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. 2019 Jul 12;431(15):2729-2746.
doi: 10.1016/j.jmb.2019.05.026. Epub 2019 May 22.

Intermolecular Interactions between Hsp90 and Hsp70

Shannon M Doyle  1 , Joel R Hoskins  2 , Andrea N Kravats  2 , Audrey L Heffner  2 , Srilakshmi Garikapati  2 , Sue Wickner  3
Affiliations

Intermolecular Interactions between Hsp90 and Hsp70

Shannon M Doyle et al. J Mol Biol. .

Abstract

Members of the Hsp90 and Hsp70 families of molecular chaperones are imp\ortant for the maintenance of protein homeostasis and cellular recovery following environmental stresses, such as heat and oxidative stress. Moreover, the two chaperones can collaborate in protein remodeling and activation. In higher eukaryotes, Hsp90 and Hsp70 form a functionally active complex with Hop (Hsp90-Hsp70 organizing protein) acting as a bridge between the two chaperones. In bacteria, which do not contain a Hop homolog, Hsp90 and Hsp70, DnaK, directly interact during protein remodeling. Although yeast possesses a Hop-like protein, Sti1, Hsp90, and Hsp70 can directly interact in yeast in the absence of Sti1. Previous studies showed that residues in the middle domain of Escherichia coli Hsp90 are important for interaction with the J-protein binding region of DnaK. The results did not distinguish between the possibility that (i) these sites were involved in direct interaction and (ii) the residues in these sites participate in conformational changes which are transduced to other sites on Hsp90 and DnaK that are involved in the direct interaction. Here we show by crosslinking experiments that the direct interaction is between a site in the middle domain of Hsp90 and the J-protein binding site of Hsp70 in both E. coli and yeast. Moreover, J-protein promotes the Hsp70-Hsp90 interaction in the presence of ATP, likely by converting Hsp70 into the ADP-bound conformation. The identification of the protein-protein interaction site is anticipated to lead to a better understanding of the collaboration between the two chaperones in protein remodeling.

Keywords: DnaJ; Hsp40; Hsp82; HtpG; Ssa1.

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Conflict of interest statement

Competing financial interests: The authors declare no competing financial interests.

