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. 1995 Jul 14;270(28):16903-10.
doi: 10.1074/jbc.270.28.16903.

Nucleotide-induced conformational changes in the ATPase and substrate binding domains of the DnaK chaperone provide evidence for interdomain communication

A Buchberger  1 , H Theyssen, H Schröder, J S McCarty, G Virgallita, P Milkereit, J Reinstein, B Bukau
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Free article

Nucleotide-induced conformational changes in the ATPase and substrate binding domains of the DnaK chaperone provide evidence for interdomain communication

A Buchberger et al. J Biol Chem. .
Free article

Abstract

Interactions of the DnaK (Hsp70) chaperone from Escherichia coli with substrates are controlled by ATP. Nucleotide-induced changes in DnaK conformation were investigated by monitoring changes in tryptic digestion pattern and tryptophan fluorescence. Using nucleotide-free DnaK preparations, not only the known ATP-induced major changes in kinetics and pattern of proteolysis but also minor ADP-induced changes were detected. Similar ATP-induced conformational changes occurred in the DnaK-T199A mutant protein defective in ATPase activity, demonstrating that they result from binding, not hydrolysis, of ATP. N-terminal sequencing and immunological mapping of tryptic fragments of DnaK identified cleavage sites that, upon ATP addition, appeared within the proposed C-terminal substrate binding region and disappeared in the N-terminal ATPase domain. They hence reflect structural alterations in DnaK correlated to substrate release and indicate ATP-dependent domain interactions. Domain interactions are a prerequisite for efficient tryptic degradation as fragments of DnaK comprising the ATPase and C-terminal domains were highly protease-resistant. Fluorescence analysis of the N-terminally located single tryptophan residue of DnaK revealed that the known ATP-induced alteration of the emission spectrum, proposed to result directly from conformational changes in the ATPase domain, requires the presence of the C-terminal domain and therefore mainly results from altered domain interaction. Analyses of the C-terminally truncated DnaK163 mutant protein revealed that nucleotide-dependent interdomain communication requires a 15-kDa segment assumed to constitute the substrate binding site.

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