Identification of the DnaK binding site and of chaperone-induced unfolding within the heat shock transcription factor sigma32.

RODRIGUEZ FERNANDA; RIST WOLFGANG; MAYER MATTHIAS P; BUKAU BERND

Vermont

Conferencia; FASEB Summer Conferences-Protein Folding in the Cell; 2004

FASEB

Central to the transcriptional control of the E. coli heat shock regulon is the stress-dependent inhibition of the sigma32 subunit of RNA polymerase by reversible association with the DnaK chaperone. This association requires the transient interaction of the DnaJ cochaperone with sigma32 whereby DnaK is targeted to sigma32. We identified the DnaK binding site within sigma32. Protease footprinting revealed a DnaK-protected site at residues 198-204 of sigma32. This site is located within the only major segment of sigma32 predicted to be unstructured. DnaK cross-linked to cysteines introduced next to this site. Mutational alterations of the site decreased the affinity of sigma32 for DnaK and RNA polymerase and perturbed cell viability and heat shock gene regulation, thus indicating a regulatory role for this site. The site exhibited high specificity for DnaK and was not recognized by other chaperones including the DnaK homologs of E. coli, HscA and HscC. DnaJ did not bind to this site either, implicating a mode of action of DnaK and DnaJ in which they bind to distinct exposed sites within sigma32. This binding site was confirmed using H/D footprinting. In analogy to protease footprinting, H/D footprinting can give information about binding interfaces in protein complexes. Amide hydrogen exchange was performed with sigma32 alone and in complex with DnaK. Two peptides, residues 183-199 and 200-208, showed protection when bound to DnaK whereas all others did not. As a positive control we used a peptide substrate of DnaK (sigma32M195-N207) which shows a similar behaviour. Furthermore, amide hydrogen exchange revealed a region that incorporated more hydrogens in the presence of DnaK than in its absence. This indicates a chaperone-induced unfolding of a secondary structure element that may facilitate degradation of sigma32 in vivo.