Selective Affimers Recognise the BCL‐2 Family Proteins BCL‐xL and MCL‐1 through Noncanonical Structural Motifs

Abstract Abstract: The BCL‐2 family is a challenging group of proteins to target selectively due to sequence and structural homologies across the family. Selective ligands for the BCL‐2 family regulators of apoptosis are useful as probes to understand cell biology and apoptotic signalling pathways, and as starting points for inhibitor design. We have used phage display to isolate Affimer reagents (non‐antibody‐binding proteins based on a conserved scaffold) to identify ligands for MCL‐1, BCL‐xL, BCL‐2, BAK and BAX, then used multiple biophysical characterisation methods to probe the interactions. We established that purified Affimers elicit selective recognition of their target BCL‐2 protein. For anti‐apoptotic targets BCL‐xL and MCL‐1, competitive inhibition of their canonical protein‐protein interactions is demonstrated. Co‐crystal structures reveal an unprecedented mode of molecular recognition; where a BH3 helix is normally bound, flexible loops from the Affimer dock into the BH3 binding cleft. Moreover, the Affimers induce a change in the target proteins towards a desirable drug‐bound‐like conformation. These proof‐of‐concept studies indicate that Affimers could be used as alternative templates to inspire the design of selective BCL‐2 family modulators and more generally other protein‐protein interaction inhibitors.

Uncleaved material and His-SUMO were removed by reapplication of the sample to a HisTrap in 20mM TRIS pH 8.0, 250mM NaCl and the flow through containing Mcl-1 or Bcl-xl collected. The proteins were then filtered before further purification on a Superdex 75 (GE healthcare) equilibrated in 20mM TRIS pH 8.0, 250mM NaCl, 0.5mM DTT, 2.5% Glycerol. Purified proteins were concentrated and stored at -80°C.

BCL-2 expression
Glutathione S-transferase (GST) tagged BCL-2 1-205 fusion protein constructs were over-expressed in E.coli BL21 (DE3) Rosetta 2, and purified. 10 ml of overnight starter culture was used to inoculate 1 L 2 xYT containing 100 μg/ml Ampicillin. Cultures were grown at 37 °C until OD600 reached 0.6 -0.8. The temperature was reduced to 18 °C and protein expression induced by adding 0.3 mM IPTG. Induced cultures were grown at 18°C overnight before harvesting by centrifugation (Beckmann JLS 8.1, 5000rpm, 15 minutes, 4°C). Cell pellets were re-suspended in lysis buffer (50mM TRIS pH 8.0, 200mM NaCl, 5mM DTT, 1mM EDTA) and lysed by sonication in the presence of 10 μL of 1 U.ml-1 DNase I per litre of over-expression culture. Cell lysate was clarified (Sorvall SS34 rotor, 18,000 rpm, 45 min, 4°C), and the supernatant was filtered (0.45 μM Minisart, Sartorius). Clarified cell lysate was added to approximately 10 mL Glutathione Superflow Resin (Generon) packed in a free-flow gravity column. The column was washed with 5 column volumes of water and equilibrated with 5 column volumes of lysis buffer. The lysate was then added to the column and placed on an analogue roller mixer (SKS science) at 4°C for 1-3 hours. Lysate was then eluted from the column under gravity, and the resin was washed first with 100 mL high salt wash buffer (50 mM Tris pH 8, 1 mM DTT, 1 mM EDTA, 500 mM NaCl, 10% glycerol, 0.01% Triton), then 100 mL low salt wash buffer (50 mM Tris pH 8, 1 mM DTT, 1 mM EDTA, 200 mM NaCl, 10% glycerol, 0.01% Triton). The resin was re-suspended in 20 mL of low salt wash buffer, supplemented with 400 μL PreScission protease to cleave the GST tag from the fusion protein. Following overnight incubation on an analogue roller mixer (SKS science) at 4°C, cleaved protein was obtained by collecting the flow through from the column. To collect all cleaved protein, the resin was washed with 50 mL of low salt wash buffer, and the flow through collected. All wash fractions were collected and analysed by SDS-PAGE. Cleaved protein was concentrated (Amicon, MWCO 10,000) to 5 ml, filtered (0.22 μM Minisart, Sartorius) and further purified by Size Exclusion Chromatography. Purified protein was concentrated and stored at -80°C, with the addition of 5% glycerol to aid long term stability.

BAK and BAX expression
Mxe intein / chitin binding domain (CBD) tagged fusion protein constructs were overexpressed in E.coli C41 (DE3) cells, and purified. 2 mL of overnight starter culture was used to inoculate 1 L LB containing 100 μg/mL Ampicillin. Cultures were grown at 37°C until OD600 reached 0.6 -0.8. For Following overnight incubation on an analogue roller mixer (SKS science) at 25°C, cleaved protein was obtained by collecting the flow through from the column. To collect all cleaved protein, the resin was washed with 50 mL of lysis buffer, then a further 100 mL of lysis buffer. All wash fractions were collected and analysed by SDS-PAGE.

