Fig. 9: Selectivity of d-DAM21/27 for the BCR::ABL1 SH2 domain.

a l-DAM27:d-Abl SH2 complex structure (PDB code: 9F00) highlighting the short Abl SH2 CD loop that enables binding to DAM27. The space where longer CD loops in other SH2 domains would be located is indicated by the dotted red circle. b Structural superposition of the Abl, Lck and Btk SH2 domains (PDB entries 3K2M, 1LKK and 6HTF, respectively). The additional four to six amino acid residues in the Lck and Btk SH2 CD loops are not compatible with binding to the DAM21 and DAM27 monobodies. c Multiple sequence alignment of the βC and βD strands of SH2 domains belonging to different SH2 domain-containing tyrosine kinase families. The βC and βD strands are colored in red and green, respectively, while the CD loop in between is black. Arg189 of ABL1, which is important for d-monobody binding via ionic interactions, is not conserved and colored in bold green to highlight the different amino acid sequences in this position. d, e Isothermal titration calorimetry (ITC) measurements of d-DAM27-XTEN titrated to the SH2 domains of (d) Btk and (e) Lck. f, g ITC measurements of split-d-DAM21 titrated to the SH2 domains of (f) Btk and (g) Lck. Each panel shows the raw heat signal of an ITC experiment (top) and the integrated calorimetric data of the area of each peak (bottom). h, i Volcano plots of identified proteins via mass spectrometry after pulldown from K562 cell lysates comparing synthetic (h) split-d-DAM21 with split-l-DAM21 and (i) d-DAM27-XTEN with l-DAM27-XTEN. Pulldowns were performed in three biological replicates for each monobody. Protein identification and statistical analysis of replicates were done with MaxQuant 2.5.1.0 and Autonomics (R package version 1.13.21)⁸⁴ resulting in FDR-corrected p-values (Benjamini-Hochberg procedure) for each identified protein where a p-value below 0.05, which corresponds to -log₁₀(p) above 1.3, was considered statistically significant (dotted line intersecting y-axis). A log₂(ratio) above 1.00 of d- vs. l-monobody correlates to a ratio above 2.00 and a higher abundance of the protein when the pulldown was performed with the d-monobody compared to its l-counterpart (dotted lines intersecting x-axis). The color coding represents the protein groups BCR and ABL1 (light red), BCR::ABL1/ABL1 interactors (blue), BCR::ABL1/ABL1 interactors with SH2 domains (green) and other proteins containing SH2 domains (purple). Plotted values of all highlighted protein groups are listed in Tables S9, S10.