Investigation of the interaction between DnaK and DnaJ by surface plasmon resonance spectroscopy

Hsp70 chaperones assist protein folding through AIP-regulated transient ass ociation with substrates. Substrate binding by Hsp70 is controlled by DnaJ co-chaperones which stimulate Hsp70 to hydrolyze Am and, consequently, to c lose its substrate binding cavity allowing trapping of substrates. We analy zed the interaction of the Escherichia coli Hsp70 homologue, DnaK, with Dna J using surface plasmon resonance (SPR) spectroscopy. Resonance signals of complex kinetic characteristics were detected when DnaK was passed over a s ensor chip with coupled DnaJ. This interaction was specific as it was not d etected with a functionally defective DnaJ mutant protein, DnaJ259, that ca rries a mutation in the HPD signature motif of the conserved J-domain. Dete ctable DnaK-DnaJ interaction required ATP hydrolysis by DnaK and was compet itively inhibited by chaperone substrates of DnaK. For DnaK mutant proteins with amino acid substitutions in the substrate binding cavity that affect substrate binding, the strength of detected interaction with DnaJ decreased proportionally with increased strength of the substrate binding defects. T hese findings indicate that the detected response signals resulted from Dna J and ATP hydrolysis-dependent association of DnaJ as substrate for DnaK. A lthough not considered as physiologically relevant, this association allowe d us to experimentally unravel the mechanism of DnaJ action. Accordingly, D naJ stimulates ATP hydrolysis only after association of a substrate with th e substrate binding cavity of DnaK. Further analysis revealed that this cou pling mechanism required the J-domain of DnaJ and was also functional for n atural DnaK substrates, and thus is central to the mechanism of action of t he DnaK chaperone system. (C) 1999 Academic Press.