Hsc66 and Hsc20, a new heat shock cognate molecular chaperone system from Escherichia coli

Abstract

The hscA and hscB genes of Escherichia coli encode novel chaperone and co-chaperone proteins, designated Hsc66 and Hsc20, respectively. We have overproduced and purified Hsc66 and Hsc2O in high yield in E. coli and describe their initial characterization including absorbance, fluorescence, and circular dichroism spectra. Immunoblot analyses of E. coli cultures using antisera to Hsc66 and Hsc20 raised in rabbits establish that Hsc66 and Hsc2O are constitutively expressed at levels corresponding to cell concentrations -20 pM and -10 pM, respectively. The levels do not change appreciably following heat shock (44"C), but a small increase in Hsc2O is observed following a shift to 10°C. Purified Hsc66 exhibits a low intrinsic ATPase activity (-0.6 min" at 37"C), and Hsc20 was found to stimulate this activity up to 3.8-fold with half-maximal stimulation at a concentration -5 pM. These findings suggest that Hsc66 and Hsc20 comprise a molecular chaperone system similar to the prokaryotic DnaK/Dnd and eukaryotic hsp70/hsp40 systems. Sequence differences between Hsc66 and Hsc20 compared to other members of this chaperone family, however, suggest that the Hsc66/Hsc20 system will display different peptide binding specificity and that it is likely to be subject to different regulatory mechanisms. The high level of constitutive expression and the lack of a major response to temperature changes suggest that Hsc66 and Hsc20 play an important cellular role(s) under non-stress conditions. Keywords: ATPase activity; chaperone; co-chaperone; constitutive expression; hsp70; hsp40; purification The hsp70, or stress-70, proteins comprise a large and widespread family of proteins -7O-kDa, which function as ATP-dependent molecular chaperones (reviewed in Gething

Contributions of the LPPVK Motif of the Iron-Sulfur Template Protein IscU to Interactions with the Hsc66-Hsc20 Chaperone System

“…Overexpression and Purification of Proteins-Recombinant Hsc66, Hsc20, and IscU were prepared as previously described (2,10). In addition, purified Hsc20 was treated to remove any contaminating IscU by affinity chromatography using IscU antiserum coupled to CNBractivated Sepharose.…”

“…ATPase Assays-Steady-state ATPase rates were determined at 23°C in HKM buffer (50 mM Hepes, pH 7.3, 150 mM KCl, 10 mM MgCl 2 ) containing 1 mM DTT and 0.5 mM ATP by measuring phosphate released using the EnzCheck coupled enzyme phosphate assay kit (20) (Molecular Probes) as previously reported (2,3,10,13,14,21). Under these conditions Hsc66 has a basal turnover number of Х0.10 min Ϫ1 .…”

“…The peptides were modeled into the substrate-binding domain and energy-minimized via simulated annealing using CNS (23). 2 …”

“…The interaction of IscU with Hsc66 is enhanced by the J domain co-chaperone Hsc20, which directly binds to both IscU and Hsc66 (2,10). Together Hsc20 and IscU synergistically stimulate the ATPase activity of Hsc66 greater than 400-fold (10).…”

Identification of a novel candidate gene in the iron-sulfur pathway implicated in ataxia-susceptibility: human gene encoding HscB, a J-type co-chaperone

“…The hscB gene in bacteria encodes a 20-kDa J-type co-chaperone, designated Hsc20, that functions with the hsp70-class molecular chaperone HscA/Hsc66 (Seaton and Vickery 1994;Kawula and Lelivelt 1994;Vickery et al 1997). In vitro studies have shown that HscB/Hsc20 regulates interactions between the Fe-S cluster assembly protein IscU and the HscA/ Hsc66 chaperone (Hoff et al 2000;Silberg et al 2001;Hoff et al 2002).…”