DnaJ-Promoted Binding of DnaK to Multiple Sites on σ32 in the Presence of ATP

• Published in: Journal of bacteriology 2014-05, Vol.196 (9), p.1694-1703
• Main Authors: Noguchi, Aki, Ikeda, Ayami, Mezaki, Moeka, Fukumori, Yoshihiro, Kanemori, Masaaki
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphatases - metabolism; adenosine triphosphate; bacteriology; binding sites; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; gel chromatography; Gene Expression Regulation, Bacterial; heat-shock protein 70; Heat-Shock Proteins - chemistry; Heat-Shock Proteins - genetics; Heat-Shock Proteins - metabolism; HSP40 Heat-Shock Proteins - genetics; HSP40 Heat-Shock Proteins - metabolism; HSP70 Heat-Shock Proteins - genetics; HSP70 Heat-Shock Proteins - metabolism; molecular weight; mutants; Protein Binding; Sigma Factor - chemistry; Sigma Factor - genetics; Sigma Factor - metabolism; transcription factors
• ISSN: 0021-9193; 1098-5530
• DOI: 10.1128/jb.01197-13

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 σ32 as a substrate in the DnaK chaperone system, we here provide new insight into the Hsp70-substrate interaction. When DnaK-σ32 complexes formed under various conditions were analyzed by gel filtration, several DnaK-σ32 complexes with different molecular masses were detected. The results indicated that multiple DnaK molecules simultaneously bind to σ32, even though it has been suggested that DnaK interacts with σ32 at a molar ratio of 1:1. Two σ32 mutants, L201D σ32 and I54A σ32, which have reduced affinities for DnaK and DnaJ (Hsp40), respectively, were used to further characterize DnaK-σ32 complex formation. Pulldown assays demonstrated that the affinity of I54A σ32 for DnaK was similar to that of wild-type σ32 in the absence of DnaJ, whereas L201D σ32 exhibited an extremely low affinity for DnaK. However, in the presence of ATP and DnaJ, the yield of DnaK eluted with L201D σ32 was much higher than that eluted with I54A σ32. These results indicate that there are multiple DnaK binding sites on σ32 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.