The vGFP cassette from pANT-9L (a kind gift from Dr Swaine Chen) was excised using NdeI and HindIII and ligated into cut pET22b (+) vector. Peptide-displaying vGFP constructs for bacterial expression/purification were subsequently made by inverse-PCR mutagenesis of pET22-vGFP using primers indicated in Supplementary Table S1. vGFP-M2 was made using primer pair 1 and 2. vGFP-M2C was made using primers 3 and 4. vGFP-M2.1 and vGFP-M2.2 were made using primer pairs 5/6 and 5/7 respectively. vGFPE4-M2 with four mutations in the Enhancer domain (S273A, R275A, S299A and F342A) was generated by successive Quickchange mutagenesis (Stratagene) of vGFP-M2 using forward and reverse primer pairs for QC-S273A (primers 8/9), QC-R275A (primers 10/11), QC-S299A (primers 12/13) and QC-F342A (primers 14/15). For the eIF4E interaction assay, vGFP-e4pep and vGFP-GSe4pep were first generated by inverse PCR using primer pairs 16/17 and 16/18 respectively. For mammalian expression of vGFP-e4pep and vGFP-GSe4pep, the constructs were subsequently cloned into EcoRI-XhoI site of pCDNA3.1 vector via infusion PCR (Clontech) using primer pair 19/20 for amplification of inserts and primer pair 21/22 for amplification of pCDNA3.1 vector. The controls vGFP-e4pepC and vGFP-GSe4pepC in pCDNA3.1 vector were then generated via Quikchange using primer-pairs 23/24 and 25/26 respectively. The control encoding vGFP only was cloned into pCDNA3.1 vector via infusion PCR using primer pair 27/28 for amplification of insert and primer pair 29/30 for amplification of pCDNA3.1 vector. For mammalian expression of vGFP-M2C and vGFP-M2, the constructs were cloned into the NheI-EcoRI sites of pCDNA3.1 vector via infusion PCR using primer pair 31/32 for amplification of inserts and primer pair 33/34 for amplification of pCDNA3.1 vector. For vGFP2-M2 and vGFP2-M2C, a (GGGGS)2 linker was introduced into the vGFP-M2 or vGFP-M2C vector respectively via inverse PCR using primer pairs 35/36 and 35/37. For vGFP3-M2 and vGFP3-M2C, Enhancer derived residues V242-E246 were added between M2 or M2C peptide and the (G4S)2 linker in vGFP2-M2/M2C constructs via inverse PCR using primer pairs 38/39 and 38/40 respectively.
Protein expression and purification.
vGFP-M2, vGFP-M2C and vGFPE4-M2 constructs were cloned as fusion proteins with C-terminal 6xHis tags. The constructs were then transformed into Escherichia coli BL21(DE3) (Invitrogen) competent cells and grown in LB medium at 37°C. At OD600 nm of 0.6, the cells were induced at 16°C overnight with 1mM IPTG (for vGFP-M2 and vGFP-M2C) or 0.5 mM IPTG (for vGFPE4-M2) before harvesting and lysis by sonication. The cell lysate was clarified and applied to a 5 mL HisTrap column (GE Healthcare) pre-equilibrated in binding buffer (50 mM Tris-HCl pH 8, 500 mM NaCl, 20 mM imidazole, 1 mM DTT),washed and eluted off the column using a linear gradient with elution buffer (50mM Tris-HCl pH 8.0, 500 mM NaCl, 500 mM imidazole) over 30 column volumes. The eluted fractions containing the protein were then pooled and dialyzed into ion-exchange binding buffer (20 mM Tris pH 8, 1mM DTT) using a HiPrep 26/10 desalting column. The protein was then loaded onto a 1 mL ion-exchange ResourceQ column (GE Healthcare) pre-equilibrated in ion-exchange binding buffer. The column was washed with binding buffer and bound protein was eluted with a linear gradient in elution buffer (20 mM Tris pH 8, 1 M NaCl, 1 mM DTT) over 60 column volumes. Protein purity was assessed by SDS-PAGE, pooled, buffer exchanged into buffer (50 mM Tris pH 8, 150 mM NaCl, 1 mM DTT) and concentrated using Amicon-Ultra (10 kDa MWCO) concentrator. The purified proteins were then used in the subsequent assays. For vGFP-M2 used for structural studies, vGFP-M2 was further purified by loading onto a Superdex 75 16/60 size exclusion column (GE Healthcare) in gel filtration buffer (50 mM Tris pH 8, 150 mM NaCl, 1 mM DTT). Protein purity was assessed by SDS-PAGE, pooled and concentrated using Amicon-Ultra (10 kDa MWCO) concentrator. Mdm2 (amino acids 6–125) was cloned as a GST-fusion protein, expressed and purified using affinity chromatography and Resource S cation exchange column as previously described35. G. pallidus RAPc8 amidase was expressed and purified as previously described45.
