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: bioRxiv

Article Title: SH2 scan : Mapping SH2 domain-ligand binding selectivity for inhibitors and degraders

doi: 10.1101/2025.07.09.663991

Figure Lengend Snippet:

Article Snippet: J.A.B. is a co-inventor on a pending patent application filed by Eurofins DiscoverX, LLC for SH2 domain competition binding assays.

Techniques: Construct, Mutagenesis, Phospho-proteomics

Journal: bioRxiv

Article Title: SH2 scan : Mapping SH2 domain-ligand binding selectivity for inhibitors and degraders

doi: 10.1101/2025.07.09.663991

Figure Lengend Snippet: a , An SH2 domain-containing protein construct (blue) fused to the NFκB DNA binding domain (pink) is tagged with an exogenous double-stranded DNA (dsDNA) probe. This construct is incubated with a capture ligand (red) immobilized on magnetic beads (green). b , In the presence of a competitor compound (yellow), less tagged protein is captured on the beads. The protein remaining on the beads is eluted using a high concentration of sodium phenyl phosphate, a generic competitor of phosphopeptide binding to SH2 domains . After elution, a lower qPCR signal is obtained at the end of the assay. In the presence of a non-competitor compound ligand (brown), more tagged protein is captured on the beads and a high qPCR signal is observed. c , Representative primary screening data for the STAT3 construct are shown. Compounds were tested at 10 μM and the hit cutoff for the compound screen was set at ≤35% of the average signal of the DMSO control wells for each construct (dashed line). Percent assay signal for each compound is expressed as the mean of at least two independent technical replicates from at least one independent experiment, ± standard deviation. d , K D data for selected validated hits for the STAT3 construct are shown. Compounds were tested in dose-response, and the data points were fit to the Hill equation (see Methods). Data are presented as mean percent assay signal from at least four independent technical replicates collected over two independent experiment, ± standard deviation.

Article Snippet: J.A.B. is a co-inventor on a pending patent application filed by Eurofins DiscoverX, LLC for SH2 domain competition binding assays.

Techniques: Construct, Binding Assay, Incubation, Magnetic Beads, Concentration Assay, Phospho-proteomics, Control, Standard Deviation

Journal: bioRxiv

Article Title: SH2 scan : Mapping SH2 domain-ligand binding selectivity for inhibitors and degraders

doi: 10.1101/2025.07.09.663991

Figure Lengend Snippet: Percent of DMSO control values were measured for each compound tested in this study and mapped as dots on a phylogenetic tree containing all 120 canonical human SH2 domains. Each dot plotted in a diagram represents a mean value collected from at least two independent technical replicates collected over at least one independent experiment, ± standard deviation. The screening results for the two SH2 domains of SYK and ZAP70 are mapped as equivalently sized dots, given that the SH2 domains for these targets are expressed in tandem constructs, and any competition observed cannot be unequivocally assigned to one or the other SH2 domain.

Article Snippet: J.A.B. is a co-inventor on a pending patent application filed by Eurofins DiscoverX, LLC for SH2 domain competition binding assays.

Techniques: Control, Standard Deviation, Construct