Conformational Properties of Aggregated Polypeptides Determine ClpB-dependence in the Disaggregation Process Agnieszka Lewandowska, Marlena Matuszewska and Krzysztof Liberek⁎ Department of Molecular and Cellular Biology, Faculty of Biotechnology, University of Gdansk, Kladki 24, 80-822 Gdansk, Poland Severe thermal stress induces massive intracellular protein aggregation. The concerted action of Hsp70 (DnaK, DnaJ, GrpE) and Hsp100 (ClpB) chaperones results in solubilization of aggregates followed by reactivation of proteins. It was shown that the Hsp70 chaperone system works at the initial step of the disaggregation reaction and is able to disentangle poly- peptides from aggregates. Studies of the protein disaggregation reaction performed in vitro showed that ClpB may be dispensable in disaggregation of certain proteins and/or aggregates of certain size. Here we focus our attention on those properties of firefly luciferase aggregates, which de- termine whether ClpB chaperone is required in the disaggregation process. We report that the size of the aggregates is not a major determinant. Instead, we postulate that certain conformational properties (in particular, beta- structures) of subunits forming these aggregates are the most important factor determining the necessity of the ClpB chaperone in the disaggrega- tion process. © 2007 Elsevier Ltd. All rights reserved. *Corresponding author Keywords: chaperone-dependent protein disaggregation; ClpB; Hsp70; protein aggregation; luciferase Introduction When a cell is challenged by a severe heat stress the intracellular aggregation of proteins takes place. 1,2 Survival under such conditions is strongly enhanced by preconditioning cells at sub-lethal tem- peratures. The resulting phenomenon of acquired thermotolerance is due to the increase of chaperone protein synthesis during the preconditioning heat shock. 3 Protein aggregates formed during the fol- lowing severe heat stress are eliminated by chaper- one proteins when the cell is returned to physio- logical conditions. 1,2,4 The removal of aggregates depends on the presence of the AAA+ (ATPase as- sociated with diverse cellular activities) chaperones, e.g. ClpB in Escherichia coli and Hsp104 in the yeast Saccharomyces cerevisiae. 1,4 AAA+ proteins form oligomeric structures and use energy from ATP hydrolysis to remodel their substrates. 5,6 The ClpB chaperone belongs to the Hsp100 family and consists of an N-terminal domain and two ATP- binding domains, which are essential for hexameri- zation and chaperone function. 7–11 The first ATP- binding domain contains an additional coiled-coil region called the “middle domain”, which is located on the outer surface of the hexamer. 12,13 The mo- bility of this region is crucial for ClpB's disaggregat- ing activity. 13 The middle domain differentiates ClpB from other Hsp100 family members (ClpA, ClpX, HslU) which function as regulatory units of proteases. 12,14 The ClpB chaperone is not able to disaggregate substrates on its own and, in this process, collabo- rates with the Hsp70 chaperone system (in E. coli: DnaK and its co-chaperones DnaJ, GrpE). 15–19 However, phenotypic effects related to protein ag- gregation that are readily observed in clpB mutants, are not easily observed in dnaK, dnaJ, or grpE mu- Abbreviations used: GuHCl, guanidine hydrochloride; ThT, thioflavin T; EM, electron microscopy; KJE, DnaK/ DnaJ/GrpE. E-mail address of the corresponding author: liberek@biotech.ug.gda.pl doi:10.1016/j.jmb.2007.05.057 J. Mol. Biol. (2007) 371, 800–811 0022-2836/$ - see front matter © 2007 Elsevier Ltd. All rights reserved.