Journal: Protein Science : A Publication of the Protein Society

Article Title: Accurate affinity models for SH2 domains from peptide binding assays and free‐energy regression

doi: 10.1002/pro.70317

Figure Lengend Snippet: Overview of concerted experimental and computational strategy for generating SH2‐peptide binding free energy models. (a) Design of peptide‐display libraries. (b) Schematic showing how a randomized bacterial display library underwent repeated bead‐based affinity selection for SH2 binding. In each selection round, the library was sequenced before and after selection. (c) Overview of the regression framework used to learn energetic binding models from the sequencing data. For each possible binding site, the energy received independent additive contributions from the residues flanking the phosphorylated tyrosine, thus controlling for the binding‐site context wherein the residues reside. These energy contributions were estimated using maximum likelihood estimation, where the likelihood of the observed sequence counts was evaluated by first computing the total affinity for each observed sequence (controlling for multiple possible binding offsets and non‐specific binding) and then computing the binomial likelihood for each round, assuming linear section. (d) Sequence logo displaying the inferred energy contributions as letters whose height reflects the magnitude of the contributions, relative to the mean for each position.

Article Snippet: To perform the single selection experiment using the phosphorylated peptide library against, 75 μL of streptavidin‐coated magnetic beads (DynabeadsTM FlowCompTM Flexi Kit, Thermo‐Fisher) were washed twice in 1 mL of SH2 binding buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 0.05% Tween 20, 1 mM TCEP) and incubated in a total of 150 μL SH2 binding buffer containing 20 μM biotinylated SH2 domain on a rotator at 4°C for 2–3 h in low protein‐binding microcentrifuge tubes (1.5 mL, Thermo ScientificTM).

Techniques: Binding Assay, Selection, Sequencing

Journal: Protein Science : A Publication of the Protein Society

Article Title: Accurate affinity models for SH2 domains from peptide binding assays and free‐energy regression

doi: 10.1002/pro.70317

Figure Lengend Snippet: Comparison of amino‐acid enrichment analysis and free‐energy regression. (a) Distribution of read counts (after down‐sampling to 500,000 reads) for sequences in the pTyrVar and X 5 YX 5 libraries, respectively, each before and after one round of affinity selection with the c‐Src SH2 domain. (b) Amino‐acid log‐enrichment due to affinity selection for c‐Src SH2, displayed as sequence logos, for the designed pTyrVar and random X 5 YX 5 library, respectively. (c) Direct comparison of log‐enrichment parameters between the two library designs. Red points indicate tyrosine, all other residues are gray. (d) Inferred free‐energy contributions (ΔΔ G /RT) at different positions within the c‐Src SH2 binding interface, displayed as sequence logos. Gray rectangles indicate position where the model was constrained to recognize (phospho)tyrosine. (e) Direct comparison of ΔΔ G/ RT parameters between the two library designs.

Article Snippet: To perform the single selection experiment using the phosphorylated peptide library against, 75 μL of streptavidin‐coated magnetic beads (DynabeadsTM FlowCompTM Flexi Kit, Thermo‐Fisher) were washed twice in 1 mL of SH2 binding buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 0.05% Tween 20, 1 mM TCEP) and incubated in a total of 150 μL SH2 binding buffer containing 20 μM biotinylated SH2 domain on a rotator at 4°C for 2–3 h in low protein‐binding microcentrifuge tubes (1.5 mL, Thermo ScientificTM).

Techniques: Comparison, Sampling, Selection, Sequencing, Binding Assay

Journal: Protein Science : A Publication of the Protein Society

Article Title: Accurate affinity models for SH2 domains from peptide binding assays and free‐energy regression

doi: 10.1002/pro.70317

Figure Lengend Snippet: Multi‐round profiling of c‐Src SH2 using the naïve and pre‐enriched X 11 libraries. (a) Binding model learned using one selection round and starting with the naïve X 11 library. (b) Scatter plot comparing the model coefficients shown in panel (a) to the coefficients of the X 5 YX 5 model shown in Figure . Red points indicate tyrosine. (c), (d) Same as (a), (b) but showing a model that was trained on data from three selection rounds. (e), (f) Same as (a), (b) but showing a model that was trained on an experiment where the input library was pre‐selected using the 4G10 antibody, followed by two rounds of c‐Src SH2 binding selection. (g), (h) Same as (a), (b) but showing a model that was trained on data from the second and third selection rounds and that was not constrained to recognize tyrosine at the central position.

Article Snippet: To perform the single selection experiment using the phosphorylated peptide library against, 75 μL of streptavidin‐coated magnetic beads (DynabeadsTM FlowCompTM Flexi Kit, Thermo‐Fisher) were washed twice in 1 mL of SH2 binding buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 0.05% Tween 20, 1 mM TCEP) and incubated in a total of 150 μL SH2 binding buffer containing 20 μM biotinylated SH2 domain on a rotator at 4°C for 2–3 h in low protein‐binding microcentrifuge tubes (1.5 mL, Thermo ScientificTM).

