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Structural insights into substrate binding by the molecular chaperone DnaK

Nature Structural Biology volume 7, pages 298–303 (2000)Cite this article

Abstract

How substrate affinity is modulated by nucleotide binding remains a fundamental, unanswered question in the study of 70 kDa heat shock protein (Hsp70) molecular chaperones. We find here that the Escherichia coli Hsp70, DnaK, lacking the entire α-helical domain, DnaK(1–507), retains the ability to support λ phage replication in vivo and to pass information from the nucleotide binding domain to the substrate binding domain, and vice versa, in vitro. We determined the NMR solution structure of the corresponding substrate binding domain, DnaK(393–507), without substrate, and assessed the impact of substrate binding. Without bound substrate, loop L3,4 and strand β3 are in significantly different conformations than observed in previous structures of the bound DnaK substrate binding domain, leading to occlusion of the substrate binding site. Upon substrate binding, the β-domain shifts towards the structure seen in earlier X-ray and NMR structures. Taken together, our results suggest that conformational changes in the β-domain itself contribute to the mechanism by which nucleotide binding modulates substrate binding affinity.

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Figure 1: In vitro studies of DnaK(1–507) allosteric function.
Figure 2: The structure of the apo β-domain and comparison with other peptide-bound forms.
Figure 3: Backbone conformation flexibility in the apo β-domain.
Figure 4: NMR titration of DnaK(393–507) with the peptide NRLLLTG.

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Acknowledgements

This work was supported by NIH grants to E.R.P.Z and to L.M.G., and a NIH fellowship to D.L.M.. The W.M. Keck Foundation, NIH, NSF and Parke-Davis/ Warner Lambert are gratefully acknowledged for financial support towards the 800 MHz NMR instrument. We thank J. Feltham for critical reading of the manuscript, and R. Sivendran for help with the assays of peptide-stimulated ATPase activity.

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Author notes

  1. Maurizio Pellecchia

    Present address: TRIAD Biotechnology, 5820 Nancy Ridge Road, San Diego, California, 92121, USA

  2. Diana L. Montgomery

    Present address: Drug Metabolism and Pharmacokinetics, Schering Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, New Jersey, 07033, USA

Authors and Affiliations

  1. Biophysics Research Division University of Michigan, 930 North University Avenue, Ann Arbor, 48109, Michigan, USA

    Maurizio Pellecchia, Shawn Y. Stevens & Erik R.P. Zuiderweg

  2. Departments of Chemistry Biochemistry and Molecular Biology, University of Massachusetts, Amherst, 01003, Massachusetts, USA

    Diana L. Montgomery, Hwa-ping Feng & Lila M. Gierasch

  3. Department of Chemistry, University of Michigan, 930 North University Avenue, Ann Arbor, 48109, Michigan, USA

    Craig W. Vander Kooi & Erik R.P. Zuiderweg

  4. Department of Biological Chemistry, University of Michigan, 930 North University Avenue, Ann Arbor, 48109, Michigan, USA

    Erik R.P. Zuiderweg

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  1. Maurizio Pellecchia
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Correspondence to Lila M. Gierasch or Erik R.P. Zuiderweg.

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Pellecchia, M., Montgomery, D., Stevens, S. et al. Structural insights into substrate binding by the molecular chaperone DnaK. Nat Struct Mol Biol 7, 298–303 (2000). https://doi.org/10.1038/74062

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