Solution structure of the Hop TPR2A domain and investigation of target druggability by NMR, biochemical and in silico approaches

Heat shock protein 90 (Hsp90) is a molecular chaperone that plays an important role in tumour biology by promoting the stabilisation and activity of oncogenic ‘client’ proteins. Inhibition of Hsp90 by small-molecule drugs, acting via its ATP hydrolysis site, has shown promise as a molecularly targeted cancer therapy. Owing to the importance of Hop and other tetratricopeptide repeat (TPR)-containing cochaperones in regulating Hsp90 activity, the Hsp90-TPR domain interface is an alternative site for inhibitors, which could result in effects distinct from ATP site binders. The TPR binding site of Hsp90 cochaperones includes a shallow, positively charged groove that poses a significant challenge for druggability. Herein, we report the apo, solution-state structure of Hop TPR2A which enables this target for NMR-based screening approaches. We have designed prototype TPR ligands that mimic key native ‘carboxylate clamp’ interactions between Hsp90 and its TPR cochaperones and show that they block binding between Hop TPR2A and the Hsp90 C-terminal MEEVD peptide. We confirm direct TPR-binding of these ligands by mapping 1H–15N HSQC chemical shift perturbations to our new NMR structure. Our work provides a novel structure, a thorough assessment of druggability and robust screening approaches that may offer a potential route, albeit difficult, to address the chemically challenging nature of the Hop TPR2A target, with relevance to other TPR domain interactors.


Materials
General analytical grade reagents of appropriate quality and purity were sourced from Sigma, BDH, Fluka or Fisher Scientific. Some stock solutions and cell growth media were sourced from the ICR central sterile supplies departments (CSSD). Screening compounds were purchased from the following suppliers: Alfa Aesar, Asinex, ChemBridge, ChemDiv, Enamine, InterBioScreen, Key Organics, Maybridge, Princeton, and Vitas M Labs. Peptides were purchased from Peptide Protein Research Ltd at >98% purity grade, as determined by HPLC. Hop TPR2A was cloned and expressed with a cleavable 6xHis-tag in-house.
Production of 13 C, 15 N-Hop TPR2A The TPR2A domain of human Hop, residues 218-350, was cloned from full-length Hop cDNA into the pTWO-E vector. This in-house plasmid is derived from pET-17b and contains a T7 promoter controlled expression site. The vector includes an N-terminal 6xHis-tag removable via a human rhinovirus 3C protease cleavage sequence. Following transformation of the expression vector into Escherichia coli BL21 (DE3) pLysS, cells were grown overnight on agar with ampicillin selection. Single colonies were selected and used to inoculate 10 ml starter cultures that in turn were used to inoculate 1 L cultures of LB medium and grown at 37 °C to an OD600 of 0.5. The 1 L cultures were pelleted by centrifugation (4,000 rpm, 10 min) and resuspended in baffled flasks with 0.5 L of M9 medium containing 0.3% 15 NH4Cl, 0.4% 13 Cglucose, Studier trace metals (Studier 2005) and 100 µg/ml ampicillin. After 30 minutes incubation at 20 °C, protein expression was induced by adding isopropyl-1-thio-β-Dgalactopyranoside (IPTG) to a final concentration of 1 mM. These cultures were incubated at 20 °C overnight with shaking to ensure high levels of medium oxygenation. Cells were harvested by centrifugation (6,000 rpm, 10 min) and resuspended in 20 mM Tris pH 8.0, 1 mM imidazole, 100 mM NaCl with protease inhibitors (Roche) and lysed by sonication. The lysate was clarified by centrifugation (20,000 rpm, 50 min, 4 °C) and incubated with TALON metal affinity resin (Clontech) for 1 hr at 4 °C with agitation in order to capture the recombinant His-tagged TPR2A. The resin was extensively washed before elution of the partially purified protein in 20 mM Tris pH 8.0, 10 mM imidazole, 100 mM NaCl. The sample was desalted and concentrated before being applied to a Source Q ion exchange column; the resultant sample was largely pure as determined by SDS-PAGE. At this stage the His-tag was cleaved by digestion with PreScission protease before a final purification step by gel filtration in 20 mM Na2HPO4, 50 mM NaCl, 1 mM DTT, at pH 6.5. The resultant protein was highly pure and was concentrated to around 1.5 mM, flash frozen and stored at -80 °C.

