Lid domain truncations reduce the refolding ability of CeHsc70.

<p>(<b>A</b>) Kinetics of firefly luciferase refolding in the presence of different chaperone combinations: CeHsc70/DNJ-13/BAG-1 (∇), CeHsc70/DNJ-13 (▪), CeHsc70/BAG-1 (♦), CeHsc70 (○), BAG-1 (□) and DNJ-13 (▾). Additionally the luminescence of a sample without chaperones and cofactors was analyzed (▴). Protein concentrations were 3.2 µM CeHsc70, 0.8 µM DNJ-13 and 0.4 µM BAG-1. Luciferase refolding assays were carried out as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033980#s4" target="_blank">Materials and Methods</a>. (<b>B</b>) Steady-state ATPase activities (black squares, left ordinate) and luciferase refolding efficiency (blue circles, right ordinate) were determined for 3.2 µM CeHsc70 and 0.8 µM DNJ-13 at different BAG-1 concentrations under standard conditions. (<b>C</b>) The luciferase refolding activity of either CeHsc70 (▪), CeHsc70-Δ545 (▾), CeHsc70-Δ512 (○) or CeHsc70-Δ384 (Δ) was determined in the presence of DNJ-13 and BAG-1. Additionally a control without chaperones and cofactors (◊) was recorded.</p

CeHsc70 truncation mutants show an altered ATP turnover.

<p>(<b>A</b>) Domain organization and amino acid identity (Id) and homology (Hom) of CeHsc70 towards bacterial, yeast and human homologs. The truncation mutants generated in this work are indicated by black arrows. (<b>B</b>) Structure of DnaK based on the PDB file 2KHO <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033980#pone.0033980-Bertelsen1" target="_blank">[93]</a>. The truncations are colored in red (CeHsc70-Δ384), red and blue (CeHsc70-Δ512) and red, blue and yellow (CeHsc70-Δ545). The lid region, which is missing in the CeHsc70-Δ545 mutant, is highlighted in orange. (<b>C</b>) The single-turnover experiments using 20 µM CeHsc70 variants were performed as outlined in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033980#s4" target="_blank">Materials and Methods</a> section in standard buffer at 25°C. Data for CeHsc70-Δ384 (▾), CeHsc70-Δ512 (▴), CeHsc70-Δ545 (•) and CeHsc70 (▪) were fit to single exponential functions. The inset shows the initial phase of the hydrolysis reaction within the first 200 s.</p

The ternary interaction of CeHsc70 with BAG-1 and DNJ-13 is affected by the lid domain truncations.

<p>(<b>A</b>) dc/dt plots were generated from sedimentation velocity experiments of 300 nM *DNJ-13 in the absence (black) or in the presence of 3 µM CeHsc70 and 4 mM ATP (blue). The influence of BAG-1 on complex formation was analyzed by addition of 15 µM BAG-1 to *DNJ-13-CeHsc70-ATP (green). (<b>B</b>) The ATPase activity of 1 µM CeHsc70 (•), CeHsc70-Δ512 (○) or CeHsc70-Δ545 (▾) was measured with increasing amounts of DNJ-13 in the presence of 2 µM BAG-1 in standard buffer at 25°C. Data analysis was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033980#s4" target="_blank">Materials and Methods</a>.</p

Biophysical and enzymatic characterization of lid domain mutants.

<p>ATPase activities were determined in standard buffer as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033980#s4" target="_blank">Materials and Methods</a> section. The K_M-determination was carried out at 2 µM protein concentration and curves showed very tight binding and full saturation at stoichiometric concentrations, implying that the K_M value is smaller than or around 2 µM. Consequently, Michaelis-Menten conditions are not maintained and a determination of an apparent K_D value is not permitted by this experimental setup (indicated by “tight”). K_D denotes the apparent affinity. The errors represent standard deviations of three independent experiments.</p

Characterization of CeHsc70.

<p>(<b>A</b>) The induction of the heat-shock response (black squares, left ordinate) was analyzed by exposing a <i>hsp-70</i>::GFP containing <i>C. elegans</i> strain to different temperatures for two hours and scoring after a recovery time of twelve hours. For this experiment nematodes at YA stage were used. The worms were grown at 20°C before shifting them to the respective heat-shock temperature. The percentage of mortality in percent of deceased animals (blue squares, right ordinate) was determined from these samples as well. The values presented are an average of three independent experiments and the error bars represent the standard deviation. (<b>B</b>) The dependence of the ATPase rate of CeHsc70 (red squares) and HsHsc70 (black circles) was determined under steady-state conditions as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033980#s4" target="_blank">Materials and Methods</a> section. The values represent the mean of three replicates with the corresponding standard deviation given as errors. (<b>C</b>) Determination of the K_M-value of CeHsc70 (•) for ATP in standard buffer at 25°C. Steady-state ATPase activities were determined for CeHsc70 at different ATP concentrations. The data were analyzed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033980#s4" target="_blank">Materials and Methods</a> section. (<b>D</b>) Single-turnover measurement of 20 µM CeHsc70 (•) in the presence of 4 µM ATP in standard buffer at 25°C. Data were analyzed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033980#s4" target="_blank">Materials and Methods</a> section.</p

Enzymatic parameters of cofactor interactions with lid domain mutants.