Figures

Fig. 1.
Fig. 1.
Hsp90Ec and DnaK residues directly interact. (a) Docked model [32] of the apo structure of Hsp90Ec (PDB ID code 2IOQ) [15] and ADP-bound DnaK (PDB ID code 2KHO) [73]. The apo conformation of the Hsp90Ec dimer is shown as a surface rendering with one protomer in gray and one protomer in wheat. DnaK in the ADP-bound conformation [73] is shown as a ribbon model with the NBD in light green and the SBD in blue. Residues in Hsp90Ec, K354 and Q358, that were mutated in this study are shown in red and orange, respectively, while residues in DnaK, E209, D211 and G328, that were mutated in this study are shown as CPK models in purple, cyan and blue, respectively. The black square represents the field enlarged in (b). (b) Enlargement of the interaction region between Hsp90Ec and DnaK shown in (a). Hsp90Ec and DnaK are colored as in (a) and residues mutated in this study are also as in (a). Yellow dashed lines indicate the pairs of Hsp90Ec and DnaK residues that were used in crosslinking studies. (a) and (b) were both rendered using PyMOL (https://pymol.org/2/). (c) Hsp90Ec-Q358C and DnaK-G328C (4 μM each) were treated with the crosslinker DTME (13.3 Å linker) alone and in a mixture together and covalent bond formation was monitored by SDS-PAGE followed by Coomassie blue staining. (d) The crosslinked product from (c) lane 3 was extracted and treated with a reducing agent and the products analyzed by SDS-PAGE followed by Coomassie blue staining. (e) Hsp90Ec mutants Hsp90Ec-Q358C and E584C (4 μM), and DnaK mutants DnaK-G328C and D45C (4 μM), were treated with BMH (13 Å linker) alone and in mixtures as indicated and covalent bond formation was monitored by SDS-PAGE followed by Coomassie blue staining. Higher molecular weight bands in lanes 2, 5, 6 and 8 are likely crosslinked dimers of Hsp90Ec-Q358C (lane 2 and 8) and E584C (lane 5 and 6); the potential Hsp90Ec-E584C dimer appears to run at a lower molecular weight than other crosslinked products likely due to it attaining a more compact conformation following crosslinking. In (c-e), (*) indicates crosslinked product of interest and the gels shown are representative of at least three independent experiments.
Fig. 2.
Fig. 2.
Specific crosslinking of a pair of Hsp90Ec and DnaK residues important for interaction. (a) The in vivo interaction between Hsp90Ec wild-type or K354C and DnaK wild-type or E209C was monitored using a bacterial two-hybrid system that measured beta-galactosidase activity in liquid culture. β-galactosidase activity is shown as mean ± SEM (n=3). (b) The in vitro interaction between biotinylated Hsp90Ec wild-type or K354C and DnaK wild-type or E209C (4 μM each) was determined using a pull-down assay in the presence of L2, CbpA and ATP as described in Materials and Methods and analyzed by SDS-PAGE followed by Coomassie blue staining. DnaK wild-type or E209C associated with biotinylated Hsp90Ec wild-type or K354C was quantified using densitometry as described in Materials and Methods and is shown as a bar graph. For each lane of the gel the amount of DnaK was corrected for non-specific binding (NSB) and then was normalized to Hsp90Ec-biotin and the ratio of DnaK mutant to wild-type is plotted as the mean ± SEM (n=6). (c) Hsp90Ec-K354C and DnaK-E209C were treated with CuCl2 (~2 Å linker) alone and in a mixture together and covalent bond formation was monitored by SDS-PAGE followed by Coomassie blue staining. (*) indicates crosslinked product of interest. A small amount of a slowly migrating species was likely the covalently linked Hsp90Ec-K354C dimer, since it was observed in reactions with and without Ssa1 (lanes 3 and 4). In (c), the gel shown is representative of at least three independent experiments.
Fig. 3.
Fig. 3.
Ssa1 residues homologous to DnaK residues important for interacting with Hsp90Ec are important for Hsp82 and Ydj1 binding. (a) Chart showing residues in S. cerevisiae Hsp70, Ssa1, that align with four residues in E. coli DnaK, that were previously shown to be in the Hsp90Ec interaction region [32]. (b) Model of Ssa1 in the ADP-bound conformation (based on the ADP-bound conformation of DnaK (PDB ID code 2KHO) [33, 73] shown as a surface rendering with the NBD in gray and the SBD in blue and generated using PyMOL (https://pymol.org/2/). Residues mutated in this study and tested for interaction with Hsp82 are shown in color and labeled. (c) The in vitro interaction between biotinylated Hsp82 (2.5 μM) and Ssa1 wild-type or mutant (8 μM) was monitored using a pull-down assay as described in Materials and Methods. The results were analyzed by SDS-PAGE followed by Coomassie blue staining. Ssa1 wild-type or mutant associated with biotinylated Hsp82 was quantified using densitometry as described in Materials and Methods and is shown as a bar graph. For each lane of the gel, the amount of Ssa1 was corrected for non-specific binding (NSB) and then was normalized to Hsp82-biotin and the ratio of Ssa1 mutant to wild-type is plotted. (d) The in vitro interaction between biotinylated Ydj1 (1.2 μM) with Ssa1 wild-type or mutant (2 μM) was monitored using a pull-down assay as described in Materials and Methods. The results were analyzed by SDS-PAGE followed by Coomassie blue staining. Ssa1 wild-type or mutant associated with biotinylated Ydj1 was quantified using densitometry as described in Materials and Methods and is shown as a bar graph. For each lane of the gel, the amount of Ssa1 was corrected for non-specific binding (NSB) and then was normalized to Ydj1-biotin and the ratio of Ssa1 mutant to wild-type is plotted. (e) Heat-inactivated luciferase reactivation by Ssa1 wild-type or mutant and Ydj1 as indicated and described in Materials and Methods. Data from three or more replicates are presented as mean ± SEM. For some points, the symbols obscure the error bars. (f) The in vitro interaction between biotinylated Sti1 (2 μM) and Ssa1 wild-type or mutant (4 μM) was monitored using a pull-down assay as described in Materials and Methods. The results were analyzed by SDS-PAGE followed by Coomassie blue staining. Ssa1 wild-type or mutant associated with biotinylated Sti1 was quantified using densitometry as described in Materials and Methods and is shown as a bar graph. For each lane of the gel, the amount of Ssa1 was corrected for non-specific binding (NSB) and then was normalized to Sti1-biotin and the ratio of Ssa1 mutant to wild-type is plotted. In (c, d and f), the gels shown are representative of at least three independent experiments and quantification of the data from three or more replicate gels are presented as mean ± SEM.
Fig. 4.
Fig. 4.
Hsp82 and Ssa1 residues directly interact. (a) Docked homology models of apo yeast Hsp82 and ADP-bound Ssa1 [33]. The apo conformation of the Hsp82 dimer is shown as a surface rendered model in light cyan and light gray and the ADP-bound conformation of Ssa1 is shown as a ribbon model with the NBD in dark gray and the SBD in wheat. Residues in Hsp82, P281, E402 and E409, that were mutated in this study are shown in red, orange and yellow, respectively, while residues in Ssa1, T219, K322 and L323, that were mutated in this study are shown as CPK models in blue, green and purple, respectively. The black square represents the field that is enlarged in (b). (b) Enlargement of the interaction region between Hsp82 and Ssa1 shown in (a). Hsp82 and Ssa1 are colored as in (a) and residues mutated in this study are also as described in (a). Yellow dashed lines indicate the pairs of Hsp82 and Ssa1 residues that were used for crosslinking experiments. (a) and (b) were both rendered using PyMOL (https://pymol.org/2/). (c) Hsp82-P281C and Ssa1-T219C (4 μM each) were treated with the crosslinker DTME (13.3 Å linker) alone and in a mixture together and covalent bond formation was monitored by SDS-PAGE followed by Coomassie blue staining. The major crosslinked species (*) is consistent with crosslinked Hsp82-P281C and Ssa1-T219C (lane 3). A small amount of a slowly migrating species was likely the covalently linked Hsp82-P281C dimer, since it was observed in reactions with and without Ssa1 (lanes 3 and 4). The minor species seen in crosslinking reactions with Hsp82-P281C and Ssa1-T219C that migrated slightly faster than the predominant species was not characterized (lane 3). (d) The predominant crosslinked species (*) from (c) lane 3 was extracted and treated with a reducing agent and the products analyzed by SDS-PAGE followed by Coomassie blue staining. (e) Hsp82-E409C and Ssa1-K322C (4 μM each) were treated with CuCl2 (~2 Å linker) alone and in a mixture together and covalent bond formation was monitored by SDS-PAGE followed by Coomassie blue staining. The Hsp82-P281C and Ssa1-T219C crosslinked species is indicated (*, lane 3) and a second species consistent with the covalently linked Hsp82-P281C dimer, since it was observed in reactions with and