Project PXD008674

PRIDE Assigned Tags:
Biological Dataset

Summary

Title

Plasticity of inter-protein contacts confers selectivity in ligand induced protein degradation dataset 2

Description

Heterobifunctional small molecule degraders that induce protein degradation through ligase-mediated ubiquitination have shown considerable promise as a new pharmacological modality. However, we currently lack a detailed understanding of the molecular basis for target recruitment and selectivity, which is critically required to enable rational design of degraders. Here we utilize comprehensive characterization of the ligand dependent CRBN/BRD4 interaction to demonstrate that binding between proteins that have not evolved to interact is plastic. Multiple X-ray crystal structures show that plasticity results in several distinct low energy binding conformations, which are selectively bound by ligands. We demonstrate that computational protein-protein docking can reveal the underlying inter-protein contacts and inform the design of the first BRD4 selective degrader that can discriminate between highly homologous BET bromodomains. Our findings that plastic inter-protein contacts confer selectivity for ligand-induced protein dimerization provide a conceptual framework for the development of heterobifunctional ligands.

Sample Processing Protocol

MM.1s cell were treated with DMSO, 1 µM dBET23, or dBET70 in biological triplicates for 5 hours and cells harvested by centrifugation. Lysis buffer (8 M Urea, 1% SDS, 50 mM Tris pH 8.5, Protease and Phosphatase inhibitors from Roche) was added to the cell pellets to achieve a cell lysate with a protein concentration between 2 – 8 mg mL-1. A micro-BCA assay (Pierce) was used to determine the final protein concentration in the cell lysate. 200 µg proteins for each sample were reduced and alkylated as previously described. Proteins were precipitated using methanol/chloroform. In brief, four volumes of methanol were added to the cell lysate, followed by one volume of chloroform, and finally three volumes of water. The mixture was vortexed and centrifuged to separate the chloroform phase from the aqueous phase. The precipitated protein was washed with one volume of ice-cold methanol. The washed precipitated protein was allowed to air dry. Precipitated protein was resuspended in 4 M Urea, 50 mM Tris pH 8.5. Proteins were first digested with LysC (1:50; enzyme:protein) for 12 hours at 25°C. The LysC digestion was diluted down in 1 M Urea, 50 mM Tris pH 8.5 and then digested with trypsin (1:100; enzyme:protein) for another 8 hours at 25°C. Peptides were desalted using a C18 solid phase extraction cartridges (Waters). Dried peptides were resuspended in 200 mM EPPS, pH 8.0. Peptide quantification was performed using the micro-BCA assay (Pierce). The same amount of peptide from each condition was labelled with tandem mass tag (TMT) reagent (1:4; peptide:TMT label) (Pierce). The 10-plex labelling reactions were performed for 2 hours at 25°C. Modification of tyrosine residue with TMT was reversed by the addition of 5% hydroxyl amine for 15 minutes at 25°C. The reaction was quenched with 0.5% TFA and samples were combined at a 1:1:1:1:1:1:1:1:1:1 ratio. Combined samples were desalted and offline fractionated into 96 fractions using an aeris peptide xb-c18 column (phenomenex) at pH 8.0. Fractions were recombined in a non-continuous manner into 24 fractions and every second fraction was used for subsequent mass spectrometry analysis. Data were collected using an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) coupled with a Proxeon EASY-nLC 1200 LC pump (Thermo Fisher Scientific). Peptides were separated on a 75 μm inner diameter microcapillary column packed with 35 cm of Accucore C18 resin (2.6 μm, 100 Å, ThermoFisher Scientific). Peptides were separated using a 3 hr gradient of 6–27% acetonitrile in 0.125% formic acid with a flow rate of 400 nL/min. Each analysis used an MS3-based TMT method as described previously53. The data were acquired using a mass range of m/z 350 – 1350, resolution 120,000, AGC target 1 x 106, maximum injection time 100 ms, dynamic exclusion of 120 seconds for the peptide measurements in the Orbitrap. Data dependent MS2 spectra were acquired in the ion trap with a normalized collision energy (NCE) set at 35%, AGC target set to 1.8 x 104 and a maximum injection time of 120 ms. MS3 scans were acquired in the Orbitrap with a HCD collision energy set to 55%, AGC target set to 1.5 x 105, maximum injection time of 150 ms, resolution at 50,000 and with a maximum synchronous precursor selection (SPS) precursors set to 10.

Data Processing Protocol

Proteome Discoverer 2.1 (Thermo Fisher) was used to for .RAW file processing and controlling peptide and protein level false discovery rates, assembling proteins from peptides, and protein quantification from peptides. MS/MS spectra were searched against a Uniprot human database (September 2016) with both the forward and reverse sequences. Database search criteria are as follows: tryptic with two missed cleavages, a precursor mass tolerance of 50 ppm, fragment ion mass tolerance of 1.0 Da, static alkylation of cysteine (57.02146 Da), static TMT labelling of lysine residues and N-termini of peptides (229.16293 Da), and variable oxidation of methionine (15.99491 Da). TMT reporter ion intensities were measured using a 0.003 Da window around the theoretical m/z for each reporter ion in the MS3 scan. Peptide spectral matches with poor quality MS3 spectra were excluded from quantitation (< summed signal-to-noise across 10 channels and < 0.5 precursor isolation specificity). Reporter ion intensities were normalized and scaled using in house scripts and the R framework. Statistical analysis was carried out using the limma package within the R framework.

Contact

Eric Fischer, Dana-Farber Cancer Institute
Eric Sebastian Fischer, Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA ( lab head )

Submission Date

15/01/2018

Publication Date

23/05/2018

Tissue

Not available

Cell Type

myeloid cell

Software

Not available

Quantification

TMT

Experiment Type

Shotgun proteomics

Publication

Publication pending