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Review
. 2013:328:99-153.
doi: 10.1007/128_2012_323.

Allostery in the Hsp70 chaperone proteins

Erik R P Zuiderweg  1 , Eric B Bertelsen, Aikaterini Rousaki, Matthias P Mayer, Jason E Gestwicki, Atta Ahmad
Affiliations
Review

Allostery in the Hsp70 chaperone proteins

Erik R P Zuiderweg et al. Top Curr Chem. 2013.

Abstract

Heat shock 70-kDa (Hsp70) chaperones are essential to in vivo protein folding, protein transport, and protein re-folding. They carry out these activities using repeated cycles of binding and release of client proteins. This process is under allosteric control of nucleotide binding and hydrolysis. X-ray crystallography, NMR spectroscopy, and other biophysical techniques have contributed much to the understanding of the allosteric mechanism linking these activities and the effect of co-chaperones on this mechanism. In this chapter these findings are critically reviewed. Studies on the allosteric mechanisms of Hsp70 have gained enhanced urgency, as recent studies have implicated this chaperone as a potential drug target in diseases such as Alzheimer's and cancer. Recent approaches to combat these diseases through interference with the Hsp70 allosteric mechanism are discussed.

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Figures

Figure 1
Figure 1. Hsp70 domain architecture in DnaK E. coli count
Figure 2
Figure 2
The Hsp70 functional cycle as an unfoldase. DnaK NBD is in red, SBD is in blue, LID is in dark green. DnaJ J-domain is in yellow, DnaJ SBD is in cyan, DnaJ GF-region is in pink. Substrate (client) is in green. NEF is in grey. The oligomeric complex with several Hsp70s and Hsp40s is unproven. Successful cycles result in unfolded protein that can spontaneously refold. Unsuccessful cycles result in protein degradation by the proteasome.
Figure 3
Figure 3
A. The relative orientation of the SBD (left) and NBD (right) for DnaK(1-605) in the ADP – NRLLLTG state in solution. NBD domain IA: yellow, IB: blue, IIA: green, IIB: red, linker: white, beta domain: cyan, LID-helix-A: magenta, LID-helix-B: pink, Lid: orange. Residues rendered in space fill are important for the NBD-SBD allosteric communication as determined by mutagenesis studies from several other workers as discussed in the text. B. Dynamical properties of the ADP-NRLLLTG state of the DnaK backbone as determined from cross-peak intensity of HNCO NMR data. High intensity indicates high mobility, low intensity indicates low mobility. Colors: NBD: red, Linker: black, SBD:green, LID: blue. C. Dynamical properties of the ATP-APO state of the DnaK(1-605)(T199A/V436F) backbone as determined from cross-peak intensity of HNCO NMR data. High intensity indicates high mobility, low intensity indicates low mobility. Colors: NBD: red, Linker: black, SBD:green, LID: blue (Bertelsen and Zuiderweg, unpublished).
Figure 3
Figure 3
A. The relative orientation of the SBD (left) and NBD (right) for DnaK(1-605) in the ADP – NRLLLTG state in solution. NBD domain IA: yellow, IB: blue, IIA: green, IIB: red, linker: white, beta domain: cyan, LID-helix-A: magenta, LID-helix-B: pink, Lid: orange. Residues rendered in space fill are important for the NBD-SBD allosteric communication as determined by mutagenesis studies from several other workers as discussed in the text. B. Dynamical properties of the ADP-NRLLLTG state of the DnaK backbone as determined from cross-peak intensity of HNCO NMR data. High intensity indicates high mobility, low intensity indicates low mobility. Colors: NBD: red, Linker: black, SBD:green, LID: blue. C. Dynamical properties of the ATP-APO state of the DnaK(1-605)(T199A/V436F) backbone as determined from cross-peak intensity of HNCO NMR data. High intensity indicates high mobility, low intensity indicates low mobility. Colors: NBD: red, Linker: black, SBD:green, LID: blue (Bertelsen and Zuiderweg, unpublished).
Figure 3
Figure 3
A. The relative orientation of the SBD (left) and NBD (right) for DnaK(1-605) in the ADP – NRLLLTG state in solution. NBD domain IA: yellow, IB: blue, IIA: green, IIB: red, linker: white, beta domain: cyan, LID-helix-A: magenta, LID-helix-B: pink, Lid: orange. Residues rendered in space fill are important for the NBD-SBD allosteric communication as determined by mutagenesis studies from several other workers as discussed in the text. B. Dynamical properties of the ADP-NRLLLTG state of the DnaK backbone as determined from cross-peak intensity of HNCO NMR data. High intensity indicates high mobility, low intensity indicates low mobility. Colors: NBD: red, Linker: black, SBD:green, LID: blue. C. Dynamical properties of the ATP-APO state of the DnaK(1-605)(T199A/V436F) backbone as determined from cross-peak intensity of HNCO NMR data. High intensity indicates high mobility, low intensity indicates low mobility. Colors: NBD: red, Linker: black, SBD:green, LID: blue (Bertelsen and Zuiderweg, unpublished).
