title: DnaK chaperone-dependent disaggregation by caseinolytic peptidase B (ClpB) mutants reveals functional overlap in the N-terminal domain and nucleotide-binding domain-1 pore tyrosine.
abstract: Protein disaggregation in Escherichia coli is carried out by ClpB, an AAA(+) (ATPases associated with various cellular activities) molecular chaperone, together with the DnaK chaperone system. Conformational changes in ClpB driven by ATP binding and hydrolysis promote substrate binding, unfolding, and translocation. Conserved pore tyrosines in both nucleotide-binding domain-1 (NBD-1) and -2 (NBD-2), which reside in flexible loops extending into the central pore of the ClpB hexamer, bind substrates. When the NBD-1 pore loop tyrosine is substituted with alanine (Y251A), ClpB can collaborate with the DnaK system in disaggregation, although activity is reduced. The N-domain has also been implicated in substrate binding, and like the NBD-1 pore loop tyrosine, it is not essential for disaggregation activity. To further probe the function and interplay of the ClpB N-domain and the NBD-1 pore loop, we made a double mutant with an N-domain deletion and a Y251A substitution. This ClpB double mutant is inactive in substrate disaggregation with the DnaK system, although each single mutant alone can function with DnaK. Our data suggest that this loss in activity is primarily due to a decrease in substrate engagement by ClpB prior to substrate unfolding and translocation and indicate an overlapping function for the N-domain and NBD-1 pore tyrosine. Furthermore, the functional overlap seen in the presence of the DnaK system is not observed in the absence of DnaK. For innate ClpB unfolding activity, the NBD-1 pore tyrosine is required, and the presence of the N-domain is insufficient to overcome the defect of the ClpB Y251A mutant.
authors: Laboratory of Molecular Biology, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors: Shannon M
authors: SM
authors: Doyle
authors: Joel R
authors: JR
authors: Hoskins
authors: Sue
authors: S
authors: Wickner
mesh_terms: D019943:Amino Acid Substitution; 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*; D020125:Mutation, Missense; D017510:Protein Folding; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D014443:Tyrosine; D049071:Endopeptidase Clp; C063842:dnaK protein, E coli; C069775:ClpB protein, E coli