Collaboration between the ClpB AAA+ remodeling protein and the DnaK chaperone system

CIpB and Hsp104, members of the AAA+ superfamily of proteins, protect cells from the devastating effects of protein inactivation and aggregation that arise after extreme heat stress. They exist as a hexameric ring and contain two nucleotide-binding sites per monomer. CIpB and Hsp104 are able to dissolve protein aggregates in conjunction with the DnaK/Hsp70 chaperone system, although the roles of the individual chaperones in disaggregation are not well understood. In the absence of the DnaK/Hsp70 system, CIpB and Hsp104 alone are able to perform protein remodeling when their ATPase activity is asymmetrically slowed either by providing a mixture of ATP and ATP[gamma]S, a nonphysiological and slowly hydrolyzed ATP analog, or by inactivating one of the two nucleotide-binding domains by mutation. To gain insight into the roles of CIpB and the DnaK system in protein remodeling, we tested whether there was a further stimulation by the DnaK chaperone system under conditions that elicited remodeling activity by CIpB alone. Our results demonstrate that CIpB and the DnaK system act synergistically to remodel proteins and dissolve aggregates. The results further show that ATP is required and that both nucleotide-binding sites of CIpB must be able to hydrolyze ATP to permit functional collaboration between CIpB and the DnaK system.