without Ssa1, was also observed. (f) Hsp82 mutants including Hsp82-P281C, E409C and Q635C (4 μM), and Ssa1-T219C (4 μM) are treated with BMH (13 Å linker) alone and in mixtures together as indicated and covalent bond formation is monitored by SDS-PAGE followed by Coomassie blue staining. Crosslinked dimers of Hsp82-P281C (lane 3 and 4), E409C (lane 5 and 6) and Q635C (lane 7 and 8) can be observed; Hsp82-Q635C runs at a lower molecular weight than other crosslinked products likely due to it attaining a more compact conformation following crosslinking. In (c-f), (*) indicates crosslinked product of interest and the gels shown are representative of at least three independent experiments.
Fig. 5.
Fig. 5.
J-protein promotes the interaction between DnaK and Hsp90Ec in the presence of ATP. (a) Hsp90Ec-Q358C (4 μM) and DnaK-G328C (4 μM) alone and in mixtures with CbpA (1 or 4 μM) (no endogenous cysteines) in the absence or presence of 4 mM ATP as indicated were treated with BMH (13 Å linker) and covalent bond formation was monitored by SDS-PAGE followed by Coomassie blue staining. (b) In the presence of ATP (4 mM), Hsp90Ec-Q358C (4 μM) and DnaK-G328C (4 μM) alone and in mixtures with increasing concentrations of CbpA as indicated were treated with BMH (13 Å linker) and covalent bond formation was monitored by SDS-PAGE followed by Coomassie blue staining. Crosslinked products were quantified using ImageJ (http://imagej.nih.gov/ij) and standardized to crosslinked products in the absence of CbpA (lane 3) and plotted as the mean ± SEM (n=3). (c) Hsp90Ec-K354C (4 μM) and DnaK-E209C or DnaKD211C (4 μM) in the presence or absence of 4 mM ATP as indicated, were treated with CuCl2 and covalent bond formation was monitored by SDS-PAGE followed by Coomassie blue staining. (*) indicates crosslinked product of interest for DnaK-E209C and Hsp90Ec-K354C (lanes 3 and 8) and (*) indicates crosslinked product of interest for DnaK-D211C and Hsp90Ec-K354C (lanes 5 and 10). (d) Crosslinked products from (c) were quantified using ImageJ (http://imagej.nih.gov/ij) and standardized to crosslinked products in the absence of ATP (-ATP) and plotted as the mean ± SEM (n=3). In (a-c), the gels shown are representative of at least three independent experiments.
Fig. 6.
Fig. 6.
J-protein promotes the interaction between Ssa1 and Hsp82 in the presence of ATP. (a) The in vitro interaction between biotinylated Hsp82 (2.6 μM) and Ssa1 (11 μM) alone and in mixtures with increasing concentrations of Ydj1 in the absence or presence of 2 mM ATP was monitored using a pull-down assay as described in Materials and Methods. (b) The in vitro interaction between biotinylated Hsp82 (1.6 μM) and Ssa1 (8 μM) with Sti1 (1 μM) and in mixtures with increasing concentrations of Ydj1 in the absence or presence of 2 mM ATP was monitored using a pull-down assay as described in Materials and Methods. In both (a) and (b) the results were analyzed by SDS-PAGE followed by Coomassie blue staining. Ssa1 associated with biotinylated Hsp82 was quantified using densitometry as described in Materials and Methods and is shown as a bar graph. For each lane of the gel, the amount of Ssa1 was corrected for non-specific binding (NSB) and then was normalized to Hsp82-biotin and the ratio of Ssa1 bound relative the amount bound in the absence of Ydj1 and ATP is plotted. The gels shown are representative of in (a) five and in (b) three independent experiments and quantification of the data from the replicate gels are presented as mean ± SEM.
Fig. 7.
Fig. 7.
Working model for the collaboration of bacterial Hsp70 and Hsp90 in protein remodeling and activation. (1) The J-protein, DnaJ in E. coli, engages inactive, non-native client. (2) DnaK/Hsp70 is recruited to the client by DnaJ/J-protein and through rounds of client binding and release prevents client misfolding or initiates protein remodeling. (3) DnaK/Hsp70 in the ADP-bound conformation stably interacts with substrate and Hsp90 is recruited via the interaction between the DnaK/Hsp70 J-protein binding site and the M-domain of Hsp90. (4) Nucleotide induced conformational changes likely in both chaperones promote client transfer from DnaK/Hsp70 to Hsp90. (5) Client is released from Hsp90 in either a partially remodeled state or in an active conformation. (6) Spontaneous refolding of partially remodeled client can occur. See Results for details of the model.
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