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J Biol Chem. 2017 Apr 7;292(14):5695-5704. doi: 10.1074/jbc.M116.768978. Epub 2017 Feb 21.

Covalently linked HslU hexamers support a probabilistic mechanism that links ATP hydrolysis to protein unfolding and translocation.

Author information

1
From the Department of Biology and.
2
Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139.
3
From the Department of Biology and bobsauer@mit.edu.

Abstract

The HslUV proteolytic machine consists of HslV, a double-ring self-compartmentalized peptidase, and one or two AAA+ HslU ring hexamers that hydrolyze ATP to power the unfolding of protein substrates and their translocation into the proteolytic chamber of HslV. Here, we use genetic tethering and disulfide bonding strategies to construct HslU pseudohexamers containing mixtures of ATPase active and inactive subunits at defined positions in the hexameric ring. Genetic tethering impairs HslV binding and degradation, even for pseudohexamers with six active subunits, but disulfide-linked pseudohexamers do not have these defects, indicating that the peptide tether interferes with HslV interactions. Importantly, pseudohexamers containing different patterns of hydrolytically active and inactive subunits retain the ability to unfold protein substrates and/or collaborate with HslV in their degradation, supporting a model in which ATP hydrolysis and linked mechanical function in the HslU ring operate by a probabilistic mechanism.

KEYWORDS:

AAA+ protease; ATP-dependent protease; ATPases associated with diverse cellular activities (AAA); HslUV; crystal structure; mixed hexameric rings; protein degradation; protein engineering; protein turnover; protein unfolding

PMID:
28223361
PMCID:
PMC5392565
DOI:
10.1074/jbc.M116.768978
[Indexed for MEDLINE]
Free PMC Article

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