Figure 4
Figure 4
A crystal structure of human Hsc70 Δ(555-646) E213A/D214A . A, Left, overall docking. Green: SBD, cyan: SBD-beta; blue LID; black NBD-SBD linker. A213 and A214 are yellow. B, Right, docking of the NBD (ribbon) on the SBD/LID/Linker (surface). The linker is at the bottom. Color coding: green: apolar, red: negative, blue: positive, white:polar, mud-green: Thr+Tyr. The mutations A213 and A214 on the NBD are in pink interact directly with a hydrophobic SBD surface.
Figure 5
Figure 5
A crystal structure of Hsp110 of S. cervisea Orientation and color coding is as in Fig 3a. (NBD domain IA: yellow, IB: blue, IIA: green, IIB: red, linker: white, beta domain: cyan, LID-helix-A: magenta, LID-helix-B: pink, Lid: orange).
Figure 6
Figure 6
In vitro studies of DnaK(1–507) allosteric function. a, ATP-induced release of peptide F-APPY in DnaK(1–507) measured by fluorescence anisotropy. The first bar represents the anisotropy value for peptide bound to 1.1 mM DnaK(1–507). The second bar represents the anisotropy value 5 min after addition of 0.44 mM ATP. The third and fourth bars represent the values for wt-DnaK under comparable conditions, and the last bar indicates the anisotropy value of free peptide. Error bars reflect the standard deviation from a mean of three measurements. b, Peptide stimulation of ATPase activity of DnaK(1–507) (P) and wt-DnaK (m). As DnaK(1–507) is titrated with the peptide NRLLLTG, the ATPase activity is stimulated in a manner similar to that of wt-DnaK. The hydrolysis rate is reported as moles of ATP hydrolyzed per minute per mole of DnaK(1–507) or wt-DnaK. The error bar on the first point reflects the standard deviation from a mean of three measurements and is valid for both assays. From ref
Figure 7
Figure 7
A: Superposition of five crystal structures for bovine Hsc70 NBD: wt-APO (2QW9.pdb), wt-ADP.PO4 (3HSC.pdb and 2QWL.pdb), wt-ADP.VO4 (2QWM.pdb) and K71M-ATP (1KAX.pdb). The N-terminus is in red, the C-terminus is in yellow. B: 15N-1H chemical shift differences between the ATP and ADP.Pi conformation of Hsc-70-NBD. Orange: significant shift, green: no shift, grey: not known. C: 15N-1H chemical shift differences between the AMPPNP and ADP.Pi conformation of TTh-NBD. Red: large shifts; orange: medium shifts; yellow: small shifts; green: no shift; grey: not known. ADP is in light blue, PO43- in dark blue.
Figure 8
Figure 8
The SBD's view of the IA / IIA interface of DnaK-TTh . A, Left: in the ADP state. B, Right: in the AMPPNP state . Color coding: hydrophobics, green; positive, blue; negative, red; polar, white. The C-terminus (residue 372) is magenta. Residue L174 (L177 in DnaK E. coli) in yellow, residue R148 (R151 in E. coli) in cyan, residue A152 (K155 in E. coli) sand, residue R164 (R167 in E. coli) in teal.
Figure 9
Figure 9
A, Left: The hypothetical “proline switch” in Hsc70 ADP.Pi. B, Right: The hypothetical “proline switch” in DnaK-E. coli without nucleotide. Following Ref .
Figure 10
Figure 10
A. Comparison of E. coli DnaK(389-605)-NRLLLTG (yellow) with DnaK(393-507)-NRLLLTG (blues), with nomenclature following ref . B. Comparison of DnaK(393-507-NRLLLTG (blue) with DnaK(393-507).apo (red) C. DnaK(389-605)-NRLLLTG. The ligand is in blue. Residue 552 is in green. L542Y, L543E on the LID are in sticks. K414, N415 and P419 are in space fill, T417 and I418 are in dots. D. The N-terminal “face” of DnaK(389-605)-NRLLLTG. Phobics are in green, positives in blue, negatives in red, polars white. The structure protruding at 3 o'clock is the residual NBD-SBD linker, with Asp393 (red) close to the SBD core.
Figure 11
Figure 11
Differences in amide-proton exchange for E. coli DnaK in the ATP and ADP state. Positive numbers indicate less protection in the ATP state. From
Figure 12
Figure 12. Chemical shifts changes and two-domain allostery
A. Chemical shift changes between the ADP-apo and ADP-NRLLLTG state in DnaK T. Thermophilus (1-501) From ref . B. Chemical shift changes between ADP-apo DnaK E. coli (1-605) and ATP-apo DnaK E. coli (1-605) (T199A/V436F) (Bertelsen and Zuiderweg, unpublished)
Figure 13
Figure 13
