(a) Schematic of the FN3 scaffold. β-Strands are labeled with A–G and loop regions diversified in the combinatorial library are in cyan. Figure generated using PyMOL (www.pymol.org). (b) Library design and loop sequences of Abl SH2-binding monobodies. X refers to a mixture of 30% Tyr (yellow), 15% Ser (red), 10% Gly (Green), 5% each of Trp, Phe and Arg (Green) and 2.5% each of all other amino acids except Cys. Z refers to a mixture of 50% Gly, 25% Tyr and 25% Ser. The numbers indicate positions for HA4. The Tyr87 position, mutated in the HA4_Y87A non-binding control, is marked with the asterisk. Because of differences in loop lengths, the numbering does not correspond to previously published monobodies. (c) SPR traces for HA4 binding to immobilized Abl SH2 domain, corrected by subtraction of the sensorgram for a blank run (gray) Parameters for the global Langmuir fit are provided, and the black lines show the best fit. Left, measurements in non-phosphate buffer. Right, measurements in phosphate buffer. (d) Left, fluorescence polarization changes of a rhodamine-labeled pY-peptide as a function of GST-Abl SH2 added to the solution. The concentration of GST-Abl SH2 required to give ∼80% maximum polarization (10 μM, indicated with the arrow) was used for HA4 competition assay shown on the right panel. Right: Fluorescence polarization of the rhodamine-labeled pY-peptide in the presence of GST-Abl SH2 is plotted versus the concentration of monobody added to the solution.

(a) Fluorescence images of SH2 chips following incubation with fluorophore-labeled HA4. Each SH2 domain is spotted in duplicate. A listing of spotted SH2 domains is provided in Supplementary Table 1. Spots for the SH2 domains shown in (b) are enclosed in the boxes. (b) The fluorescence signal strengths of individual SH2 domain spots are plotted as a function of HA4 concentration. The curves show the best fit of a 1:1 binding model, and the estimated dissociation constants are provided.

(a) The asymmetric unit containing two copies of the complex. β-strand A and the N-terminal extension, forming an intermolecular β-sheet, are labeled. (b) The HA4/Abl SH2 interface. Left, cartoon representation of the complex. Right, orthogonal view, where the SH2 domain is shown as a surface model, HA4 residues as stick models with residues in the BC, DE, and FG loops in yellow, cyan and green, respectively. Amino acids that differ from the library template are underlined. SH2 surfaces contacted by FG loop ‘finger’ residues are in pink, and ‘palm’ contacts are in brown. A putative inorganic phosphate in the pY-binding pocket is shown (red mesh). (c) Backbone traces comparing HA4 FG loop residues near the pY pocket with the typical position of a pY-peptide (PDB ID: 1LCJ).39 The side chains of Tyr87 and Trp80 are shown for HA4, and the pY and Y+3 residues of the pY-peptide. (d) Polar interactions (dashed lines) made by ‘finger’ residues (green sticks with black labels) near the pY pocket. SH2 residues are shown as white sticks and labeled in red. (e) Polar contacts made by ‘palm’ residues. Residues in the monobody scaffold are shown in cyan. Tyr35 is in yellow. Residues are labeled as in (d). (f) Changes in the binding free energy (ΔΔG_binding) for alanine mutants of HA4 residues. Asterisks indicate the lower limit of ΔΔG_binding for mutants for which no binding was detected.

(a) Amino-acid sequence alignment of Abl, Abl2 and Src family SH2 domains. Residues within 5Å of the HA4 interface are colored as follows: gray, residues where the consensus amino acid among the Src family members is identical to that of Abl; yellow, conservative substitutions; and red, non-conservative substitutions. Residues in the SH2 CD loop are in the blue box. Residues in the peptide-binding interface, inferred from the Lck structure39 are indicated with asterisks. (b) Cartoon model of the Lck SH2 domain structure with a bound peptide (1LCJ).39 The phosphopetide (sticks) lies across the central strand (βD). The pY-binding pocket and the Y+3 pocket are on either side of βD. (c) Conservation of HA4-interacting residues shown on the surface of the Lck SH2 domain. A phosphopeptide (sticks) highlights the overlap between phosphopeptide-binding and HA4-binding interfaces. (d) HA4/Abl complex (left) and hypothetical HA4/Lck complex (right, modeled by aligning Lck SH2 with Abl SH2 in the HA4/Abl SH2 complex), emphasizing SH2 CD-loop residues (blue). The two SH2 domains are viewed from an equivalent direction. HA4 is shown as a cartoon model, with the DE loop shown as orange sticks and gray mesh. The CD loop of the Lck SH2 domain that creates a protruding knob and is predicted to clash with the DE loop of HA4 is enclosed in the solid circle. The equivalent region in the Abl SH2 domain is marked with the dotted circle.

(a) Left, an overlay of the HA4/Abl SH2 complex (blue; only HA4 is shown for clarity) and autoinhibited Abl (1OPL)36 highlighting steric clash (boxed) between the HA4 FG loop (black mesh) and the Abl αI-αI′ helix (red mesh). The SH3, SH2 and kinase domains of Abl are colored in red, yellow and green, respectively. In the blow-up, SH2 is omitted for clarity. (b) Mutually exclusive interactions of two SH2 residues (Arg153 and Arg189) between the two structures. Residues in the SH2 and kinase domains of the autoinhibited Abl structure are shown with the carbon atoms in cyan and green, respectively. Residues in the SH2 domain and HA4 in the HA4/SH2 complex are shown with the carbon atoms in yellow and white, respectively. Polar interactions involving Arg153 and Arg189 of the SH2 domain are shown with dashed lines. SH2 residues are labeled in red. (c) Effects of HA4 on Abl activation. Left, the activity of the Abl kinase is plotted as a function of HA4 (solid black), phosphopeptide (red), and HA4_Y87A (open squares) concentrations. The activity was normalized relative to that in the absence of monobody or peptide. Right, the normalized activity of an autoinhibition-defective mutant of Abl kinase (G2APP: open boxes, dashed line) and the activity of the highly activated Bcr-Abl fusion protein (solid boxes, solid line) plotted as a function of HA4 concentration.

(a) Schematic of constructs used to monitor Abl-mediated processive phosphorylation of paxillin in HEK293 cells. The active Abl mutant, G2APP, was cotransfected with HA4 (or HA4_Y87A) and paxillin, which contains multiple phosphorylation sites. Paxillin and HA4/HA4_Y87A were expressed as HA-tag (gray oval) fusions to facilitate detection in Western analysis. (b) Western blots of lysates from HEK293 cells transfected with the indicated amounts of DNA. The putative positions of Abl kinase and paxillin are indicated by arrows. HA4-mediated disruption of paxillin processive phosphorylation is observed, with a more pronounced effect with lower levels of paxillin (c) Histograms showing the numbers of K562 cells transfected with a HA4-green fluorescent protein (GFP) fusion vector (line) or untreated K562 cells (gray area) as a function of GFP fluorescence intensity as measured by flow cytometry. The cells were binned in three classes, low, medium and high, based on GFP fluorescence as indicated. (d) Flow cytometric analysis of tyrosine phosphorylation of STAT5 in transfected K562 cells. Histograms depict signal intensities of cells stained with anti-phospho-Stat5 antibody. The fusion proteins used for transfection are indicated. The three panels show results of the three GFP-gated classes as shown in (c).