Protein expression and purificationPTPN1 and PTPN2 constructs were synthesized by Quintara Biosciences with a C-terminal Avitag and 8xHis tag in a pET28a vector.The constructs were expressed in BL21 DE3 Escherichia coli cells. The cultures were grown to an OD600 (optical density at 600 nm) of 0.6–0.9 at 37 °C in LB containing kanamycin (100 μg/ml) and was induced with 1 mM isopropyl-β-d-thiogalactoside. The expression was allowed to proceed overnight at 18 oC, the cells were harvested the following day by centrifuging at 17,568 g (Sorvall LYNX 6000) and the pellet was snap-frozen in liquid nitrogen. The frozen pellet was resuspended in buffer containing 25 mM Tris (pH 7.5), 250 mM NaCl, 10 mM imidazole, 1 mM tris(2-carboxyethyl) phosphine (TCEP) and PierceTM protease inhibitor tablet (Thermo Fisher). The pellet was lysed by sonication (five 1 min cycles with 5 s ON and 10 s OFF) and the pellet was clarified by centrifuging at 33,746 g. The lysate was incubated with HisPur™ Ni-NTA Resin (Thermo Fisher) equilibrated in lysis buffer for 1 hour. The beads were separated from the lysate by flowing through an Econo-Pac® gravity flow column (Bio-Rad) and washed with 3–5 column volumes of wash buffer (25 mM Tris (pH 7.5), 250 mM NaCl, 40 mM imidazole and 1 mM tris(2-carboxyethyl) phosphine (TCEP)). The protein was eluted in buffer containing 25 mM Tris (pH 7.5), 250 mM NaCl, 400 mM imidazole and 1 mM tris(2-carboxyethyl) phosphine (TCEP). The eluent was concentrated in a 10,000–molecular weight cutoff (MWCO) Amicon Concentrator (Millipore) to <0.5 ml. For constructs containing Avitag, the protein was subjected to biotinylation for 3 h at 37 °C using the BirA biotin-protein ligase standard reaction kit (Avidity LLC). The protein was then injected onto a Superdex 200 10/300 GL Increase size exclusion column (GE Healthcare) equilibrated in 1xPBS. Following size exclusion, the protein was concentrated, frozen, and stored at −80 °C.Full-length (amino acids 1–442) human CRBN was cloned into pFastBac1 vector with an N-terminal StrepII tag. Full-length (amino acids 1–1140) human DDB1 was cloned into pFastBac1 vector with an N-terminal 8× His tag. The baculoviruses of CRBN and DDB1 were prepared using the Bac-to-Bac system. Baculoviruses were prepared in Sf9 insect cells and subsequently used for co-infection in Sf21 insect cells. Expression was carried out for 48 h at 27 ˚C. Cell pellet was resuspended in a lysis buffer containing 50 mM Tris-HCl (pH 8.0), 200 mM NaCl, 1 mM TCEP, 1 mM PMSF with complete protease inhibitor (Roche) and lysed by sonication. The proteins were first purified by affinity chromatography using Strep-Tactin ® XT column (IBA Lifesciences). The 50 mM biotin elution sample was desalted into buffer of 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM TCEP, 1 mM PMSF, and loaded onto Mono Q column (Cytiva). The proteins were eluted with a linear gradient of 100 mM – 1 M NaCl in buffers. The appropriate fractions were pooled and further purified by size-exclusion chromatography on a Superdex 200 column (Cytiva) equilibrated with the gel filtration buffer containing 50 mM HEPES (pH 7.5), 200 mM NaCl, 0.25 mM TCEP. The final proteins were stored as aliquots at −80 °C.Cell cultureB16F10 mouse melanoma cells (ATCC) and HEK 293 T human kidney cells (ATCC) were cultured in Dulbecco’s Modified Eagle’s Media (DMEM, MilliporeSigma, Burlington) plus 10% heat-inactivated fetal bovine serum (FBS, MilliporeSigma, Burlington).pSTAT1 HTRFB16F10 cells were incubated overnight with Cmpd-1 and Cmpd-2 in dose-response. Cells were stimulated with 100 ng/mL IFNγ (R&D Systems, Minneapolis) for 10 min. Staurosporine was added at 3 µM and incubated for 1 additional hour. Assay media was removed, and cells were lysed in 50 µL of 1x lysis buffer (kit provided). The lysate phosphorylation status of STAT1 was assessed using Phospho-STAT1 (Tyr701) Cellular HTRF kit (Cisbio, Bedford). Data is reported as a fold change of HTRF ratio with PTPN2 degrader over DMSO control.Western blottingIn vitro Western blots samples were generated by treating B16F10 mouse cells with a dose-response of Cmpd-1 and Cmpd-2 for 24 h. When noted, Bortezomib and Lenalidomide (all MilliporeSigma, Burlington) were added at 10 µM and 100 µM, respectively, two hours prior to Cmpd-1/Cmpd-2 addition. Treatment media was