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. 2006 Feb;15(2):223-33.
doi: 10.1110/ps.051732706. Epub 2005 Dec 29.

The allosteric transition in DnaK probed by infrared difference spectroscopy. Concerted ATP-induced rearrangement of the substrate binding domain

Fernando Moro  1 , Vanesa Fernández-Sáiz, Arturo Muga
Affiliations

The allosteric transition in DnaK probed by infrared difference spectroscopy. Concerted ATP-induced rearrangement of the substrate binding domain

Fernando Moro et al. Protein Sci. 2006 Feb.

Abstract

The biological activity of DnaK, the bacterial representative of the Hsp70 protein family, is regulated by the allosteric interaction between its nucleotide and peptide substrate binding domains. Despite the importance of the nucleotide-induced cycling of DnaK between substrate-accepting and releasing states, the heterotropic allosteric mechanism remains as yet undefined. To further characterize this mechanism, the nucleotide-induced absorbance changes in the vibrational spectrum of wild-type DnaK was characterized. To assign the conformation sensitive absorption bands, two deletion mutants (one lacking the C-terminal alpha-helical subdomain and another comprising only the N-terminal ATPase domain), and a single-point DnaK mutant (T199A) with strongly reduced ATPase activity, were investigated by time-resolved infrared difference spectroscopy combined with the use of caged-nucleotides. The results indicate that (1) ATP, but not ADP, binding promotes a conformational change in both subdomains of the peptide binding domain that can be individually resolved; (2) these conformational changes are kinetically coupled, most likely to ensure a decrease in the affinity of DnaK for peptide substrates and a concomitant displacement of the lid away from the peptide binding site that would promote efficient diffusion of the released peptide to the medium; and (3) the alpha-helical subdomain contributes to stabilize the interdomain interface against the thermal challenge and allows bidirectional transmission of the allosteric signal between the ATPase and substrate binding domains at stress temperatures (42 degrees C).

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Figures

Figure 1.
Figure 1.
(A) Interaction of caged-ATP with DnaK. Equimolar amounts of DnaK and ATP or caged-ATP were mixed and their free concentration was estimated after centrifugation of the mixture in microconcentration filters. (B) Kinetics of ATP-induced conformational change in DnaK. Time dependence of the absorbance change at 1636 cm−1. Protein concentration was ~1.4 mM and the nucleotide:protein molar ratio was 1. Experiments were performed in H2O-based buffer at 25°C. (Inset) Absorbance changes during the first 25 sec after ATP release.
Figure 2.
Figure 2.
ATP binding to DnaK induces absorbance changes in the protein IR spectrum. (A) Time dependence of the IR difference spectrum of DnaK during the first 2 min after ATP release to an ADP-saturated DnaK sample. The final ATP/protein molar ratio was ~1. Protein concentration was 1.4 mM, the buffer was prepared in H2O, and measurements were carried out at 25°C. For the purpose of comparison, a baseline spectrum recorded with the protein sample and without flash (baseline) and the photolysis spectrum (phot) measured without protein are also shown. Average of 13 independent experiments. (B) Kinetics of the absorbance changes at 1650 cm−1 (circles) and 1636 cm−1 (squares), corresponding to the spectra depicted in A. The best fits obtained with single exponentials are shown in solid lines.
Figure 3.
Figure 3.
Assignment of the ATP-binding event. Comparison of the IR difference spectrum of wild-type apo-DnaK obtained during the first 10 sec after ATP release to the medium (upper trace) with the spectrum corresponding to an ATPase defective protein mutant (T199A) (lower trace). Experiments were performed in H2O-based buffer, at a protein concentration of 1.4 mM and 25°C. The spectrum corresponding to the photolysis reaction is shown in dotted line. The nucleotide:protein molar ratio was 1. Average of 10 (wild-type DnaK) and 6 (T199A DnaK) experiments.
Figure 4.
Figure 4.
Assignment of absorbance changes due to interdomain interaction. (A) (Upper panel) ATP-binding spectrum of wild-type DnaK, DnaK(1–507), and the ATPase domain of the protein, recorded in H2O medium at 25°C. Average of 10 (wild-type DnaK), 7 (DnaK1–507), and 13 (ATPase) independent experiments. Protein concentration was 1.4 mM and the nucleotide:protein molar ratio was 1. Spectra were recorded during the first 10 sec after nucleotide release. (Lower panel) ADP-binding spectra of the same samples recorded in H2O-based buffer under the same experimental conditions. Average of seven (wild-type DnaK), six (Dnak1–507), and eight (ATPase) different experiments. (B) (Upper panel) ATP-induced absorbance changes in the IR difference spectrum of wild-type DnaK, DnaK1–507, and the ATPase domain of the protein. Protein concentration was 1 mM and equimolecular amounts of nucleotide were released to the medium. Experiments were performed in D2O-based buffer at 25°C and are the average of five (wild-type DnaK), four (DnaK1–507), and seven (ATPase) different experiments. (Lower panel) ADP-binding spectra of the same samples using the same experimental conditions. Average of 12, 5, and 4 independent experiments for wild-type DnaK, DnaK1–507, and ATPase domain, respectively. The photolysis spectra are shown in dotted lines. Other details are as in Figure 2.
Figure 5.
Figure 5.
Thermal stability of the interdomain interface. (A) ATP-binding IR difference spectra of wild-type DnaK (upper traces) and DnaK1–507 (lower traces) recorded at 25°C (solid line) and 42°C (broken line) in H2O medium. Spectra were recorded during the first 10 sec after ATP release into samples containing ~1.4 mM DnaK or DnaK1–507 saturated with equimolecular amounts of ADP. ADP was added to stabilize the ATPase domain of the protein so that it can stand stress temperatures (e.g., 42°C). The concentration of ATP released from the cage was 1.4 mM and 4.2 mM at 25°C and 42°C, respectively, to account for the temperature-induced activation of the protein. Spectra at 25°C are taken from Figure 4. Average of seven (wild-type) and seven (DnaK1–507) independent experiments at 42°C. (B) Peptide-induced stimulation of the ATPase activity of wild-type DnaK (upper panel) and DnaK1–507 (lower panel) measured at 25°C (empty symbols) and 42°C (filled symbols). Relative values are the average of at least two independent measurements of the ATPase activity of the proteins in the absence (Act0) and in the presence of different concentrations of NR peptide (Act+NR). The best fits obtained, as described in Materials and Methods, are shown in solid (25°C) and broken (42°C) lines.
Figure 6.
Figure 6.
ATP hydrolysis relaxes DnaK to an ADP-like conformation. Time evolution of the IR difference spectrum of DnaK at times >10 sec after ATP release. DnaK concentration was 1.4 mM. Measurements were performed at a nucleotide:protein molar ratio of 1 at 25°C in H2O-based buffer. The lower spectrum corresponds to the photolysis reaction in the absence of protein. Spectra were recorded at the indicated times after the photolysis flash. Average of 10 independent experiments. For the sake of comparison, the difference spectrum corresponding to the ADP-bound state is shown in the upper panel.
Figure 7.
Figure 7.
Kinetics of absorbance changes detected during ATP hydrolysis. The samples contained equimolar amounts of ATP and protein, prepared in H2O- (A) and D2O-based (B) buffers, as detailed in Materials and Methods. Absorbance changes in H2O- and D2O-based buffers are respectively assigned to α-helix (1650 and 1645 cm−1), β-structure (1636 and 1631 cm−1), carboxylate groups (1391 and 1389 cm−1), and phosphate vibrations (1245 and 1250 cm−1).
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