AlphaScreen assay
AlphaScreen assays were carried out in 384-well white PerkinElmer OptiPlates (Cat. 6007299) with a total assay volume of 25 µl per well. Assay buffer (20 mM HEPES pH 7.4, 100 mM NaCl, 1 mM DTT, 0.1% Tween-20) was prepared from stocks on the day of use. AlphaScreen reagents were purchased as a kit from PerkinElmer containing streptavidin coated donor beads and nickel chelate acceptor beads (Cat. 6760619). Final assay composition was 8 nM Biotin-Hsp90α peptide, 50 nM Hop TPR2A and 5 µg/ml each of donor and acceptor beads.
Protein, peptides and AlphaScreen reagents were prepared at 5-fold final concentration in assay buffer. Assay components were added to the plate as follows: 5 µl/well assay buffer (with compound or DMSO where required), 5 µl/well peptide, and 5 µl/well protein. Assay plates were then centrifuged for 30 s at 1000 rpm. After a 10 min incubation period at room temperature, nickel chelate acceptor beads were added at 5 µl/well. Finally, in subdued light conditions, 5 µl/well of streptavidin donor beads were added giving a total volume of 25 µl/well, the plate was then sealed, centrifuged (30 s, 600 rpm) and incubated in the dark for 2 hours. Luminescent output was read on the Envision plate reader (Perkin Elmer).

Chemical shift perturbation (CSP) experiments and Kd determination
Changes in chemical shifts were determined by tracking ligand shifted HSQC peaks in NMRView and exporting the resultant peak lists to Excel. In Excel, using appropriate lists of control peaks the change in chemical shift (Dd) was calculated for each backbone assigned N-H peak using Equation S1.
Where Δ ! ! is the change in proton chemical shift, and Δ # is the change in nitrogen chemical shift. Using a scaling factor (0.2) normalises the difference in magnitude of chemical shifts at these two nuclei, various scaling factors are used in the literature and a value of 0.2 has been suggested by Ziarek et al.
In which Δ is the adjusted chemical shift change, Δ &'( is the chemical shift change at saturation, [ ] 7 the total protein concentration, 8 the equilibrium dissociation constant, and [ ] 7 the total ligand concentration. Here the Δ &'( was taken to be the observed Δ at the highest [ ] 7 . Except in cases where this was clearly far from saturation, when this variable was not constrained during the curve fit. Determining dissociation constants using CSP titrations requires that the experimental [P] is high enough that the signal to noise of the experiment is sufficient while falling into a suitable range for accurate curve fitting using equation S2. These considerations are discussed in two references, Williamson 2013 and Markin and Spyracopoulos 2012. They suggest that [P] should fall in the range 0.1 -10 fold Kd to produce usable data. Our experimental data do fall into this range with the tightest binding ligand (the Hsp90 MEEVD peptide) and the weakest (9) at either limit.

