Endoplasmic reticulum (ER) resident molecular chaperones are essential for maintaining the integrity of the proteome during times of ER stress. Failure of these chaperone systems results in protein misfolding and subsequent aggregation, which have been linked to neurodegenerative diseases such as Alzheimer's, ALS, and Parkinson's disease. The ER Hsp70, BiP, has a well characterized chaperone cycle that is facilitated by co-chaperones to transition between high and low affinity states for misfolded client proteins; J-domain proteins stimulate BiP’s ATP hydrolysis rates and deliver misfolded clients, while nucleotide exchange factors facilitate BiP’s exchange of ADP to enable ATP rebinding. Additionally, it is well established that the BiP chaperone system collaborates with the ER Hsp90 chaperone, Grp94, in protein remodeling. The canonical client remodeling mechanism for BiP and Grp94 is conserved with other Hsp70-Hsp90 chaperones, where Hsp70 and cochaperones initiate client remodeling and recruit Hsp90 for enhanced remodeling. Previous studies from our lab investigating chaperone complexes formed during client remodeling showed correlations between the amount of BiP and J-protein in complex with Grp94. Binary complexes between BiP and Grp94 have been reported in numerous studies from our lab and others. Studies with cytosolic chaperones have indicated that Hsp70, J-proteins, and Hsp90 form ternary complexes during protein remodeling. Furthermore, J-proteins have been shown to interact with Hsp90s in the absence of Hsp70. These previous findings support the basis for our prediction that this interaction is further conserved in human ER chaperones. Pulldown assays were performed to confirm the interactions between Grp94 and a J-protein. Chemical crosslinking was carried out to further clarify the characteristics of the complex and its stoichiometry. Analysis of the crosslinked gels by mass spectrometry will identify direct points of physical interaction between the two chaperones. Together, these results will allow for improved understanding of these chaperones and their collaboration in protein remodeling. This information is helpful in rational drug design targeting protein-protein interactions.

Purification of DnaJB11

DnaJB11 plasmid was transformed into origami cells with three antibiotics, kanamycin, tetracycline, and carbenicillin, on an agar plate to be grown into colonies overnight. The resulting colonies were then incubated in terrific broth media at 37 °C to an optical density (OD) of 0.6 before inducing with Isopropyl B-D-1-thiogalactopyranoside (IPTG) and temperature shifting to 18 °C overnight. A 10% polyacrylamide gel was run to confirm the induction of all cultures. The cells of these cultures were then collected and suspended in a 50 mM Hepes, 500 mM NaCl, 10% glycerol and 20 mM imidazole buffer before centrifuging and lysing in a French Press pressure lysing system. The resulting lysate was centrifuged into a pellet and supernatant containing the protein of interest. The collected supernatant was filtered prior to loading onto a 50 mL SuperLoop and Histrap column for separation by affinity chromatography. The resulting fractions, load, flow-through, and wash were run on a 10% polyacrylamide gel to confirm presence of DnaJB11 in the fractions before proceeding. Appropriate fractions were then pooled and subject to dialysis for cleavage of the 6-HisSumo tag overnight with Tobacco Etch Virus (TEV) protease in a 1:80 ratio and 1 mM DTT in a 50 mM Hepes-NaOH, 500 mM NaCl, 10% glycerol, and 20 mM imidazole dialysis buffer. Following dialysis, the pooled fractions were loaded onto a Hisfrontal column, after which fractions and a load sample were run on a gel to confirm appropriate cleavage of the 6-HisSumo tag during dialysis. Prior to loading the confirmed fractions onto size exclusion pooled fractions were concentrated to a final volume of 5 mL or less. The concentrated sample volume was then loaded onto a S200 size exclusion column in a 50 mM Hepes-NaOH, 10% glycerol, and 500 mM NaCl buffer. Appropriate fractions were collected and run on a final gel to confirm the concentration and presence of DnaJB11. Fractions were stored at -80 °C for future use.