removed, cells washed with phosphate buffered saline (PBS) and lysed directly in 2x NuPAGE LDS sample buffer, 1x NuPAGE Sample Reducing Agent and Halt™ Protease Inhibitor Cocktail (all ThermoFisher Scientific, Waltham). Lysates were boiled for 5 min prior to loading gels.Two hours post-final dose, C57Bl/6 mouse spleens were harvested and pushed through a 70 µM strainer to generate single cell suspensions. Splenocytes were red blood cell lysed with eBioscience™ 1x RBC lysis buffer (ThermoFisher Scientific, Waltham) preceding cell lysis with Pierce™ RIPA Lysis Buffer and Halt™ Protease Inhibitor Cocktail (all ThermoFisher Scientific, Waltham). Protein was quantified and normalized using Pierce™ 660 nm Protein Assay kit (ThermoFisher Scientific, Waltham) before addition of 4x NuPAGE LDS sample buffer and 10x NuPAGE Sample Reducing Agent at final concentrations of 1x. Lysates were boiled for 5 min before loading gels.Western lysates were loaded on NuPAGE 4–12% Bis-Tris gels in NuPAGE MES SDS running buffer (ThermoFisher Scientific, Waltham) and transferred onto PVDF membranes using Trans-Blot Turbo (Bio-Rad, Hercules). Membranes were blocked for 2 h with Intercept (PBS) blocking buffer (LI-COR, Lincoln), incubated overnight at 4 °C with primary antibodies diluted in blocking buffer, and washed with phosphate buffered saline plus 0.1% Tween 20 (PBS-T). Membranes were incubated with secondary antibodies diluted in blocking buffer for 1 h at room temperature and washed with PBS-T. Blots were imaged using the LI-COR Odyssey CLx (LI-COR, Lincoln) and densitometry analyzed using Image Studio v5.2 (LI-COR, Lincoln). PTPN2 and PTPN1 signals were normalized to vinculin loading control. Data are reported as percent of protein remaining as it relates to the DMSO control. Primary antibodies used include anti-TCPTP (TC45) (D7T7D) Rabbit mAb (Cell Signaling, Danvers, 58935), anti-mouse PTP1B goat pAb (R&D Systems, Minneapolis, AF3954), and anti-vinculin mouse mAb (MilliporeSigma, Burlington, SAB4200080). Secondary antibodies used include IRDye 800CW Donkey anti-rabbit IgG (LI-COR, Lincoln, 925-32213), IRDye 800CW Donkey anti-goat IgG (LI-COR, Lincoln, 925-32214), and IRDye 680RD Donkey anti-mouse IgG (LI-COR, Lincoln, 925-68072).HiBiT degradation assayPTPN2 degradation was determined based on quantification of luminescent signal using Nano-Glo® HiBiT Lytic Assay kit (Promega, Madison, WI, USA). Test compounds were added to the 384-well plate from a top concentration of 10 µM with 11 points, half log titration in duplicates. B16F10 cells and 293 T cells ectopically expressing PTPN2 with N-terminal HiBit fusion tag were added into 384-well plates at a cell density of 5000 cells per well. The plates were kept at 37 °C with 5% CO2 for 24 h. The cells treated in the absence of the test compound were the negative control and the cells without Nano-Glo® HiBiT Lytic reagent were the positive control. After 24 h incubation, Nano-Glo® HiBiT Lytic Assay reagents were added to the cells. Luminescence was acquired on EnVision™ Multilabel Reader (PerkinElmer, Santa Clara, CA, USA).Selectivity assayAssay performed and analyzed as previously described (4). Briefly, 0.5 nM of 16 different phosphatases purchased from Eurofins were pre-incubated with a dose-response of Cmpd-1 before 30 min incubation with 5 µM DiFMUP (ThermoFisher) substrate at room temperature. After quenching assay with 100 µM bpV(Phen) (Enzo Life Sciences), fluorescence signal was quantified using an EnVision microplate reader (Perkin-Elmer) at 340 nm excitation and 450 nm emission wavelengths.Biolayer InterferometryBiolayer interferometry was performed using Octet Red96E (Sartorius). For assays to measure affinity to the degrader. Super Streptavidin (SSA) biosensors (Sartorius item no.: 18-5057) were equilibrated in 1x kinetics buffer (Sartorius item no.: 18-1105) for 60 s following which biotinylated PTPN1/2 at 25 µg/mL was immobilized on the sensors for 180 s. The sensors were blocked by dipping in biocytin solution at 20 µg/mL for 120 s. The sensor was dipped in a kinetics buffer for 60 s to remove unbound biocytin. Association with the degrader was recorded by dipping in two-fold serially diluted solutions of Cmpd-1 or Cmpd-2 (1000 to 15.6 nM final) for 180 s while dissociation was recorded by dipping in kinetics buffer for 240 s. To