Cartoon representation of the allosteric changes between the ADP.sub state (top) and ATP-apo state (bottom). Color coding is (as in Fig 1): NBD subdomain IA, yellow; IB, blue; IIA, green; IIB, red; linker, gray; beta domain, cyan; LID-helix-A, magenta; Lid, orange. Relative domain orientations of NBD, SBD and LID in the top cartoon are representative of reality (see Fig 1). The relative domain orientations of those domains in the bottom cartoon are based on the hypotheses reviewed herein. Also see Table 4.
Figure 14
Figure 14
NBD-NEF complexes. The NBD's were superposed and all colored blue. GrpE (1DKG) in red; Hsp110 (3C7N) in magenta, BAG-1 (1HX1) in green; BAG-2 (3CQX) in yellow. The NBD of HSC without NEF (3HSC) is shown in cyan.
Figure 15
Figure 15
Hypothetical GrpE-DnaK complexes. A. Model of a functional E. coli DnaK-GrpE complex based on the structures of DnaK ADP.NRLLTG (2KHO) and DnaK-NBD + GrpE 40-197 (1DKG.pdb) in which the NBD's were superposed. GrpE is in red. The GrpE N-terminus (residue 40) is at the left. B. Chemical shifts occurring on DnaK T. Thermophilus (1-501) upon addition of GrpE T. Thermophilus. Model composed as in Fig 15a. Deep and Zuiderweg, unpublished.
Figure 16
Figure 16
The solution structure of E. coli DnaJ(2-76) in context of DnaJ(2-108) (1XBL). The mutationally sensitive residues Y25 (yellow), K26 (red), R26(magenta) , H33(green) ,P34 (blue) ,D35 (cyan) and F47 (orange) are high-lighted.
Figure 17
Figure 17
A: Overlay of a crystal structure of the peptide-binding and dimerization domain of human HDJ1 in yellow, with that of yeast YDJ1, in cyan. The HDJ1 substrate (GPTIEEVD) is in red, the YDJ1 substrate (GWLYEIS) is in blue. The CA positions in the SBD (193-240 and 205-252 for HDJ1 and YDJ1, respectively) were superposed. YDJ1's Zn domain is at the left, HDJ1's dimerization helices are at the right (the latter were deleted in the crystallization construct of YDJ1). B: detail of the hydrophobic cleft of HDJ1, composed of residues M183, I185, L204, I206, F220, I235 and F237 and ligand GPTIEEVD.
Figure 18
Figure 18
Hypothetical binding modes between DnaK (Red: NBD; blue: SBD), DnaJ (Yellow: J-domain; magenta: G-F; cyan: SBD; brown: dimerization domain and substrate (green) in the ADP state.
Figure 19
Figure 19. The location of residues on E. coli DnaK that affect DnaK-DnaJ interaction as deduced from mutagenesis experiments (see text)
Figure 20
Figure 20
Crystal contacts in the covalent adduct of human Auxilin (DnaJ homologue) and the NBD human Hsc70 . The NBD and J-domain of the PDB-deposited adduct are in light and dark green, respectively. ADP is in white. The NBD and J-domain of the neighbor in the crystal are in red and magenta, respectively. The “green” J-domain has a 372 Å2 interface with its disulfide-linked Hsc70 NBD partner (light green). However, a symmetry-related NBD (red) has a 582 Å2 interface with the same “green” J-domain (using www.ebi.ac.uk/msd-srv/prot_int/pistart.html).) This figure was prepared using the Swiss PDB viewer.
Figure 21
Figure 21
The crystal structure of the covalent adduct of Auxilin-J-domain (orange) and the NBD of human Hsc70 (yellow) superposed on the solution conformation for the non-covalent complex of DnaJ-J-domain (white), DnaK NBD (yellow), NBD-SBD linker (black) and DnaK SBD (cyan). On Auxilin, the HPD loop and its covalent link to the NBD are in purple and the positive residues on helix II are red. On DnaJ, the HPD loop is green, and the positive residues on helix II are blue. Val210, in the center of the DnaK interface, is in blue-gray.
Figure 22
Figure 22. Potential sites for interference with the Hsp70 chaperone function. Purple: primary sites, Cyan: allosteric sites, blue: co-chaperone interaction sites
Figure 23
Figure 23
The NMR-determined binding sites of recently discovered Hsp70 inhibitors. Left, 115-7C; middle, myricetin right, MKT-077. Note that neither compound binds to a Hsp70 primary site, hence the compounds affect Hsp70 function by affecting allostery or co-chaperone interaction.
Figure 24
Figure 24
A. Chemical shifts seen for the interaction of MKT-077 (cyan) with human Hsc70 in the ADP state. B. Chemical shifts seen for the interaction of MKT-077 with human Hsc70 in the AMP-PNP state
Figure 25
Figure 25
A. The MKT-077 binding site (blue) projected on human Hsc70 NBD in the open state (complexed with NEF; 3C7N.pdb). B. The MKT-077 binding site (blue) projected on Hsc70 NBD in the closed state (3HSC.pdb)
Figure 26
Figure 26
The MKT-077 Hsc70 NBD interaction in detail. A. Collection of poses for the binding of MKT077 to Hsc70 (3C7N.pdb) obtained from NMR restrained AUTODOCK. Blue: NMR shifts. B. Final best binding pose of MKT077 (cyan) on Hsc70 (3C7N.pdb) obtained with AMBER. Green: no shifts; red: shifts; grey: undecided.
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