Screening for Affimers
BCL-2 family proteins were biotinylated using EZ-link NHS-SS-biotin (Pierce), according to the manufacturer's instructions. Biotinylation was confirmed using streptavidin conjugated to horseradish peroxidase (HRP). Biotin-BCL-2 family proteins were added and incubated on pre-blocked steptavdin plate, the plate was then washed using a KingFisher robotic platform (ThermoFisher) and 10 12 cfu of the prepanned phage library was added and incubated for 2.5 h with shaking. Wells were wash ten times and eluted with 100 µL 0.2 M glycine (pH 2.2) for ten minutes neutralized with 15 µL 1 M Tris-HCl (pH 9.1), further eluted with triethylamine 100 mM for 6 min, and neutralised with 1 M Tris-HCl (pH 7). Eluted phage were used to infect ER2738 cells for 1 h at 37 °C and 90 rpm then plated onto LB agar plates with 100 µg/ml carbenicillin and grown overnight. All colonies were scrapped into 5 mL of 2XYT with carbenicillin (10 µg/mL) and 1 x 10 9 M13K07 helper phage were added. After an overnight incubation phage were precipitated with 4 % polyethylene glycol 8000, 0.3 M NaCl and resuspended in 1 ml of 10 mM Tris, pH 8.0, 1 mM EDTA (TE buffer). 2 µL phage suspension was used for the second round panning round using streptavidin magnetic beads as opposed to streptavidin plates (Invitrogen); otherwise the second pan was conducted in the same way as the first pan. The third pan was conducting using neutravidin high binding capacity plates (Pierce). After the final pan colonies were picked, an ELISA was conducted to select positive clones (in the same way as the enrichment ELISA) which were sent for Sanger sequencing.

Overexpression and purification of Affimers
The Affimers were subcloned from the phage display vector into pET11a then expressed and purified from E. coli strain Rosetta 2. 10 ml of overnight starter culture was used to inoculate 1 L 2 x YT containing 125 μg/ml Ampicillin Cultures were grown at 37 °C until OD600 ~ 0. The purified protein was concentrated to ~ 6 mg/ml and stored at -80 °C with the addition of 5% Glycerol.

Isothermal Titration Calorimetry
Kinetic information of the Affimer interactions with the Bcl-2 family proteins was established

Co-crystallisation
BCL-xL was incubated with an excess of BCL-xL-AF7 overnight, before co-purification in 200mM NaCl, 50mM TRIS pH 8.0, 0.5mM DTT via gel filtration on a Superdex75 column.
Crystals grew in 12% PEG 1500, 0.1M Sodium Acetate pH 5.5, 2.5 M NaCl, 1.5% MPD at 20°C at 5mg/ml using the sitting drop vapour diffusion method. The crystals were cryoprotected in 20% glycerol and data collected at the Diamond Light Source on beamline i04-1 to 2.24 Å resolution at 100K. The diffraction images were integrated, scaled and reduced using the suite of program XIA2 4 with five percent of the reflections selected at random and excluded from the refinement using FREERFLAG. 5 The unit cell parameters for the crystal are a=68.3Å, b=87.3Å, c=112.2Å, α=90.0°, β=96.2°, γ=90.0° in space group P21 with four BCL-xL-AF7/BCL-XL complexes in the asymmetric unit cell. The data processing statistics are shown in Table 2. The structure was determined by molecular replacement using the program PHASER 6 with the human BCL-XL structure (PDB code 1R2D), 7 ) and the truncated Affimer (PDB code 4N6T, 8 ) as the search models. Manual inspection of electron density maps with iterative cycles of model building and refinement were carried out using COOT 9 and REFMAC5. 10, 11 During the course of model building structural validations were carried out using the program MOLPROBITY. 12 All refinement statistics are shown in Table 2 The crystals were cryoprotected with 20% glycerol and data was collected at the Diamond Light Source beamline i03 to 1.90 Å resolution at 100K. The unit cell parameters for the crystal are a=71.9Å, b=71.9Å, c=204.2Å, α=90.0°, β=90°, γ=90.0° in space group P41 2 2 with two BclXL:BCL-xL-AF6 complexes in the asymmetric unit cell. The structure was solved using similar strategy as detailed above for BclXL:BCL-xL-AF7 complex.
Mcl-1 was incubated with an excess of MCL-1-AF11 at room temperature for 3 hours. The      Figure S4. Sample 2Fo-Fc electron density at the final stage of refinement for crystal structures. (a) The interface between Affimer BCL-xL-AF7 and BCL-xL; (b) interface between Affimer BCL-xL-AF6 and BCL-xL; (c) interface between Affimer MCL-AF11 and MCL-1. Despite slightly higher than usual Rwork and Rfree (for BCL-xL-AF7 and MCL-1-AF11, probably because of a high overall Wilson B-factor for this crystal), the electron density is absolutely unambiguous. Figure generated using Coot 2 . a c b Figure S5. Overlay of Bcl-xL when bound in different complexes. Overlay of Bcl-xL when bound to Affimer (dark green) BIM peptide (purple) or WEHI-539 (light blue). Note the position of the BCL-xL helix to the left of peptide/compound (arrow): the helix binding groove is wider when bound to peptide than to WEHI-539 or BCL-xL-AF7.