Mdm2 (6-125) pull-down assay
The purified vGFP-M2, vGFP-M2C, vGFPE4-M2 proteins (10 mM) were incubated with Mdm2 (6-125) at a molar ratio of 1:9 at 4 oC for 3 hours, diluted using 1x Binding/Wash buffer (1 x TE + 500 mM NaCl). The mixture was then incubated with the Dynabeads His-tag isolation system (Thermo Fisher Scientific) at 4oC for 30 minutes. Beads were washed and bound protein was eluted by boiling in SDS buffer and analysed by SDS-PAGE.
Formation of vGFP-M2 -Mdm2 (6-125) complex for structural studies.
Purified vGFP-M2 was incubated with purified Mdm2 (1: 2.5 molar ratio) at 4oC for 4 hours. The protein mixture was then filtered and resolved onto a Superdex 75 16/60 size exclusion column (GE Healthcare) in gel filtration buffer (50 mM Tris pH 8, 150 mM NaCl, 1 mM DTT). Protein fractions were then assessed by SDS-PAGE, pooled and concentrated using Amicon-Ultra (10 kDa MWCO) concentrator (Millipore).
Crystallization and structure determination.
All crystals were grown at 16oC using the sitting drop vapour diffusion method and clarified by centrifugation before setting up the crystallization trials. MDM2-vGFP-PM2 complex was concentrated to approximately 8.8 mg/ml. Crystals of the complex were grown by mixing the complex with the reservoir solution (25 % w/v polyethylene glycol 3500, 100 mM Bis-Tris pH 5.5, 200 mM sodium chloride) in a ratio of 1:1. Single crystals were briefly soaked in mother liquor as cryo-protectant and then flash frozen under liquid nitrogen. Diffraction data were collected up to 3 Å resolution at National Synchrotron Radiation Research Center (NSRRC, Taiwan) at beamline BL13B1 at 1 Å wavelength. Data were indexed, integrated, and scaled with the HKL2000 program package (HKL research). Molecular replacement was performed using 3K1K as a search model for GFP and 4UMN for mdm2 in PHASER46.
Restrained refinement with TLS was carried out using REFMAC47 in the CCP4 suite48 and model building was done in COOT49. Data collection and refinement statistics are shown in Supplementary Table S2. Structure figures were generated using PyMOL (Delano Scientific LLC). Crystal structure coordinates are deposited in the Protein Data Bank with PDB access code 5WTS.
Thermal stability assay
vGFP variants (10 µM) were incubated alone or with Mdm2/Amidase (100 µM) and Nutlin compounds (1mM) at room temperature for 30 minutes in PBS buffer and placed into 0.2 mL thin-wall PCR tubes. Thermal ramping was carried out on a BioRad CFX96 Real Time System and fluorescence was measured over a temperature range between 35oC and 100oC at 0.5oC increments.
P53-reporter gene assay in T22 cells
T22 reporter cells (stably transfected with pRGCd-Fos-lacZ carrying a p53-driven β-galactosidase gene) 31 were maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% (v/v) fetal calf serum (FCS) and 1% (v/v) penicillin/streptomycin. Transfection was carried out using Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific) according to manufacturer’s instructions. For intracellular targeting activity in T22 reporter cells, luciferase expression plasmid was co-transfected with respective vGFP expression constructs at 1:1 ratio, 1.0mg total plasmid DNA.
eIF4E interaction assay in HEK 293 cells
Twenty-four hours prior to transfection, HEK 293 cells (Thermo Fisher Scientific) were seeded at a cell density of 1 x106 per well of a 6-well plate. Transfections in 6-well plates were performed using Lipofectamine 3000 (Thermo Fisher Scientific) with 0.5 μg of plasmid vectors per well according to the manufacturer’s instructions. After 48 hrs, cells were lysed and m7GTP pull down were performed as described50. Briefly, 300 µg of cell lysates were incubated with 20 μl of m7GTP (Jena Bioscience) agarose beads for 3 hrs at 4 °C on a rotator. Beads were then washed four times with lysis buffer and vGFP-eIF4E complexes eluted by boiling the beads for 5 min at 95 °C in presence of Laemmli buffer. Samples were resolved by SDS/PAGE and transferred onto PVDF membrane using the Trans-Blot Turbo system (Bio-Rad) according to the manufacturer’s protocol. Blocked membranes were blotted with 3H9 anti GFP antibody (Chromotek) followed by incubaton with the secondary goat anti-rat for 1 hour at room temperature. Chemiluminescence was then detected with a Licor system (Licor Biosciences).
Live cell spinning disk confocal fluorescence microscopy.
For live cell imaging, T22 cells grown on 27 mm diameter Nunc glass bottom dish (Thermofisher) were transfected 24 hrs prior to image acquisition. Images were acquired on Nikon Eclipse Ti inverted microscope, using a 40x oil immersion objective (NA 1.3). Z-stacks were acquired for all images and sum slices projection was applied to all the stack images using ImageJ Software.