1. Molecular Mechanism of J-Domain-Triggered ATP Hydrolysis by Hsp70 Chaperones 
Roman Kityk, Jürgen Kopp, Matthias P. Mayer 
Molecular Cell 
Volume 69, Issue 2, Pages e4 (January 2018)
DOI: /j.molcel
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2. Molecular Cell 2018 69, 227-237.e4DOI: (10.1016/j.molcel.2017.12.003)
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3. Figure 1
Crystal Structure of DnaK·ATP in Complex with the J-Domain of DnaJ
(A) JDPs in synergism with substrates stimulate the ATPase activity of Hsp70 proteins, leading to efficient substrate trapping.
(B) Structure of Hsp70·ATP (PDB: 4B9Q) and Hsp70·ADP (PDB: 2KHO) with bound substrate (dark blue spheres).
(C) Structure of DnaK in complex with ATP·Mg2+ and the J-domain of DnaJ. NBD in dark teal (lobe I) and cyan (lobe II), linker in yellow, SBDβ in dark red, SBDα in orange, the J-domain in purple (PDB: 5NRO), and the HPD motif in green spheres.
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4. Figure 2
The Structure of JD-DnaKcryst Overlays Well with Previous Structures of DnaK·ATP and Is Completely Consistent with Previous Genetic, Biochemical, and Biophysical Data
The structure of JD-DnaKcryst aligns well with the structure of DnaK·ATP in the absence of a J-domain (PDB: 4B9Q; Kityk et al., 2012; with a root-mean-square deviation [RSMD] = over 430 atoms) (PDB: 4JN4; Qi et al., 2013; RMSD = over 442 atoms).
(A) Ribbon representation of an overlay of the NBD plus linker of JD-DnaKcryst (brown, red, yellow) with the NBD plus linker of DnaK·ATP (blue, PDB: 4B9Q; Kityk et al., 2012). Arrowheads point to small deviations within the structure most likely caused by direct contact with the J-domain.
(B) Ribbon representation of an overlay of the linker plus SBD of JD-DnaKcryst (yellow, dark red, and orange) with the linker plus SBD of DnaK·ATP (blue, PDB: 4B9Q; Kityk et al., 2012).
(C) Cartoon representation of an overlay of the J-domain of JD-DnaKcryst (purple) with the NMR structure of the J-domain of E. coli DnaJ (green, PDB: 1XBL; Pellecchia et al., 1996). The HPD motifs of both structures are shown as sticks. The helices of the J-domain are numbered (JαI–JαIV).
(D) Residues of the J-domain contacting DnaK are consistent with previous genetic, biochemical, and structural data. Top: J-domain colored according chemical shift perturbations found in nuclear magnetic resonance (NMR) experiments by titrating the NBD of DnaK to 13C-15N-labeled J-domain (magenta and red; Greene et al., 1998) and according to intensity quenching observed in NMR paramagnetic relaxation enhancement experiments (red and orange residues are within 5–15 Å of position 210 in DnaK; Ahmad et al., 2011). Middle: J-domain colored according to possible polar (<3.5 Å) and non-polar contacts (<4 Å) in our structure. Bottom: J-domain with residues colored according to growth defects observed in E. coli upon replacement by alanine or glycine (Genevaux et al., 2002).
(E) Surface representation of the NBD (residues 2–385) plus linker (residues 386–393; lavender) colored according to mutagenesis (magenta and red; Gässler et al., 1998) and NMR experiments (red and orange residues are within 5–15 Å of position 30 in the J-domain; Ahmad et al., 2011). The J-domain is shown in cartoon representation for orientation.
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5. Figure 3
The Universally Conserved HPD Motif Has a Functional Role in the Stimulation of ATP Hydrolysis by DnaK
(A) Zoom into the JD-DnaKcryst structure, highlighting hydrophobic interactions. Indicated residues in space-filling representation in atom colors with carbon in black (linker), purple (J-domain), green (HPD-motif), and dark red (SBDβ).
(B) Zoom into the JD-DnaKcryst structure, highlighting polar interactions of HPD motif His33 and Asp35 and Arg36 of the J-doman with linker, NBD and SBDβ. Residues colored in atom colors with carbon in dark teal (NBD lobe I), black (linker), purple (J-domain), green (HPD-motif), and dark red (SBDβ). Black dashed lines, potential hydrogen bonds (distance 2.2–3.4 Å).
(C) Single-turnover ATPase rate of DnaKwt and DnaK-Q378A in the absence of cofactors and in the presence of DnaJ (50 nM), σ32 (1 μM), or both (mean ± SEM, n = 3).
(D) Refolding of chemically denatured luciferase in the absence of chaperones (black) and in the presence of DnaKwt (dark blue) or DnaK-Q378A (dark red; 800 nM), DnaJ (40 nM), and GrpE (400 nM) (mean ± SEM, n = 3).
(E) Complementation of the temperature-sensitivity phenotype of a ΔdnaK E. coli strain. Tenfold serial dilutions of an overnight culture of a ΔdnaK strain containing an empty vector or expressing dnaKwt or Q378A were grown at 30°C or 40°C (for protein levels, see Figure S2).