<p>ATPase activities were determined in standard buffer as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033980#s4" target="_blank">Materials and Methods</a> section. DNJ-13 stimulation or BAG-1 inhibition were not observed in some experiments (denoted by “n.d.”). Consequently an apparent K_D cannot be deduced. The semi-quantitative value “tight” points to the fact that in the respective experiment, quantitative binding appeared substoichiometric. Consequently, no reasonable data fitting can be performed, using the normal absorption isotherm. K_D denotes the apparent affinity. The errors represent standard deviations of three independent experiments.</p

DNJ-13 interacts with CeHsc70 in presence of ATP and is released by BAG-1.

<p>(<b>A</b>) Domain organization of DNJ-13 homologs from <i>C. elegans</i>, <i>E. coli</i>, <i>S. cerevisiae</i> and <i>H. sapiens</i>. Percentages relate to identical (Id) and homolog (Hom) residues in respect to the nematode protein. (<b>B</b>) Steady-state ATPase activities were measured in the presence of increasing amounts of DNJ-13 for either CeHsc70 (•), CeHsc70-Δ512 (○) or CeHsc70-Δ545 (▾). Data were analyzed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033980#s4" target="_blank">Materials and Methods</a> section. (<b>C</b>) Single-turnover measurements of 10 µM CeHsc70-Δ384 (∇), CeHsc70-Δ512 (○), CeHsc70-Δ545 (▾) and CeHsc70 (•) in the presence of 15 µM DNJ-13. All data points were fit to single exponential functions. (<b>D</b>) dc/dt plots were generated from sedimentation velocity experiments of 300 nM *DNJ-13 in the absence (black) or in the presence of 3 µM CeHsc70 (pink). The influence of nucleotides was analyzed by addition of 4 mM of either ADP (gold), AMP-PNP (red), ATPγS (turqoise) or ATP (blue) to 300 nM *DNJ-13 and 3 µM CeHsc70. (<b>E</b>) dc/dt profiles of sedimentation velocity experiments of 300 nM *DNJ-13 in the presence of either 3 µM CeHsc70 (blue), CeHsc70-Δ384 (red), CeHsc70-Δ512 (black) or CeHsc70-Δ545 (green) in the presence of ATP.</p

The function of the NEF BAG-1 is conserved in context with CeHsc70.

<p>(<b>A</b>) Domain organization of BAG-1 homologs from <i>C. elegans</i>, <i>H. sapiens</i> and <i>S. cerevisiae</i>. Percentages relate to identical (Id) and homolog (Hom) residues in respect to the nematode protein (Ubl: ubiquitin-like domain, Bag: BAG-domain, NLS: nuclear localization signal, TRSEEX: region containing multiple repetitions of the pentapeptide TRSEEX. (<b>B</b>) dc/dt plots were generated from sedimentation velocity experiments of 300 nM *BAG-1 in the absence (black) or presence of 3 µM CeHsc70 (blue) or 3 µM of the isolated ATPase domain CeHsc70-Δ384 (red), the full lid-deletion CeHsc70-Δ512 (green) and the half-lid deletion construct CeHsc70-Δ545 (pink). (<b>C</b>) dc/dt plots were generated from sedimentation velocity experiments of 300 nM *BAG-1 alone (black) or in the presence of 3 µM CeHsc70 (blue). The influence of nucleotides was analyzed by addition of 4 mM ATP (green) to 300 nM *BAG-1 and 3 µM CeHsc70. Data were analyzed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033980#s4" target="_blank">Materials and Methods</a> section.</p

A model for the regulation of CeHsc70's ATPase by the lid domain.

<p>A structural hypothesis for the regulation of the CeHsc70 ATPase cycle may be formulated based on the structures 2KHO of DnaK (<i>74</i>) and 3D2E of the Hsp70-homolog protein Sse1 (<i>75</i>). After initial binding of ATP to the NBD of CeHsc70 (Step A→B), conformational changes result in a hydrolysis competent conformation (Step B→C). This reaction is favored in CeHsc70, as evident from the observation, that hydrolysis is not rate-limiting. The helical lid likely regulates the equilibrium or the kinetics of the B→C transition, as this reaction appears to be much slower in CeHsc70-Δ545. DNJ-13 (red) accelerates the formation of the hydrolysis-competent conformation and thus promotes ATP hydrolysis. ATP hydrolysis likely is irreversible (Step C→D). After hydrolysis, DNJ-13 leaves the complex and Hsc70 returns to its open conformation (Step D→E). BAG-1 (yellow) acts to displace the nucleotide (Step E→A). Based on this model, simultaneous BAG-1 and DNJ-13 binding to CeHsc70 would be mutually exclusive, although several intermediate steps might exist during this sophisticated cycle.</p