Hsp70 proteins bind Hsp100 regulatory M domains to activate AAA+ disaggregase at aggregate surfaces

Abstract

Bacteria, fungi and plants rescue aggregated proteins using a powerful bichaperone system composed of an Hsp70 chaperone and an Hsp100 AAA+ disaggregase. In Escherichia coli, the Hsp70 chaperone DnaK binds aggregates and targets the disaggregase ClpB to the substrate. ClpB hexamers use ATP to thread substrate polypeptides through the central pore, driving disaggregation. How ClpB finds DnaK and regulates threading remains unclear. To dissect the disaggregation mechanism, we separated these steps using primarily chimeric ClpB-ClpV constructs that directly recognize alternative substrates, thereby obviating DnaK involvement. We show that ClpB has low intrinsic disaggregation activity that is normally repressed by the ClpB middle (M) domain. In the presence of aggregate, DnaK directly binds M-domain motif 2, increasing ClpB ATPase activity to unleash high ClpB threading power. Our results uncover a new function for Hsp70: the coupling of substrate targeting to AAA+ chaperone activation at aggregate surfaces.

Single-molecule spectroscopy reveals chaperone-mediated expansion of substrate protein

“…Indeed, earlier studies (13) suggested that additional chaperones, such as the chaperonin system GroEL/GroES, are required to refold denatured rhodanese. Our results therefore support the model that the DnaK/DnaJ/GrpE system passes substrate proteins on to downstream chaperones (11,56). We note, however, that with an excess of substrate present, the cellular DnaJ and DnaK concentrations may become limiting, resulting in a less pronounced expansion or even a bypassing of the DnaK/DnaJ/GrpE system (51).…”

Non-native, N-terminal Hsp70 Molecular Motor Recognition Elements in Transit Peptides Support Plastid Protein Translocation

“…Considering the possible dual involvement of Hsp93 and Hsp70, one can envision two modes of function as follows: (a) they bind TPs sequentially and independently or (b) they bind TPs simultaneously and synergistically (81). Recent work has shown some Hsp100 are intimately associated with DnaK, and it is now clear that disaggregation function of ClpB involved interaction with DnaK (88,89). This observation would support synergistic model B.…”

Single-molecule Analyses of the Dynamics of Heat Shock Protein 104 (Hsp104) and Protein Aggregates

“…7, 21). Alternatively, previous in vivo imaging using GFP fusions showed "snapshots" of the individual interactions of Hsp104/ ClpB with the substrates (15,19). However, so far, no attempts have been made to visualize and analyze the dynamics of the interaction of Hsp104 with the aggregates.…”

“…How can we reconcile these single-molecule findings with previous biochemical data? A recent detailed analysis of E. coli ClpB revealed that the activation of ClpB is regulated by the binding of DnaK (19). The conformations of ClpB exist in an equilibrium between the "repressed" and "derepressed" states, which are associated with the low-and high-activity states, respectively (42).…”