Albany 2015:Book of Abstracts

Albany 2015
Conversation 19
June 9-13 2015
©Adenine Press (2012)

Interaction and collaboration between DnaK and ClpB during protein disaggregation

The DnaK chaperone system and ClpB collaboratively disaggregate protein aggregates and reactivate inactive proteins. A specific interaction between the M-domains of hexameric ClpB and the DnaK nucleotide-binding domain (NBD) has been established (Miot et al., 2011; Sielaff et al., 2010). To identify the specific site on E. coli DnaK that interacts with ClpB, we substituted amino acid residues throughout the DnaK NBD (Doyle et al., 2014). We found that several variants with substitutions in subdomain IB and IIB of the DnaK NBD were defective in ClpB interaction in vivo in a bacterial two-hybrid assay and in vitro in a fluorescence anisotropy assay. The DnaK subdomain IIB mutants were also defective in the ability to disaggregate protein aggregates with ClpB, DnaJ and GrpE, although they retained some ability to reactivate proteins with DnaJ and GrpE in the absence of ClpB. We observed that GrpE, which also interacts with subdomains IB and IIB of DnaK, inhibited the interaction between ClpB and DnaK in vitro, suggesting competition between ClpB and GrpE for binding DnaK.

Computational modeling of the DnaK-ClpB hexamer complex was utilized to validate the interacting residues identified biochemically as well as suggest additional regions and residues important for DnaK and ClpB interaction and collaboration. The model and the experiments support a common and mutually exclusive GrpE and ClpB interaction region on DnaK. Interestingly, our modeling study of the DnaK-ClpB hexamer complex predicts that one DnaK monomer contacts two adjacent ClpB protomers simultaneously, a result with potential functional implications. Biochemical studies to test this prediction are underway and will be discussed. Altogether, these results provide insight into the molecular mechanism of collaboration between the DnaK system and ClpB for protein disaggregation.


This research was supported by the National Science Foundation CAREER grant no. MCB-0952082 (to G.S.) and the Intramural Research Program of the NIH, NCI, Center for Cancer Research.

    Miot, M. et al. Species-specific collaboration of heat shock proteins (Hsp) 70 and 100 in thermotolerance and protein disaggregation. (2011) Proc Natl Acad Sci U S A 108, 6915-20.

    Sielaff, B. & Tsai, F.T. The M-domain controls Hsp104 protein remodeling activity in an Hsp70/Hsp40-dependent manner. (2010) J Mol Biol 402, 30-7.

    Doyle, S. et al. Interplay between E. coli DnaK, ClpB and GrpE during protein disaggregation. (2014) doi:10.1016/j.jmb.2014.10.013.

Shannon M. Doyle1
Andrea N. Kravats1
Joel R. Hoskins1
Yu-Hsuan Shih2
George Stan2
Sue Wickner1

1 Laboratory of Molecular Biology
Bethesda, MD 20892
2 Department of Chemistry
University of Cincinnati
Cincinnati, OH 45221

Ph: (301) 402-9282 doyles@mail.nih.gov