correct for any reference binding, sensors without immobilized protein were dipped similarly in corresponding concentrations of degrader and the signal was subtracted. Affinities were calculated using steady state kinetics by plotting response as a function of degrader concentration and determining the concentrations at which half the maximum signal was obtained.For binding to CRBN, High Precision Streptavidin (SAX) biosensors (Sartorius item no.: 18-5117) were equilibrated in 1x kinetics buffer for 60 s following which biotinylated PTPN1/2 at 8 µg/mL was immobilized on the sensors for 180 s. The sensors were then dipped in a solution containing 20 µg/mL biocytin and 1 µM Cmpd-1 or Cmpd-2 to simultaneously block the sensor and load the immobilized protein with degrader. Association with the CRBN was recorded by dipping in two-fold serially diluted solutions of CRBN-DDB1 (800 to 12.5 nM final) for 180 s while dissociation was recorded by dipping in kinetics buffer for 240 s. To correct for any reference binding, sensors with immobilized PTPN1/PTPN2 without degrader were dipped similarly in 800 nM CRBN-DDB1 and the signal was subtracted. Affinities were calculated by globally fitting the obtained curves to a 1:1 binding model. The qualities of fits were assessed and those above an R2 value of 0.90 were considered for evaluation. Two measurements were taken from the same samples using different sensors.Analytical SECAnalytical size exclusion chromatography was performed using Agilent 1200 Series Infinity II HPLC. 1 µM of PTPN1 or PTPN2, 1 µM of CRBN-DDB1 and 1.5 µM of Cmpd-1 or Cmpd-2 were mixed in a 150 µL solution of 1xPBS. 100 µL of this solution was injected onto a Superdex 200 Increase 5/150 GL column (Cytiva item no.:28990945) equilibrated in PBS and the absorbance of the eluent at 280 nm was recorded. The eluent was collected as 50 µL fractions and samples from fractions corresponding to 4.5 to 6.25 s were run on a reduced SDS-PAGE gel (Invitrogen item no.: NP0322BOX). The bands were visualized by staining the gel in Coomassie staining solution (Abcam item no.: ab119211).Isothermal Titration Calorimetry (ITC)Interaction between CRBN-DDB1 and Cmpd-1 was measured with a MicroCal PEAQ-ITC (Malvern Panalytical Inc.). Experiments were performed in 1xPBS with 2% DMSO at 20 °C. Cmpd-1 concentration in the syringe was 220 μM, and 2 μL aliquots were injected into cells containing 300 μL CRBN-DDB1 at a concentration of 10 μM. The final titration curve was fitted using MicroCal ITC data analysis software, assuming a single binding site per monomer.DSFThermal shift assay was performed on Prometheus Panta NT.48 from NanoTemper technologies by measuring the intrinsic dual-UV fluorescence change in tryptophan and tyrosine residues in proteins at emission wavelengths of k = 330 and 350 nm. The ratio of the recorded emission intensities (Em350 nm/Em330 nm), which represents the change in TRP fluorescence intensity as well as the shift of the emission maximum to higher wavelengths (“red-shift”) or lower wavelengths (“blue-shift”) was plotted as a function of the temperature. The fluorescence intensity ratio and its first derivative were calculated and determined to be the melting temperature (Tm), with the manufacturer’s software (PR.Panta Control and PR.Panta Analysis). The samples were loaded using capillaries in a volume of 15 µL on Prometheus Panta NT.48 from NanoTemper Technologies. PTPN2 constructs were diluted to 1.5 mg/mL and then subjected to thermal change from 25–85 °C with a ramp rate of 1 °C/min. The experiment was performed in duplicate with two samples for each protein and the mean Tm was determined.X-ray crystallographyCrystals of PTPN2 with Cmpd-2 were obtained by co-crystallization using sitting drop vapor diffusion technique. A 100 mM compound stock in 100% DMSO was added to the purified protein in 7:1 molar ratio. Crystals were obtained in 0.49 M NaH2PO4 and 0.91 M K2HPO4, pH6.9 at 23 °C. Crystals were cryo-protected in mother liquor containing 15% glycerol and flash-frozen into liquid nitrogen. Diffraction data were collected with 1.000 Å wavelength at 100 K using a Pilatus 6 M detector on the IMCA-CAT beamline 17ID at Argonne National Laboratory. Diffraction data w indexed with autoPROC and scaled with XScale. Crystals diffracted to 1.93 Å and belong to space group C2221 with unit cell dimensions of 77.2, 126.5, and 150.2 Å for a, b, and c axis respectively. Initial structure was solved by molecular replacement using MOLREP. Structure was corrected manually and refined using Coot and Phenix respectively. The final refined structure had the following Ramachandran statistics: 96.92% favored, 2.72% allowed and 0.36% outliers. Structure figures were prepared using PyMOL. Structure and associated data have been deposited with RCSB with accession code 8U0H.Cryo-EM specimen preparation and data acquisitionSamples were prepared at NanoImaging Services. 