title: Covalently linked HslU hexamers support a probabilistic mechanism that links ATP hydrolysis to protein unfolding and translocation.
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.
authors.affiliation: From the Department of Biology and.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Baytshtok
authors.affiliation: From the Department of Biology and.
authors.forename: Jiejin
authors.initials: J
authors.lastname: Chen
authors.affiliation: From the Department of Biology and.
authors.forename: Steven E
authors.initials: SE
authors.lastname: Glynn
authors.affiliation: From the Department of Biology and.
authors.forename: Andrew R
authors.initials: AR
authors.lastname: Nager
authors.affiliation: From the Department of Biology and.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Grant
authors.affiliation: From the Department of Biology and.|Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139.
authors.forename: Tania A
authors.initials: TA
authors.lastname: Baker
authors.affiliation: From the Department of Biology and bobsauer@mit.edu.
authors.forename: Robert T
authors.initials: RT
authors.lastname: Sauer
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D049071:Endopeptidase Clp / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D058767:Protein Unfolding
chemical_list: C483566:ClpYQ protease, E coli; D029968:Escherichia coli Proteins; D000255:Adenosine Triphosphate; D049071:Endopeptidase Clp
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