how the AAA+ ring docks with ClpP may be necessary for optimal function.

Keywords: ClpAP; ClpXP; E. coli; aaa+ atpase; aaa+ protease; biochemistry; chemical biology; molecular biophysics; structural biology.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cross-Linking Reagents
  • Endopeptidase Clp / metabolism*
  • Escherichia coli / enzymology*
  • Escherichia coli Proteins / metabolism*
  • Models, Molecular
  • Protein Conformation
  • Protein Folding
  • Proteolysis

Substances

  • Cross-Linking Reagents
  • Escherichia coli Proteins
  • ClpA protease, E coli
  • ClpP protease, E coli
  • Endopeptidase Clp