3 μL of purified DDB1-CRBN-Cmpd-d-PTPN2 at ~10 mg/mL were mixed with 0.75 mM fluorinated Fos-Choline-8 (Anatrace) to glow-discharged (PELCO EasiGlow system) Quantifoil 300 mesh R 0.6/1.0 UltrAuFoil Holey Gold Films. The grids were prepared with a FEI Vitrobot Mark IV (Thermo Fisher Scientific) with the environmental chamber set at 95% humidity, 4 °C. The grids were blotted for 10 s with a force of 10 and then flash-frozen in liquid ethane and stored in liquid nitrogen. Grids were screened using a Thermo Fisher Scientific (Hillsboro, Oregon) Glacios Cryo Transmission Electron Microscope (Cryo-TEM) operated at 200 kV and equipped with a Falcon 4 direct electron detector to select those which were best suited for high-resolution data collection.The best two grids were imaged on a FEI Titan Krios (Hillsboro, Oregon) transmission electron microscope operated at 300 kV and equipped with a Gatan Quantum 967 LS imaging filter and Gatan K3 Direct Detection Camera. Vitreous ice grids were clipped into cartridges, transferred into a cassette and then into the Krios autoloader, all while maintaining the grids at cryogenic temperature (below −170 °C). Automated data-collection was carried out using Leginon software38, where high magnification movies were acquired at 105,000x nominal magnification with a calibrated pixel size of 0.832 Å, at a dose rate of 36.09 e-/Å2/s with a total exposure of 1.40 s for an accumulated dose of 50.53 e-/Å2. Intermediate frames were recorded every 0.04 s for a total of 35 frames per micrograph. A total of 16,136 images were collected at a nominal defocus range of − 1.0 to −2.0 μm (Figure S6).Cryo-EM data processing, and atomic model building and refinementData processing was performed first in cryoSPARC live and continued in cryoSPARC v3.3. Dose-weighted movie frame alignment was performed using patch motion correction39,40 to account for stage drift and beam-induced motion. The contrast transfer function was estimated for each micrograph using patch CTF41,42. Micrographs with poor contrast transfer function fits were removed. Automated particle picking was performed live with the blob-based picker, in which resulted in a total of four million particles extracted with a box size of 360 pixel (~299 Å2). Several rounds of 2D classifications and select 2D classes were performed for initial particle clean up, yielding 838,773 particles. The selected particles were used in three rounds of hetero-refinement with four decoys followed by an homogenous refinement. The 666,084 particles resulted in a very flexible 3D reconstruction model. Therefore, two rounds of 3D variability jobs were used to resolve the sample continuous heterogeneity clustering the particles in five discrete conformational states. A cluster of 69,542 particles generated a map with the highest resolution, and that map was selected for further refinement using CTF local and global refinement followed by local resolution refinement. The estimated resolution is 3.3 Å based on gold standard Fourier shell correlation of 0.143. A full summary of the processing workflow could be found in Supplementary Fig. 9.The post-processed map was optimized with PHENIX autosharpen map and local anisotropy sharpen to maximize the detail and the connectivity43.Model building and refinement were initiated with a published model for CRBN ~DDB1 (PDB ID: 4CI1) and an internal crystal structure for the PTPN2. The two structures were placed into the sharpened density map using ChimeraX fit in map44. The generation of Cartesian coordinates and geometry restraints of the ligand were carried out in eLBOW a program module of the PHENIX suite45. Iterative rounds of model building and refinement were performed in PHENIX 1.19.2 and COOT46,47. The