FLAG co-immunoprecipitation
Immunoprecipitation from lysates containing FLAG-Hsp90 was used to investigate the effects of small molecules and peptides on Hsp90 complexes. All steps of this protocol were carried out at 4°C. Cell pellets of FLAG-Hsp90 expressing HCT116 cells were harvested and lysed in HNTG buffer, 50 mM HEPES, 150 mM NaCl, 1% Triton, 10% glycerol, 1 mM MgCl2, 1 mM EDTA, plus protease and phosphatase inhibitor tablets (Roche), on ice for 20 min. Lysed samples were centrifuged at 13,000 rpm to pellet the cellular debris and the protein concentration of the resultant lysate was determined by BCA.
While sample lysis was ongoing anti-FLAG conjugated agarose beads (Sigma) were washed four times in HNTG buffer. Between each wash beads were pelleted by centrifugation at 5500 g for 1 min and the buffer was aspirated with a pipette and discarded, taking great care not remove beads in the process. Lysate was added to 20 μl bead aliquots (40 μl of supplier's suspension) in a volume equivalent to 1 mg of protein per sample. HNTG buffer was added to each sample up to a total volume of 250 μl, compounds or peptides were added at this stage to probe their effects on Hsp90-cochaperone interactions. Samples were placed on a rotating mixer for 2 hours.
After allowing the anti-FLAG antibody to capture FLAG-Hsp90 complexes, the samples were centrifuged and the remaining lysate aspirated and retained. Beads and the associated Hsp90 complexes were washed 3 times with HNTG buffer to remove nonspecific interactors. Compounds or peptides were alternatively added at this stage. The remaining complexes were eluted from the beads using a FLAG peptide with high-affinity for the antibody (Sigma) in 20 μl per sample at 1 mg/ml. The beads were centrifuged for a final time and the final sample aspirated (20 μl) and transferred to a clean Eppendorf. Following the addition of Laemmli buffer and boiling for 5 min samples were stored at -80°C for Western blot analysis.
Western blotting was used for the detection of specific protein levels in immunoprecipitated samples. Following determination of the protein concentration, samples were prepared of equal volume and equal total protein content, usually between 15-30 μg protein/lane depending on lowest sample abundance. Laemmli buffer (60 mM Tris-Cl pH 6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol, 0.01% bromophenol blue) was added to each sample before boiling for 5 min, in order to denature proteins.
Samples were separated by SDS-PAGE on Novex Tris-glycine 4-20% gels (Invitrogen) alongside the SeeBlue Plus2 marker (Invitrogen) in running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS). Gels and nitrocellulose membranes (0.2 μm pore size, Invitrogen) were soaked in transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol) and sandwiched between filter paper in a cassette. Sample transfer from gel to membrane was achieved by passing a constant current of 150 mA across the cassettes for 2 hours.
Membranes were blocked with casein buffer (10 mM Tris pH 7.4, 150 mM NaCl, 0.5% w/v casein, 0.5 mM thiomersal) for 1 hour. Primary antibodies, diluted to an appropriate concentration in casein buffer, were incubated with the blocked membranes overnight at room temperature. Excess and unbound primary antibody was removed by three 10 min washes with PBST. Secondary horseradish peroxidise (HRP) conjugated antibodies from the appropriate species were diluted in casein buffer and incubated with the membrane for 1 hour. Excess antibody was again removed by three PBST or TBST washes. Following a brief 1 min incubation in a HRP substrate (SuperSignal West Pico Chemiluminescent Substrate, Pierce) the resulting chemiluminescent signal was detected in a dark room using Hyperfilm ECL (GE). Films were developed using an automated developer and scanned for electronic manipulation and storage.  Figure Figure S9: Sequence alignment of TPR domains. Showing conservation of the carboxylateclamp residues (marked in black boxes above the alignment). The residues are coloured by physicochemical properties (blue=positively charged, red=negatively charged, pink is hydrophobic and the remainder are polar residues). Some differences between the different proteins are seen in key interaction residues. Figure S11: Co-immunoprecipitation Western blot of FLAG-tagged Hsp90 complexes from HCT116 human colon cancer cell line lysate. A: Comparison of Hsp90 complex composition following addition of exogenous Hsp90 peptide MEEVD or small molecules to cell lysate. TPR cochaperone association is reduced by the presence of the MEEVD peptide but not compounds 4, 11, or an inactive control compound (NC). B: Comparison of Hsp90 complex composition following addition of Hsp90 peptide MEEVD or small molecules to purified Hsp90 complexes. The MEEVD peptide and compound 11 reduce TPR cochaperone association but not 4 or an inactive control. N = 1.

Supplementary Figures
*Full, uncropped blot images are shown in Appendix A.    Image of full Western Blot film scan for Hsp70. Orange box indicates specific blot and exposure used in Supplementary Figure S11. Simultaneously blotted with p23 (lower band -not visible in exposure indicated by orange box).