Purification of Grp94

Grp94 plasmid was transformed into BL21 cells with carbenicillin antibiotic on an agar plate to be grown overnight. The resulting colonies were grown and incubated as described above. Collected cells were suspended in a 50 mM Tris, 500 mM NaCl, 20 mM imidazole, 1 mM BME buffer before undergoing lysing and filtering as described previously. Pooled fractions from the preceding Histrap column were subject to dialysis for cleavage overnight with TEV protease in a 1:100 ratio and 25 mM Tris, 150 mM NaCl, 1 mM BME, 5% glycerol buffer. Hisfrontal and size exclusion were executed as previously described with a 50 mM Hepes, 150 mM KCl, 10 mM MgCl2, and 5% glycerol size exclusion buffer. Appropriate fractions were collected and run on a final gel to confirm the concentration and presence of Grp94. Fractions were stored at -80 °C for future use.

Chemical Crosslinking

 Proteins were purified as described previously and prepared in a solution of 20 mM Hepes-KCl, 75 mM KCl, and 10 mM MgCl2 buffer with disuccinimidyl sulfoxide (DSSO) crosslinker (catalog number: A33545) at a final volume of 10 μm. Protein and crosslinker reactions were allowed to incubate for 30 minutes at 23 °C with mixing at the halfway point before addition of 1 μl of 200 mM Tris to terminate the reaction. Quenching was left to progress for 15 minutes before the reactions were run on SDS-PAGE, followed by staining with Blazin’ Blue™ gel stain for analysis. Crosslinking performed with disuccinimidyl suberate (DSS) crosslinker (catalog number: 21655) utilized a 20 mM Hepes-KCl reaction buffer and was allowed to progress for the same incubation and quenching period as crosslinking with DSSO. 

Biotin Labeling

Fractions of intended proteins were buffer exchanged into a 25 mM Hepes, 0.1 M NaCl buffer using 7K Zeba™ Spin Desalting Columns. Once in the appropriate buffer, 1.5 molar excess of 20 mM NHS-PEG4-biotin was added and allowed to incubate for 2 hours on ice. Following this incubation, excess unreacted biotin was removed by 7K Zeba™ Spin Desalting Columns.

Streptavidin Pulldown

Streptavidin beads were prepared by agitation until beads had dispersed through the storage solution. 25 μl of bead were pipetted into labeled centrifuge tubes. The centrifuge tubes were placed on a DynaMag™-2 Magnet and allowed for the solution to clear for 2-4 minutes before proceeding. Remaining liquid was pipetted out, and the beads were then introduced to the prepared reaction mixture. Following introduction of the reaction mixture, each tube was then washed three times with 500 μl of 25 mM Hepes KOH, 200 mM KCl, 10% glycerol, 0.01% triton-X, and 2 mM DTT buffer, followed by a fourth wash with 400 μl of the same buffer. Protein reactions were prepared with increasing concentration of the WT protein from 1 to 9 μm, 0.5 μm of the biotin-labeled protein, and addition of a 50 mM Tris, 75 mM KCl, 0.01% triton-X reaction buffer. Proteins were biotin-labeled according to the procedure described above. Reactions were incubated at 30 °C for 10 minutes after addition of all components and before introduction to beads. Once beads had been washed, samples were run on a 4-12% SurePAGE™ Bis-Tris gel and stained with Blazin’ Blue™ gel stain.

• DnaJB11 and Grp94 interact without the presence of BiP. 

• Due to DnaJB11s tetramer structure, analysis of crosslinked species will require the use of monomeric or dimeric DnaJB11 by mutation without the dimerization domain.

• There will be further analysis of crosslinked bands by mass spectrometry for identification of direct structural interactions in the future.

• Characterization of how the dimerization and J-domain contribute to DnaJB11's interactions with Grp94

1(Amankwah et al., 2022)

2(Wickramaratne et al., 2023)

3(Pobre et al., 2018)

4(Chen et al., 2017)