BPB did not have a clear density to correctly assign side-chains and backbone. Thus, we decided to not model this propeller into the map. The final model was validated against the half-maps and its quality assessed by MolProbity48.Structural modeling (docking) of PTPN1-Cmpd-1 or Cmpd-2We used our x-ray structure of PTPN2-Cmpd-2 as an initial reference. However, prior to employing this construct for calculations, we carried out refinement and preparation steps using the MOE QuickPrep module49. This involved adjusting the protonation states of all atoms and performing structure optimization of the X-ray construct using the Amber10:EHT force field, which is integrated into the MOE-2022.02 software package12. During the minimization procedure, we implemented MOE default tether restraints on the protein atoms and meticulously monitored the root-mean-square (RMS) gradient of the potential energy to ensure it remained below 0.1 kcal mol−1 Å−2. To include PTPN1 in the ternary complex model, we utilized the crystal structure of PTPN1 bound to an inhibitor (PDB ID: 2QBP). Similar to the aforementioned steps, we prepared and refined this x-ray construct using the MOE software to ensure its suitability for integration into the overall model.MD simulationBefore commencing MD simulations, we performed refinement of the cryo-EM PTPN2-Cmpd-1/CRBN-DDB1 complex using the MOE QuickPrep module. The resulting ternary complex was then immersed in a water/ion box with dimensions of 161.0 Å3 × 161.0 Å3 × 161.0 Å3, containing excess ions at a concentration of 150 mM to mimic the physiological conditions. Overall, the system consisted of 415 K atoms, accounting for the protein complex, water molecules, and ions present in the simulation box. To optimize this system, we initiated energy minimization using the steepest descents algorithm in GROMACS, consisting of 1000 steps. The refinement was followed by an MD simulation in a canonical ensemble, where the system was heated gradually from 0 K to 310 K in 200 ps, followed by MD simulations in an isobaric-isothermal ensemble for an aggregated 800 ps, during which the pressure was maintained at 1 bar to relax the simulation box. Throughout these pre-equilibration steps, the positional restraints were placed on all heavy (non-H atoms) atoms of proteins and Cmpd-1, gradually reduced to 0 kcal mol−1 Å−2 for the final equilibration step. To establish a reference for monitoring the displacement of PTPN2 on the surface of CRBN, we placed positional harmonic restraints on the backbone atoms of the DDB1 protein during the subsequent simulations, with a force constant of 10 kcal mol−1 Å−2. Finally, we optimized the PTPN2-Cmpd-1-CRBN-DDB1 constructs by removing the positional restraints and performed MD simulations for sufficient duration to reach equilibrium. The equilibration progress was monitored by observing the root-mean-square deviation (rmsd) of protein Cα atoms, as depicted in Fig. 4b. We replicated this procedure three times to generate three independent replicates for subsequent analysis.During the simulations, the AMBER-14 force field parameter set50 was employed to describe the PTPN2, DDB1, CRBN proteins, and ions. The Cmpd-1 degrader was parameterized using the Antechamber program with the Generalized AMBER force field (GAFF)51. The TIP3P model was utilized to represent the water molecules.The temperature was maintained at 310 K using a velocity-rescale thermostat52 with a damping constant of 1.0 ps for temperature coupling and the pressure was controlled at 1 bar using a Parrinello-Rahman barostat algorithm53 with a 5.0 ps damping constant for the pressure coupling. Isotropic pressure coupling was used during this calculation. The Lennard-Jones cutoff radius was 12 Å, where the interaction was smoothly shifted to 0 after 10 Å. Periodic boundary conditions were applied to all three directions. The Particle Mesh Ewald algorithm54 was used to calculate long-range coulombic interactions with a real cutoff radius of 10 Å and a grid spacing of 1.2 Å. A compressibility of 4.5 × 10−5 bar−1 was used to relax the box volume. In all the above simulations, water OH bonds were constrained by the SETTLE algorithm55. The remaining H-bonds were constrained using the P-LINCS algorithm56. All MD simulations were carried out using GROMACS57.Chemical synthesisThe