Journal: bioRxiv

Article Title: Mechanistic modeling identifies emergent properties of tandem SH2 domain interactions

doi: 10.1101/2025.05.13.653723

Figure Lengend Snippet: Experimental testing of the PTPN11:GAB1 interaction. A) SISA-KiT was used to phosphorylate both pTyr sites within the GAB1 region expressed using SRC kinase catalytic domain. The secondary interaction used in SISA-KiT to enhance phosphorylation of the substrate is the ABL SH3 domain with the p40 polyproline sequence (APTYSPPPPP). B) The degree of phosphorylation and confirmation of multisite phosphorylation on purified GAB1 was evaluated by running native PAGE and immunoblotting with MYC and confirming by pan-specific phosphoantibody. GAB1 WT refers to the GAB1 region with both tyrosines and the Y1F and Y2F mutate one and both tyrosines to phenylalnine, respectively, as a control for where on the gel the species run. C) Purified, phosphorylated GAB1 was biotinylated using BirA ligase and streptavidin BLI tips were used to measure the kinetics of ligand with SH2 domain. Shown here are the background corrected on- and off-kinetics and the fit of the BLI data with a 1:1 kinetic model. Depending on the degree of effective binding affinity, we adjusted the serial dilution series to avoid saturating the system and the specific concentrations of SH2 domain in the experiment are indicated. Where fit was sufficiently reliable in the experiment, we report an effective affinity (K D,eff ). We used the double Y2F mutant as a negative control. D) Chimeras were produced by swapping out the PTPN11 N and PTPN11 C SH2 domains with RASA1 N and PLC γ 1 C , respectively. Simulations of these chimeras interacting with GAB1 were performed in the model, and BLI experiments were performed to calculate K D,eff for each new species.

Article Snippet: Experiments were run on the Gator® Pilot BLI instrument using Streptavidin (SA) probes (Gator SKU 160002), MAX plates (Gator SKU 130062) for probe loading, and black flat plates (Gator SKU 130150) for sample loading.

Techniques: Phospho-proteomics, Sequencing, Purification, Clear Native PAGE, Western Blot, Control, Binding Assay, Serial Dilution, Mutagenesis, Negative Control, Produced