Disruption of Ionic Interactions between the Nucleotide Binding Domain 1 (NBD1) and Middle (M) Domain in Hsp100 Disaggregase Unleashes Toxic Hyperactivity and Partial Independence from Hsp70

Abstract

Background: Hsp100 chaperones cooperate with Hsp70 chaperones to disaggregate and reactivate heat-denatured proteins. Results: Mutations in the interface region between NBD1 and M domains of Hsp100 result in a hyperactive protein toxic to the cell. Conclusion:The interaction between M and NBD1 domains is crucial for regulation of Hsp100 activity. Significance: A novel important aspect of the Hsp100 mechanism of action is described.

Spatially Organized Aggregation of Misfolded Proteins as Cellular Stress Defense Strategy

“…The Hsp70-dependent binding of Hsp104 to amorphous and amyloidogenic aggregates is directly linked to its targeted activation, enabling the AAA+ chaperone to extract misfolded polypeptides from the aggregates via their ATP-dependent threading through the central pore ( Figure 2) [62,[65][66][67][68]. The disaggregation activity of the bi-chaperone system is essential for thermotolerance development [69] and propagation of prions [38,70] in yeast.…”

Disaggregases, molecular chaperones that resolubilize protein aggregates

“…2; Lee et al 2003, Schirmer et al 2004. The mobility of the M-domain is critical for the disaggregase function of ClpB (Lee et al 2003) and Hsp70 has a role in shifting the M-domain away from the NBD1 that increases both the ATPase activity and the disaggregase activity of Hsp100 (Oguchi et al 2012, Lipińska et al 2013. Additionally, some mutations in the M domain of Hsp104 can potentiate its disaggregase and ATPase activities (Jackrel et al 2014).…”

Chaperone-assisted protein aggregate reactivation: Different solutions for the same problem

“…Crosslinking residues of M domain motif-2 and NBD1 of ClpB Tth promoted stimulation of the NBD2 even in the absence of nucleotide binding to the NBD1, indicating that the M domain is implicated in inter-ring communication [66]. In stark contrast, single point mutations that stabilize the motif 2-NBD1 interaction repressed ClpB Eco /Hsp104, while its destabilization generated (hyper)stimulated variants that were toxic in vivo [30,75,109,110]. This observation indicates that the role of the M domain in allosteric regulation of the ATPase activity might be different in ClpB Tth .…”

“…Indeed, hyperactive ClpB/Hsp104 variants that display an increased disaggregase activity are able to unfold stable protein domains, in contrast to their wild type counterparts [75], and protect yeast cells from proteins associated to neurodegenerative disorders such as α-synuclein [135]. However, evolution has not selected them because they are highly toxic at high temperatures or when they are overexpressed, most likely due to an uncontrolled substrate threading activity that stresses the importance of a stringent regulation of ClpB/Hsp104 activity [63,75,109]. This interpretation is supported by the fact that potentiated Hsp104 variants do not require the Hsp70 system for disaggregation [136].…”