synthesis of the molecules could be found as described in Veits, G.K. et al.58.Hydrogen-deuterium exchange mass spectrometryHDX-MS was performed by UVic-Genome BC Proteomics Centre, Victoria, Canada.HDX-MS sample preparationHDX reactions comparing apo PTPN2 to PTPN2 incubated with Cmpd-1 and PTPN2 incubated with Cmpd-1 and CRBN/DDB1 complex were carried out in a 44.75 µl reaction volume containing 15 pmol of PTPN2, 20 pmol of CRBN/DDB1 and 1 µM of Cmpd-1. The exchange reactions were initiated by the addition of 34.75 µL of D2O buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 1 mM TCEP 93.3% D2O (V/V)) to 5.25 µL of protein (final D2O concentration of 81.05%). Reactions proceeded for 3 s on ice and 3 s, 30 s, 300 s and 3000 s at 20 °C before being quenched with ice cold acidic quench buffer, resulting in a final concentration of 0.6 M guanidine HCl and 0.9% formic acid post-quench. All conditions and timepoints were created and run in independent triplicate. Samples were flash frozen immediately after quenching and stored at −80 °C until injected onto the ultra-performance liquid chromatography (UPLC) system for proteolytic cleavage, peptide separation, and injection onto a QTOF for mass analysis, described below.Protein digestion and MS/MS data collectionProtein samples were rapidly thawed and injected onto an integrated fluidics system containing a HDx-3 PAL liquid handling robot and climate-controlled (2 °C) chromatography system (LEAP Technologies), a Waters Acquity UPLC I-Class Series System, as well as an Impact HD QTOF Mass spectrometer (Bruker). The samples were run over an immobilized pepsin column (Affipro; Enzymate Protein Pepsin Column, 2.1 mm × 20 mm) at 200 µL/min for 3 min at 2 °C. The resulting peptides were collected and desalted on a C18 trap column (Acquity UPLC BEH C18 1.7 µm column (2.1 × 5 mm); Waters 186004629). The trap was subsequently eluted in line with an ACQUITY 300 Å, 1.7 μm particle, 100 × 2.1 mm BEH C18 UPLC column (Waters), using a gradient of 3–10% B (Buffer A 0.1% formic acid; Buffer B 100% acetonitrile) over 1.5 min, followed by a gradient of 10–25% B over 4.5 min, followed by a gradient of 25–35% B over 5 min, finally after 1 min at 35% B a gradient of 35–80% B over 1 min was used. Mass spectrometry experiments acquired over a mass range from 150 to 2200 m/z using an electrospray ionization source operated at a temperature of 200 °C and a spray voltage of 4.5 kV.Peptide identificationPeptides were identified from the non-deuterated samples of PTPN2 using data-dependent acquisition following tandem MS/MS experiments (0.5 s precursor scan from 150–2000 m/z; twelve 0.25 s fragment scans from 150–2000 m/z). MS/MS datasets were analysed using FragPipe v19.1 and peptide identification was carried out by using a false discovery-based approach using a database of purified proteins and known contaminants. MSFragger was utilized, and the precursor mass tolerance error was set to −20 to 20 ppm. The fragment mass tolerance was set at 20 ppm. Protein digestion was set as nonspecific, searching between lengths of 4 and 50 aa, with a mass range of 400 to 5000 Da.Mass analysis of peptide centroids and measurement of deuterium incorporationHD-Examiner Software (Sierra Analytics) was used to automatically calculate the level of deuterium incorporation into each peptide. All peptides were manually inspected for correct charge state, correct retention time, appropriate selection of isotopic distribution, etc. Deuteration levels were calculated using the centroid of the experimental isotope clusters. Results are presented as relative levels of deuterium incorporation and the only control for back exchange was the level of deuterium present in the buffer (81.0%). Differences in exchange in a peptide were considered significant if they met all three of the following criteria: ≥5.5% change in exchange, ≥0.45 Da difference in exchange, and a p-value < 0.01 using a two tailed student t-test. Samples were only compared within a single experiment and were never compared to experiments completed at a different time with a different final D2O level. The data analysis statistics for all HDX-MS experiments are in Supplementary Table 1 according to published guidelines59.Reporting summaryFurther information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
