The Lid Domain of Caenorhabditis elegans Hsc70 Influences ATP Turnover, Cofactor Binding and Protein Folding Activity

Abstract

Hsc70 is a conserved ATP-dependent molecular chaperone, which utilizes the energy of ATP hydrolysis to alter the folding state of its client proteins. In contrast to the Hsc70 systems of bacteria, yeast and humans, the Hsc70 system of C. elegans (CeHsc70) has not been studied to date.We find that CeHsc70 is characterized by a high ATP turnover rate and limited by post-hydrolysis nucleotide exchange. This rate-limiting step is defined by the helical lid domain at the C-terminus. A certain truncation in this domain (CeHsc70-Δ545) reduces the turnover rate and renders the hydrolysis step rate-limiting. The helical lid domain also affects cofactor affinities as the lidless mutant CeHsc70-Δ512 binds more strongly to DNJ-13, forming large protein complexes in the presence of ATP. Despite preserving the ability to hydrolyze ATP and interact with its cofactors DNJ-13 and BAG-1, the truncation of the helical lid domain leads to the loss of all protein folding activity, highlighting the requirement of this domain for the functionality of the nematode's Hsc70 protein.

The Balanced Regulation of Hsc70 by DNJ-13 and UNC-23 Is Required for Muscle Functionality

“…PCR products were inserted into the pET28b vector (Merck) and sequenced (GATC Biotech AG, Konstanz, Germany). Expression and purification were performed as described previously (7). Hsc70 was produced in a BioStat C fermenter culture of 7 liters (B. Braun Biotech, Melsungen, Germany).…”

“…Harvested bacteria were frozen at Ϫ80°C, and 50 g of the bacterial pellet was used for each purification. The purification of Hsc70 was carried out as described previously (7).…”

“…Proteins were dialyzed in 40 mM potassium phosphate, pH 7.5, in concentrations of 0.2 mg/ml and measured as described previously (7). Spectra were measured with 10 accumulations, and the molar ellipticity (⌰ MRW ) was calculated.…”

“…ATPase Measurements-The ATPase activity of Hsc70 was determined using an enzyme-coupled assay described previ-ously (7,30). For the activity determination without J domaincontaining proteins 3 M Hsc70 and 5 M NEF (BAG-1 or UNC-23 and the corresponding fragments) were used.…”

“…NEFs, like BAG domain-containing proteins, compete with J domain proteins and trigger nucleotide release (3,6,7). If both cofactors are present, the ATP turnover is activated strongly.…”