Techniques: Binding Assay, Selection

Journal: Protein Science : A Publication of the Protein Society

Article Title: Accurate affinity models for SH2 domains from peptide binding assays and free‐energy regression

doi: 10.1002/pro.70317

Figure Lengend Snippet: Flanking specificity of the c‐Src, Grb2 and Fyn SH2 domains. (a) Energy logos for the c‐Src SH2, Fyn SH2 and Grb2 SH2 binding models. (b) Scatter plots comparing the predictions from the binding models in (a) with competitive fluorescence polarization measurements. Vertical bars indicate standard error. Dashed black lines (and accompanying model expressions and r 2 values) indicate linear regression fits to the log‐transformed K D ‐values. ( c ) Comparison of the c‐Src and Fyn binding models from (a) using an energy logo (top, showing the difference − ∆ ∆ ∆ G / RT between the model coefficients) and a scatter plot (bottom). (e), (d) AlphaFold 3 models of the c‐Src and Fyn SH2 domains (shown as surfaces in the central panels) bound to a high‐affinity phospho‐peptide (GHH‐pY‐EEIG, shown as purple sticks). Residues on the SH2 domains colored in beige are sites where c‐Src and Fyn diverge. A key divergent site (N201 in c‐Src and H199 in Fyn) is shown in teal. The zoom‐in panels highlight key residues in a cationic pocket on the SH2 domain that interacts with the ±1 residue on the peptide ligand.

Article Snippet: To perform the single selection experiment using the phosphorylated peptide library against, 75 μL of streptavidin‐coated magnetic beads (DynabeadsTM FlowCompTM Flexi Kit, Thermo‐Fisher) were washed twice in 1 mL of SH2 binding buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 0.05% Tween 20, 1 mM TCEP) and incubated in a total of 150 μL SH2 binding buffer containing 20 μM biotinylated SH2 domain on a rotator at 4°C for 2–3 h in low protein‐binding microcentrifuge tubes (1.5 mL, Thermo ScientificTM).

Techniques: Binding Assay, Fluorescence, Transformation Assay, Comparison, Residue

Journal: Protein Science : A Publication of the Protein Society

Article Title: Accurate affinity models for SH2 domains from peptide binding assays and free‐energy regression

doi: 10.1002/pro.70317

Figure Lengend Snippet: Flanking specificity for the Lyn, Yes and Blk SH2 domains. (a) Energy logos showing binding models for Lyn, Yes, and Blk. The models were trained on two‐round experiments using the X 5 YX 5 starting library. (b) Scatter plots comparing model predictions and validation measurements for the Lyn SH2 domain, shown as in Figure .

Article Snippet: To perform the single selection experiment using the phosphorylated peptide library against, 75 μL of streptavidin‐coated magnetic beads (DynabeadsTM FlowCompTM Flexi Kit, Thermo‐Fisher) were washed twice in 1 mL of SH2 binding buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 0.05% Tween 20, 1 mM TCEP) and incubated in a total of 150 μL SH2 binding buffer containing 20 μM biotinylated SH2 domain on a rotator at 4°C for 2–3 h in low protein‐binding microcentrifuge tubes (1.5 mL, Thermo ScientificTM).

Techniques: Binding Assay, Biomarker Discovery

Journal: Protein Science : A Publication of the Protein Society

Article Title: Accurate affinity models for SH2 domains from peptide binding assays and free‐energy regression

doi: 10.1002/pro.70317

Figure Lengend Snippet: Distribution of the predicted quantitative impact of missense variants in SH2 binding sites in the human proteome. Scatterplot of allelic effect of missense variation in SH2 binding sites documented in the PTMVar database of human phosphorylation site variants (Hornbeck et al., ), colored by the direction of the effect. The x ‐value corresponds to the greater of the predicted affinities of the two alleles, where relative affinity score is inversely proportional to the K D ; the y ‐value corresponds to the ratio of predicted affinities between the two alleles.

Article Snippet: To perform the single selection experiment using the phosphorylated peptide library against, 75 μL of streptavidin‐coated magnetic beads (DynabeadsTM FlowCompTM Flexi Kit, Thermo‐Fisher) were washed twice in 1 mL of SH2 binding buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 0.05% Tween 20, 1 mM TCEP) and incubated in a total of 150 μL SH2 binding buffer containing 20 μM biotinylated SH2 domain on a rotator at 4°C for 2–3 h in low protein‐binding microcentrifuge tubes (1.5 mL, Thermo ScientificTM).

