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The hsp70 chaperone DnaK is a secondary amide peptide bond cis-trans isomerase

Nature Structural Biology volume 9, pages 419–424 (2002)Cite this article

Abstract

Peptidyl prolyl cis-trans isomerases can enzymatically assist protein folding, but these enzymes exclusively target the peptide bond preceding proline residues. Here we report the identification of the Hsp70 chaperone DnaK as the first member of a novel enzyme class of secondary amide peptide bond cis-trans isomerases (APIases). APIases selectively accelerate the cis-trans isomerization of nonprolyl peptide bonds. Results from independent experiments support the APIase activity of DnaK: (i) exchange crosspeaks between the cis-trans conformers appear in 2D 1H NMR exchange spectra of oligopeptides (ii) the rate constants for the cis-trans isomerization of various dipeptides increase and (iii) refolding of the RNase T1 P39A variant is catalyzed. The APIase activity shows both regio and stereo selectivity and is stimulated two-fold in the presence of the complete DnaK/GrpE/DnaJ/ATP refolding system. Moreover, known DnaK-binding oligopeptides simultaneously affect the APIase activity of DnaK and the refolding yield of denatured firefly luciferase in the presence of DnaK/GrpE/DnaJ/ATP. These results suggest a new role for the chaperone as a regioselective catalyst for bond rotation in polypeptides.

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Figure 1: DnaK shows APIase activity using dipeptide substrates.
Figure 2: cis-Alanine methyl region of the 2D exchange 1H NMR spectrum of Ala-Ala-Tyr-Ala-Ala at 5°C with the corresponding 1D spectra along F2 taken at the F1 position of the correlated trans signals (solid line).
Figure 3: Rate acceleration of the Tyr 38-Ala 39 trans-to-cis isomerization limited refolding of the RNase T1 P39A variant in the presence of 2 μM DnaK (2), kobs = 0.0011 s−1, in comparison to the time course of the spontaneous refolding (1), kobs = 0.00043 s−1.
Figure 4: Reactivation of guanidinium-Cl-denatured firefly luciferase (1.2 nM) by DnaK (485 nM), GrpE (137 nM) and DnaJ (171 nM).

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Acknowledgements

We thank F.U. Hartl for providing the DnaK plasmid, K.-P. Rücknagel for amino acid sequence analysis, D. Wildemann and G. Jahreis for syntheses and B. Korge, K. Walther, R. Steuding and M. Seidel for excellent technical assistance. We are grateful to J. Page for critical reading of the manuscript.

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Author notes

  1. Cordelia Schiene-Fischer, Judith Habazettl and Franz X. Schmid: These authors contributed equally to the work.

Authors and Affiliations

  1. Max Planck Research Unit for Enzymology of Protein Folding, Weinbergweg 22, Halle/Saale, D-06120, Germany

    Cordelia Schiene-Fischer, Judith Habazettl & Gunter Fischer

  2. Laboratorium für Biochemie, Universität Bayreuth, Universitätsstrasse, Bayreuth, D-95440, Germany

    Franz X. Schmid

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  1. Cordelia Schiene-Fischer
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  2. Judith Habazettl
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  3. Franz X. Schmid
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  4. Gunter Fischer
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Correspondence to Gunter Fischer.

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Schiene-Fischer, C., Habazettl, J., Schmid, F. et al. The hsp70 chaperone DnaK is a secondary amide peptide bond cis-trans isomerase. Nat Struct Mol Biol 9, 419–424 (2002). https://doi.org/10.1038/nsb804

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