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. 2011 Nov 22;108(47):18966-71.
doi: 10.1073/pnas.1111220108. Epub 2011 Nov 7.

Heat shock protein 70 kDa chaperone/DnaJ cochaperone complex employs an unusual dynamic interface

Atta Ahmad  1 , Akash Bhattacharya, Ramsay A McDonald, Melissa Cordes, Benjamin Ellington, Eric B Bertelsen, Erik R P Zuiderweg
Affiliations

Heat shock protein 70 kDa chaperone/DnaJ cochaperone complex employs an unusual dynamic interface

Atta Ahmad et al. Proc Natl Acad Sci U S A. .

Abstract

The heat shock protein 70 kDa (Hsp70)/DnaJ/nucleotide exchange factor system assists in intracellular protein (re)folding. Using solution NMR, we obtained a three-dimensional structure for a 75-kDa Hsp70-DnaJ complex in the ADP state, loaded with substrate peptide. We establish that the J domain (residues 1-70) binds with its positively charged helix II to a negatively charged loop in the Hsp70 nucleotide-binding domain. The complex shows an unusual "tethered" binding mode which is stoichiometric and saturable, but which has a dynamic interface. The complex represents part of a triple complex of Hsp70 and DnaJ both bound to substrate protein. Mutagenesis data indicate that the interface is also of relevance for the interaction of Hsp70 and DnaJ in the ATP state. The solution complex is completely different from a crystal structure of a disulfide-linked complex of homologous proteins [Jiang, et al. (2007) Mol Cell 28:422-433].

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Chemical shift perturbations in the 1H-15N TROSY heteronuclear single quantum correlation of DnaJ(1–70) upon titration with DnaK(1–605), in the presence of ADP. The chemical shift changes are depicted on PDB ID code 1XBL. Helix II is at the right, helix III at the left, the HPD-containing loop faces forward. Amide protons are represented as spheres. In all panels, green indicates no effect, gray, no information. (Left) In magenta and red, 1HN chemical shift changes > 0.006 ppm in the spectrum of DnaJ(1–108) upon addition of 2∶1 DnaK(1–605). In red, shifts remaining after addition of NR. (Center) DnaJ(1–70) in red, 1HN chemical shifts > 0.006 ppm and in orange, 0.004 < 1HN < 0.006; (Right) Reduction in signal height of the DnaJ(1–70) NH cross-peaks after adding 1.2∶1 DnaK(1–605)V210C-MTSL (in the presence of NR).
Fig. 2.
Fig. 2.
Cartoons conceptualizing possible DnaK–DnaJ interactions. (A) DnaK (Hsp70) is on top, with NBD in red and SBD in blue. The LID domain is not shown. A DnaJ dimer is at the bottom, with the J domains in yellow, the GF regions in magenta, the SBDs in cyan, and the dimerization helices in brown. Ellipses represent constructs used in this study. (B) Bivalent interaction of a DnaJ monomer with DnaK. The area in the ellipse corresponds to the complex between DnaJ(1–108) and DnaK. (C) The trans-complex between DnaK and DnaJ with substrate peptide (NR). The area in the ellipse corresponds to the complex between DnaJ(1–108) and DnaK with NR. (D) The cis-triple complex between DnaK, DnaJ, and substrate protein. The area in the ellipse corresponds to the complex between DnaJ(1–70) and DnaK(1–605) with NR determined in this work (Fig. 4). (E) As in D, but in context of a hypothetical oligomeric complex involving DnaJ dimers.
Fig. 4.
Fig. 4.
The average position of DnaJ(1–70) with respect to DnaK(1–605) in the presence of ADP and NR as obtained from a molecular dynamics simulation constrained by PRE distance constraints. DnaK NBD is in yellow, DnaK SBD in cyan, and DnaJ(1–70) in white. (A) Location of spin labels as discussed in the text. DnaJ M30C-MTSL (blue) affects the HN resonances on DnaK shown in red. DnaJ R19C-MTSL (green) and K41C-MTSL (green) had no effect. DnaK V210C-MTSL (red), D326C-MTSL (orange), and T417C-MTSL (orange) affect, to different extents, the resonances of the residues on DnaJ indicated in blue. D148C-MTSL, R166C-MTSL, and K421C-MTSL (all in green) had no effect. (B) A superposition of 64 MD snapshots, 0.5-ps apart, showing NBD (yellow) and DnaJ (white) only. Note that the NMR relaxation data show that DnaJ(1–70) is dynamically tethered to DnaK with S2 = 0.37, so each of the J positions is a possible dynamic average. (C) Location of functional residues, discussed in the text, in the complex (color coding as in A).
Fig. 3.
Fig. 3.
Change of the average of the amide proton line width in the 15N1H TROSY heteronuclear single quantum correlation spectrum of DnaJ(1–70) as a function of the addition of DnaK(1–605) (●). The thin line represents the function R2 = ffree × R2free + fbound × R2bound, where ffree and fbound were calculated for KD = 16 μM for the protein concentrations in the experiment. The heavy line is a fit with the same KD but allowing for chemical exchange broadening due to a koff of 14 s-1. (see SI Text)
Fig. 5.
Fig. 5.
Reduction in signal height of the DnaJ(1–70) NH TROSY heteronuclear single quantum correlation cross-peaks due to the presence of spin-labeled DnaK. (Top) DnaK(1–605)V210C-MTSL and NR (DnaJ∶DnaK ratio: blue, 1∶0; green, 1∶0.5; red 1∶1.2). (Middle) As above, but with DnaK(1–605)D326C-MTSL and NR (DnaJ∶DnaK ratio: blue, 1∶0; red 1∶1.2). (Bottom) With DnaK(1–605)K421C-MTSL and NR (DnaJ∶DnaK ratio: blue, 1∶0; red 1∶1.2)
Fig. 6.
Fig. 6.
The crystal structure of the covalent adduct (14) of Auxilin–J domain (orange) and the NBD of human Hsc70 (yellow) superposed on the solution conformation for the noncovalent complex of DnaJ (white), DnaK NBD (yellow), NBD-SBD linker (black), and DnaK SBD (cyan). Different functional residues as discussed in text are labeled. V210 is the center of the J-interaction interface on DnaK.
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