Techniques: Binding Assay, Phospho-proteomics

Journal: The Journal of Biological Chemistry

Article Title: Signaling adaptor ShcD suppresses extracellular signal-regulated kinase (Erk) phosphorylation distal to the Ret and Trk neurotrophic receptors

doi: 10.1074/jbc.M116.770511

Figure Lengend Snippet: Shp2 binds the ShcD SH2 domain and regulates CH1 region tyrosine phosphorylation, influencing distal signaling. A, the potential for Shp2 to associate with ShcD was first pursued via co-immunoprecipitation analyses from cells transfected with a combination of TrkB-HA, ShcD-FLAG, and Shp2. An ShcD-Shp2 interaction was detected in the presence and absence of the RTK, and Shp2 was found to oppose TrkB-mediated ShcD phosphorylation. B, to determine the Shp2 docking site on ShcD, the PTB*, SH2*, and Y6F ShcD mutants were co-expressed with TrkB-HA and Shp2. ShcD-FLAG immunoprecipitation followed by Shp2 immunoblotting demonstrated that disabling the ShcD SH2 domain reduced Shp2 binding. C, to further validate the interaction, GST fusions of the isolated PTB, SH2, and SH2* domains of ShcD were incubated with glutathione beads and cell lysates from Shp2 alone or Shp2 + TrkB-HA-transfected cells. The wild type ShcD SH2 domain successfully co-precipitated Shp2 with a stronger association noted in the presence of TrkB. Interactions with Shp2 were substantially reduced by the SH2* mutant. D, to confirm the effects of Shp2 on ShcD phosphorylation, wild type or SH2* ShcD was coexpressed with TrkB-HA with and without Shp2 wild type or dominant-negative Cys-to-Ser (C/S) mutants. ShcD phosphorylation decreased in the presence of Shp2 and increased with the Cys-to-Ser mutant. Conversely, Erk activation was higher in the presence of Shp2 and substantially reduced with the Cys-to-Ser mutant, demonstrating that Shp2 positively regulates Erk activation by dephosphorylating ShcD. E, pErk/Erk intensity ratios relative to ShcD WT as determined from immunoblot densitometry performed on D confirm the visual observations. One-way ANOVA (n ≥ 3; p = 0.0152) followed by Tukey's multiple comparison test yielded the following multiplicity-adjusted p values for significant pairs: −Shp2 versus +Shp2, p = 0.0206; +Shp2 versus +Shp2 Cys-to-Ser, p = 0.0349. Error bars denote S.E. *, p ≤ 0.05. IP, immunoprecipitation; PD, pulldown; IB, immunoblot.

Article Snippet: Following removal of supernatant, beads were washed three times with 800 μl of PLC plus buffer, and protein complexes were eluted in 2× SDS loading buffer by boiling at 100 °C for 3 min. GST fusion proteins for ShcD-PTB, ShcD-SH2, and disabled ShcD-SH2 (SH2*) were expressed in Escherichia coli BL21 cells by overnight induction with 0.5 m m isopropyl β- d -1-thiogalactopyranoside and purified using glutathione-Sepharose TM 4B beads (GE Healthcare).

Techniques: Immunoprecipitation, Transfection, Western Blot, Binding Assay, Isolation, Incubation, Mutagenesis, Dominant Negative Mutation, Activation Assay

Journal:

Article Title: Negative regulation of FAK signaling by SOCS proteins

doi: 10.1093/emboj/cdg503

Figure Lengend Snippet: Fig. 3. Y397 in FAK regulates the FAK–SOCS interaction. (A) COS-7 cells were transiently transfected with the indicated plasmids (0.2 µg of HA-FAK or HA-FAK-Y397F, 0.5 µg of Myc-SOCS-1/3). Forty-eight hours after transfection, cell lysates were immunoprecipitated with anti-HA antibody, and the precipitates were analyzed by immunoblotting with antibodies against Myc, pTyr or HA. Total cell lysates were subjected to immunoblotting with anti-Myc antibody to confirm SOCS protein expression levels. (B) Denatured lysates of HA-FAK or HA-FAK-Y397F-transfected COS-7 cells were incubated with purified GST or with GST–SOCS-3-SH2 fusion proteins conjugated to glutathione–Sepharose beads. Associated proteins were examined by anti-HA immunoblotting (top panel). Ponceau S staining of the same blot is shown on the bottom panel. (C) GST fusion proteins coding for the N-terminal domain (amino acids 1–406) of wild-type or Y397-mutant of FAK were produced in the bacterial TK strain expressing an active tyrosine kinase. The produced proteins were separated by SDS–PAGE and transferred onto PVDF membrane and analyzed by immunoblotting with the indicated antibodies (upper left panel), or by far-western blotting with the indicated fusion proteins as described in Materials and methods (lower panel). Coomassie Blue staining of the fusion proteins used as probes is shown on the upper right panel.

Article Snippet: The membranes were blocked with binding buffer (10 mM Tris–HCl pH 7.4, 1 mM EDTA, 150 mM NaCl, 1 mM DTT and 0.5% BSA) for 2 h. The membranes were probed for 4 h at room temperature in the binding buffer with 1 µg/ml EZ-Link™ Sulfo-NHS-LC-Biotin (Pierce)-labeled GST–SH2 fusion proteins of either Src ( Vuori et al ., 1996 ), SOCS-3 or SOCS-3-R71E.

Techniques: Transfection, Immunoprecipitation, Western Blot, Expressing, Incubation, Purification, Staining, Mutagenesis, Produced, SDS Page, Far Western Blot