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Abstract

The molecular chaperones ClpB (Hsp104) and DnaK (Hsp70) co-operate in the ATP-dependent resolubilization of aggregated proteins. A sequential mechanism has been proposed for this reaction; however, the mechanism and the functional interplay between both chaperones remain poorly defined. Here, we show for the first time that complex formation of ClpB and DnaK can be detected by using various types of affinity chromatography methods. The finding that the DnaK chaperone of Escherichia coli is not co-operating with ClpB from Thermus thermophilus further strengthens the specificity of this complex. The affinity of the complex is weak and interaction between both chaperones is nucleotide-dependent. The presence of ADP, which is shown to cause dissociation of ClpB(Tth), as well as ClpB deletion mutants incapable of oligomer formation prevent ClpB-DnaK complex formation. The experiments presented indicate a correlation between the oligomeric state of ClpB and its ability to interact with DnaK. The chaperone complex described here might facilitate transfer of intermediates between ClpB and DnaK during refolding of substrates from aggregates.