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6. Figure 4
The J-Domain Enhances Substrate-Mediated Signal for ATP Hydrolysis
(A and B) Zoom into the JD-DnaKcryst structure, highlighting the contacts of the J-domain with the NBD (A) and the SBDβ (B). See also Figure S3.
(C) Complementation of the temperature sensitivity of a ΔdnaK E. coli strain. Tenfold serial dilutions of an overnight culture of a ΔdnaK strain containing an empty vector or expressing dnaKwt, dnaK-T420A, or dnaK-D477A were grown at 30°C or 40°C (for protein levels, see Figure S4).
(D) Refolding of chemically denatured luciferase in the absence of chaperones (black) and the presence of DnaKwt (dark blue) or DnaK-D477A (dark red; 800 nM), DnaJ (40 nM), and GrpE (400 nM) (mean ± SEM, n = 3; data for DnaKwt and no chaperones are the same as in Figure 2D).
(E) Single-turnover ATPase rates of DnaKwt and DnaK-D477A in the absence of cofactors and in the presence of DnaJ (50 nM), σ32 (1 μM), or both (mean ± SEM, n = 3).
(F) DnaK-D477A has a lower affinity for substrates. Equilibrium titration of the fluorescent-labeled peptide σ32-Q132-Q144-C-IAANS (Mayer et al., 2000) with DnaKwt and DnaK-D477A. Inset, KD values (mean ± SEM, n = 3; ∗∗∗∗p < ; determined by F-test).
(G) Single-turnover ATPase rates of DnaKwt, DnaK-M404A (Mayer et al., 2000), or DnaK-D477A in the presence of DnaJwt or DnaJ-K48A (20 nM) and increasing concentrations of σ32 (mean ± SEM, n = 3).
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7. Figure 5
Residues Involved in J-Domain-Hsp70 Interactions Are Conserved in Evolution
(A) Top: relative identity (same residue as in E. coli DnaJ, indicated below the columns; darker colors) or similarity (as indicated in parenthesis below the columns; lighter colors) as derived by a BLAST search using class A JDPs E. coli DnaJ, S. cerevisiae Ydj1 and human DnaJA1, and class B JDPs E. coli CbpA, S. cerevisiae Sis1, and human DnaJB1 as query against the UNIPROT90 and vertebrate databases and subsequent Clustal Ω sequence alignment. Bottom: same as in the top panel, except that the sequences of E. coli DnaK, S. cerevisiae Ssa1, and human Hsc70 were used as a query. The arrows between the two panels connect the pairs of residues that interact with each other. The brackets indicate polar interaction with the backbone carbonyl or amide proton. Of note, because of a short (4-residue) deletion in eukaryotic Hsp70s corresponding to residues 211–214 of DnaK, the Clustal Ω sequence alignment does not detect homologs of residues 206 and 211, which in our structure interact with Arg22JD and Arg27JD.
(B) Single-turnover ATPase rates of wild-type and mutant human Hsc70 (HSPA8) in the absence and presence of human Hdj1 (DNAJB1, 2 μM) (mean ± SD, n = 3; ∗∗∗∗p < ; ∗∗∗p = , ANOVA Sidak’s multiple comparisons test).
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8. Figure 6
Model for the Synergistic Stimulation of the ATPase Activity of DnaK by DnaJ and Substrate as Basis for Efficient Trapping of Substrates by Hsp70 Chaperones
(A) After ATP binding, the SBDβ docks onto the rotated lobes of the NBD by interaction between Lys414 and Asp329 and between Asp481 with Arg167 and Ile168, preventing back rotation to an optimal position for ATP hydrolysis. In the absence of substrate and a J-domain, basal ATPase activity is very low (path 1). Mutating Lys414 or Asp481 increases ATPase activity by 25- to 80-fold (Kityk et al., 2015). The addition of substrate (path 2) leads to dissociation of the SBDβ from the NBD through a signal propagated through the SBDβ to the NBD (hatched arrows) (Zhuravleva et al., 2012), stimulating ATPase activity, albeit by only 2- to 5-fold due to complete release of the SBDβ, which allows too much freedom to the lobes of the NBD (green arrows) and dissociation of the linker from the lower crevice (blue arrow). The inefficient stimulation of ATPase activity traps substrates inefficiently (transparent blue substrate symbol on DnaK), and some substrates may dissociate before closure of the substrate-binding pocket (arrow with transparent blue substrate symbol). In the simultaneous presence of DnaJ and a substrate (path 3), the J-domain binds at the interface between SBDβ and NBD, stimulates ATPase activity directly through the interaction network from the linker to the catalytic center and indirectly by making DnaK more sensitive to the substrate signal (solid arrows in the SBDβ), and prevents dissociation of SBDβ and linker, leading to a very high ATPase rate and effective trapping of the substrate.
(B) Structure of the DnaK-J-domain complex highlighting as spheres His33 and Asp35 of the J-domain, residues involved in SBDβ docking onto the NBD, and residues involved in signal transmission from the substrate to the NBD (Val440, Leu484, and Asp148; Kityk et al., 2015).
(C) Schematic representation of the network of polar and non-polar contacts connecting the J-domain to the catalytic center of the NBD. Arrows indicate polar contacts, and hatched lines non-polar contacts.
See also Movie S2.
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