title: Interplay between E. coli DnaK, ClpB and GrpE during protein disaggregation.
abstract: The DnaK/Hsp70 chaperone system and ClpB/Hsp104 collaboratively disaggregate protein aggregates and reactivate inactive proteins. The teamwork is specific: Escherichia coli DnaK interacts with E. coli ClpB and yeast Hsp70, Ssa1, interacts with yeast Hsp104. This interaction is between the middle domains of hexameric ClpB/Hsp104 and the DnaK/Hsp70 nucleotide-binding domain (NBD). To identify the site on E. coli DnaK that interacts with ClpB, we substituted amino acid residues throughout the DnaK NBD. We found that several variants with substitutions in subdomains IB and IIB of the DnaK NBD were defective in ClpB interaction in vivo in a bacterial two-hybrid assay and in vitro in a fluorescence anisotropy assay. The DnaK subdomain IIB mutants were also defective in the ability to disaggregate protein aggregates with ClpB, DnaJ and GrpE, although they retained some ability to reactivate proteins with DnaJ and GrpE in the absence of ClpB. We observed that GrpE, which also interacts with subdomains IB and IIB, inhibited the interaction between ClpB and DnaK in vitro, suggesting competition between ClpB and GrpE for binding DnaK. Computational modeling of the DnaK-ClpB hexamer complex indicated that one DnaK monomer contacts two adjacent ClpB protomers simultaneously. The model and the experiments support a common and mutually exclusive GrpE and ClpB interaction region on DnaK. Additionally, homologous substitutions in subdomains IB and IIB of Ssa1 caused defects in collaboration between Ssa1 and Hsp104. Altogether, these results provide insight into the molecular mechanism of collaboration between the DnaK/Hsp70 system and ClpB/Hsp104 for protein disaggregation.
authors.affiliation: Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Shannon M
authors.initials: SM
authors.lastname: Doyle
authors.affiliation: Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Shankar
authors.initials: S
authors.lastname: Shastry
authors.affiliation: Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Andrea N
authors.initials: AN
authors.lastname: Kravats
authors.affiliation: Department of Chemistry, University of Cincinnati, Cincinnati, OH 45221, USA.
authors.forename: Yu-Hsuan
authors.initials: YH
authors.lastname: Shih
authors.affiliation: Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Marika
authors.initials: M
authors.lastname: Miot
authors.affiliation: Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Joel R
authors.initials: JR
authors.lastname: Hoskins
authors.affiliation: Department of Chemistry, University of Cincinnati, Cincinnati, OH 45221, USA.
authors.forename: George
authors.initials: G
authors.lastname: Stan
authors.affiliation: Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. Electronic address: wickners@mail.nih.gov.
authors.forename: Sue
authors.initials: S
authors.lastname: Wickner
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D003198:Computer Simulation; D049071:Endopeptidase Clp; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D066329:Protein Aggregates; D054730:Protein Interaction Domains and Motifs; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques
chemical_list: D029968:Escherichia coli Proteins; C489400:GrpE protein, E coli; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D066329:Protein Aggregates; D029701:Saccharomyces cerevisiae Proteins; C070972:HsP104 protein, S cerevisiae; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases; C063842:dnaK protein, E coli; C069775:ClpB protein, E coli; C095973:SSA1 protein, S cerevisiae
keywords: GrpE; Hsp40; molecular chaperone; nucleotide exchange factor; protein disaggregation