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. 2013 Aug;69(Pt 8):1540-52.
doi: 10.1107/S0907444913010640. Epub 2013 Jul 19.

Combining crystallography and EPR: crystal and solution structures of the multidomain cochaperone DnaJ

Thomas R M Barends  1 , Richard W W Brosi, Andrea Steinmetz, Anna Scherer, Elisabeth Hartmann, Jessica Eschenbach, Thorsten Lorenz, Ralf Seidel, Robert L Shoeman, Sabine Zimmermann, Robert Bittl, Ilme Schlichting, Jochen Reinstein
Affiliations

Combining crystallography and EPR: crystal and solution structures of the multidomain cochaperone DnaJ

Thomas R M Barends et al. Acta Crystallogr D Biol Crystallogr. 2013 Aug.

Abstract

Hsp70 chaperones assist in a large variety of protein-folding processes in the cell. Crucial for these activities is the regulation of Hsp70 by Hsp40 cochaperones. DnaJ, the bacterial homologue of Hsp40, stimulates ATP hydrolysis by DnaK (Hsp70) and thus mediates capture of substrate protein, but is also known to possess chaperone activity of its own. The first structure of a complete functional dimeric DnaJ was determined and the mobility of its individual domains in solution was investigated. Crystal structures of the complete molecular cochaperone DnaJ from Thermus thermophilus comprising the J, GF and C-terminal domains and of the J and GF domains alone showed an ordered GF domain interacting with the J domain. Structure-based EPR spin-labelling studies as well as cross-linking results showed the existence of multiple states of DnaJ in solution with different arrangements of the various domains, which has implications for the function of DnaJ.

Keywords: cross-linking; crystal dehydration; electron paramagnetic resonance; hybrid structure determination; molecular chaperones; radiation-damage-induced phasing with anomalous scattering; single-wavelength anomalous diffraction.

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Figures

Figure 1
Figure 1
Super-sharpened origin-removed isomorphous difference Patterson section (w = 2/3) calculated between the final 120° and the first 120° of data of a 360° data set of the mercury derivative, showing the effect of radiation damage on the mercury derivative. Contour interval = 1σ. This figure was prepared with XPREP (Schneider & Sheldrick, 2002 ▶).
Figure 2
Figure 2
(a) DnaJ sequence, showing the J (grey), GF (blue) and C-terminal (green) domains, as well as the polyproline region and the deletion in the DnaJΔ108–114 mutant (red). (b) Structure of the isolated J/GF domain. The HPD motif and the polyproline region are shown as sticks. EPR spin-labelling and cross-linking sites (Thr18, Ser58, Ser86 and Glu95) are indicated. (c) Interface between the J and GF domains, showing the J-domain surface (coloured by atom type: C, grey; N, blue; O, red) and the GF-domain phenylalanines. All figures use the colour scheme in (a). (d) Stereo image showing the final refined 2mF o − DF c electron-density map of the 1.64 Å resolution DnaJTth114 structure at 1σ. As an example, the polyproline region (residues 75–80) of chain B is shown. All molecular-structure figures were prepared using PyMOL (DeLano, 2002 ▶)
Figure 3
Figure 3
(a) Structure of a dimer of DnaJΔ108–114 from the 2.9 Å resolution crystal structure. The relative orientations of the J/GF domains and the C-­terminal domains differ in the four monomers in the asymmetric unit. (b) 2mF o − DF c density at 1.5σ of the 3.8 Å resolution WT data after molecular replacement with the C-terminal domain of DnaJΔ108–114. No density for the N-terminal domains was found and sufficient space for these domains is present in the packing of the molecules.
Figure 4
Figure 4
(a) Lanes from SDS–PAGE of cysteine mutants of DnaJΔ108–114 without (−) and with (+) cross-linking by BMB after the removal of tetramers by gel filtration (note that the lanes marked * are the same marker lane shown twice for clarity). (b) Luciferase-refolding activity of DnaK assisted by DnaJ. DnaJ variants with mutations in the GF domain show particularly strong modulation of refolding activity. Experiments were conducted in triplicate.
Figure 5
Figure 5
(a–c) X-band pELDOR measurements of DnaJ double mutants (a) T18C/S86C, (b) S86C/E95C and (c) I50C/V90C: background-corrected time traces and their Tikhonov regularization fits (left; regularization parameter α = 10) and corresponding distance distributions superimposed on their respective rotamer distribution predicted from the crystal structure (right). (d) X-band pELDOR measurements of DnaJ single mutants S86C (black) and S58C (blue) using a long time window for measurements of large distances. Dipolar evolution functions with Tikhonov regularization fit (inset; α = 100) and corresponding distance distributions (right). Colour codes above the distributions signify reliability intervals; the reliability of the mean distance of the calculated distribution peaks is shown from dark blue (highest reliability) to red (lowest reliability).
Figure 6
Figure 6
Refolding assays show that SeMet DnaJΔ108–114/4M is functional.
Figure 7
Figure 7
Room-temperature continuous-wave EPR results.
Figure 8
Figure 8
Cross-linking feasibility simulation results.
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