DnaJ-Promoted Binding of DnaK to Multiple Sites on [sigma]^sup 32^ in the Presence of ATP

The Escherichia coli DnaK chaperone system is a canonical heat shock protein 70 (Hsp70) chaperone system comprising Hsp70, Hsp40, and a nucleotide exchange factor. Although Hsp40 is known to facilitate the effective binding of Hsp70 to substrates, the role of Hsp40 in Hsp70-substrate interactions ha...

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Bibliographic Details
Published inJournal of bacteriology Vol. 196; no. 9; p. 1694
Main Authors Noguchi, Aki, Ikeda, Ayami, Mezaki, Moeka, Fukumori, Yoshihiro, Kanemori, Masaaki
Format Journal Article
LanguageEnglish
Published Washington American Society for Microbiology 01.05.2014
Subjects
Adenosine triphosphatase
Bacteriology
Binding sites
E coli
Molecules
Proteins
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Summary:The Escherichia coli DnaK chaperone system is a canonical heat shock protein 70 (Hsp70) chaperone system comprising Hsp70, Hsp40, and a nucleotide exchange factor. Although Hsp40 is known to facilitate the effective binding of Hsp70 to substrates, the role of Hsp40 in Hsp70-substrate interactions has not yet been fully elucidated. Using the E. coli heat shock transcription factor ... as a substrate in the DnaK chaperone system, we here provide new insight into the Hsp70-substrate interaction. When DnaK-... complexes formed under various conditions were analyzed by gel filtration, several DnaK-... complexes with different molecular masses were detected. The results indicated that multiple DnaK molecules simultaneously bind to ..., even though it has been suggested that DnaK interacts with ... at a molar ratio of 1:1. Two s32 mutants, L201D ... and I54A ..., which have reduced affinities for DnaK and DnaJ (Hsp40), respectively, were used to further characterize DnaK-s32 complex formation. Pulldown assays demonstrated that the affinity of I54A ... for DnaK was similar to that of wild-type ... in the absence of DnaJ, whereas L201D ... exhibited an extremely low affinity for DnaK. However, in the presence of ATP and DnaJ, the yield of DnaK eluted with L201D ... was much higher than that eluted with I54A ... These results indicate that there are multiple DnaK binding sites on ... and that DnaJ strongly promotes DnaK binding to any site in the presence of ATP, regardless of the intrinsic affinity of DnaK for the site. (ProQuest: ... denotes formulae/symbols omitted.)
ISSN:0021-9193
1098-5530