Structural insights into substrate binding by the molecular chaperone DnaK

Maurizio Pellecchia, Diana L. Montgomery, Shawn Y. Stevens, Craig W. Vander Kooi, Hwa Ping Feng, Lila M. Gierasch, Erik R.P. Zuiderweg

Research output: Contribution to journalArticlepeer-review

182 Scopus citations

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 coil 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.

Original languageEnglish
Pages (from-to)298-303
Number of pages6
JournalNature Structural Biology
Volume7
Issue number4
DOIs
StatePublished - Apr 2000

Bibliographical note

Funding Information:
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.

ASJC Scopus subject areas

  • Structural Biology
  • Biochemistry
  • Genetics

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