NDLI: Functional Defects of the DnaK756 Mutant Chaperone ofEscherichia coli Indicate Distinct Roles for Amino- and Carboxyl-terminal Residues in Substrate and Co-chaperone Interaction and Interdomain Communication

Content Provider	Journal of Biological Chemistry (JBC)
Author	Buchberger, Alexander Gässler, Claudia S. Büttner, Martina McMacken, Roger Bukau, Bernd
Abstract	The first discovery of an Hsp70 chaperone gene was the isolation of an Escherichia coli mutant,dnaK756, which rendered the cells resistant to lytic infection with bacteriophage λ. The DnaK756 mutant protein has since been used to establish many of the cellular roles and biochemical properties of DnaK. DnaK756 has three glycine-to-aspartate substitutions at residues 32, 455, and 468, which were reported to result in defects in intrinsic and GrpE-stimulated ATPase activities, substrate binding, stability of the substrate-binding domain, interdomain communication, and, consequently, defects in chaperone activity. To dissect the effects of the different amino acid substitutions in DnaK756, we analyzed two DnaK variants carrying only the amino-terminal (residue 32) or the two carboxyl-terminal (residues 455 and 468) substitutions. The amino-terminal substitution interfered with the GrpE-stimulated ATPase activity. The carboxyl-terminal mutations (i) affected stability and function of the substrate-binding domain, (ii) caused a 10-fold elevated ATP hydrolysis rate, but (iii) did not severely affect domain coupling. Surprisingly, DnaK chaperone activity was more severely compromised by the amino-terminal than by the carboxyl-terminal amino acid substitutions both in vivoand in vitro. In the in vitro refolding of denatured firefly luciferase, the defect of the DnaK variant carrying the amino-terminal substitution results from its inability to release, upon GrpE-mediated nucleotide exchange, bound luciferase in a folding competent state. Our results indicate that the DnaK-DnaJ-GrpE chaperone system can tolerate suboptimal substrate binding, whereas the tight kinetic control of substrate dissociation by GrpE is essential.
Related Links	http://www.jbc.org/content/274/53/38017.abstract
Ending Page	38026
Starting Page	38017
Page Count	10
File Format	HTM / HTML PDF
ISSN	00219258
Journal	Journal of Biological Chemistry (JBC)
Issue Number	53
Volume Number	274
DOI	10.1074/jbc.274.53.38017
e-ISSN	1083351X
Language	English
Publisher	American Society for Biochemistry and Molecular Biology
Publisher Date	1999-12-31
Access Restriction	Open
Subject Keyword	2-[4′-(iodoacetamido)anilino]naphthalene-6-sulfonic acid () PROTEIN CHEMISTRY AND STRUCTURE
Content Type	Text
Resource Type	Article
Subject	Cell Biology Biochemistry Molecular Biology

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