Substrate Interaction with the Fes Kinase Domain
(A) Details of the substrate peptide (IYESL) interaction with the kinase domain.
(B) Structure of the Fes-substrate complex showing a detail of the location of the peptide (shown in sticks). The surface has been colored by electrostatic potential between −10 and +10 kcal/mol.
(C) 2F_o – F_c electron density map contoured at 2σ around the substrate peptide residues.

Cartoon Representation of Fes Activation
In its unligated and unphosphorylated state, the Fes SH2 domain (blue), αC (red), and activation segment (purple) are significantly disordered (left). Binding of a primed peptide (yellow) stabilizes the SH2 domain, leading to a productive orientation of the SH2 domain, with respect to the kinase domain, and stable positioning of αC (middle). Phosphorylation of the activation segment at Y713 and binding of the substrate molecule to the kinase domain stabilizes the activation segment in a conformation suitable for catalysis (right).

Structural Overview of Human Fes
(A) Domain architecture. The locations of the F-BAR, SH2, and kinase domains are indicated. The F-BAR domain contains the FCH and coiled-coil (CC) sequences. The crystallized construct is highlighted by arrows.
(B) Ribbon diagram representing the overall structure of the SH2-kinase domain unit. The main secondary structure elements are labeled.
(C) Surface representation of active Fes. The surface is colored by electrostatic potential between −10 and +10 kcal/mol.

Stabilization of the Fes SH2 Domain by Ligand Binding
(A) Coordination of a sulphate ion in the SH2 phosphotyrosine-binding site in active Fes and 2F_o – F_c electron density map contoured at 2σ around residues that coordinate the sulfate ion.
(B) Superimposition of active Fes (green) and Fes phosphorylated at the activation segment Y713 with an unligated SH2 domain (gray). Structural changes associated with ligand binding to the SH2 domain are indicated by arrows.
(C) Effect of the R/M mutation in the Fes SH2 domain measured by NMR. Overlay of ¹H-¹⁵N HSQC spectra of wild-type (black) and mutant R/M (green) isolated Fes SH2 domain, with regions of the spectra relating to residues located in the SH2-kinase domain interface indicated in the right two panels (R483^∗: side chain resonance). Residues that showed significant chemical shift changes in the spectra of the R/M mutant SH2 domain, as compared to wild-type, are depicted in green on the Fes SH2 domain surface in the left panel. Blue indicates the position of R483. Helix αC and the loop between strands β4 and β5 of the kinase domain are shown in orange.

Conformation of the Fes Activation Segment
(A) Superimposition of the activation segment in active Fes (gray) and the phosphorylated Fes-substrate complex. Hydrophilic interactions formed by the phosphate moiety and the substrate tyrosine are indicated by yellow dots. The antiparallel β sheet formed at the tip of the activation segment is labeled as β-A-loop, and the substrate peptide is shown as a red ribbon. The induced β sheet present in the substrate complex (βI) is also shown.
(B) Interactions of the activation segment Y713 in unphosphorylated (left), active (ligating a sulphate ion; middle), and phosphorylated (right) Fes. A 2F_o – F_c electron density map around the interacting residues is also shown contoured at 2σ.
(C) Schematic drawing of the Fes main chain. Regions with high temperature factors are shown by an increasing radius of the backbone. The activation segment is highlighted in purple; αC, in pink; the SH2 domain, in green; and the substrate peptide (in the phosphorylated Fes structure), in orange. Note the absence of ordered loop regions in the SH2 domain and kinase N lobe of phosphorylated Fes.

Activation of Fes Kinase Activity by SH2 Domain Interactions
(A) The effect of mutations in the SH2 domain on Fes kinase activity: HEK293T cells were cotransfected with Flag-tagged Fes and GST-cortactin. Cell lysates were immunoprecipitated with anti-Flag and blotted with anti-pTyr to reveal autophosphorylated (p) Flag-Fes (top panel) or with anti-Flag (lower panel) as a control. WT indicates wild-type Fes, and mutants are described in the text.
(B) GST-cortactin was affinity purified from cells coexpressing WT or mutant Fes proteins and blotted either with anti-pTyr antibodies to measure Fes-induced phosphorylation (p) (top panel) or with anti-GST (bottom panel).
(C) Comparison of the N-terminal SH2 domain sequence of Fps/Fes orthologs. Insertions originally used to identify the SH2 domain in v-Fps are indicated (RX15m and AX9m) (Sadowski et al., 1986; Stone et al., 1984). Conserved residues are highlighted in red and similar residues in yellow.
(D) Details of the Fes SH2-kinase interface. Residues important for the SH2 domain-kinase interaction are conserved in Fps/Fes family members but not in other tyrosine kinases. Residues mutated in this study are indicated by a green asterisk. Interactions of interface residues and the involved secondary structure elements are shown in the structure of active Fes. The alignment of human sequences is colored as in (C).

The Abl SH2 Domain Stimulates Kinase Activity
(A) Ribbon representation of the active conformation of Abl (PDB entry 1OPL chain B) and close-up view of SH2-kinase domain interface. Critical interface residues (I164, T291, and Y331) are shown. R171 highlights the location of the pTyr-binding site in the SH2 domain.
(B) SH2-kinase interface residues are required for in vivo Abl activity. HEK293 cells were transfected with c-Abl (WT) or the activated Abl variants PP (^∗) or G2A/PP (^∗∗) or with the corresponding ITY/EEA mutants, as indicated. Cells were lysed, and anti-Abl immunoprecipitates were analyzed by anti-Abl and anti-pTyr (pY) immunoblotting (lower panels). The histograph shows the in vitro kinase activity (mean of two experiments done in duplicate, black bars) and levels of autophosphorylation (mean of two immunoprecipitations, gray bars) of the Abl constructs relative to c-Abl and corrected for endogenous c-Abl levels.
(C) I164 in the Abl SH2 domain is required for efficient kinase activity (WT = c-Abl, ^∗ = Abl-PP; corresponding mutants are indicated).
(D) The SH2 domain is necessary and sufficient to stimulate in vitro Abl kinase activity. Purified Abl proteins (see also Figures S9D and S9E) were evaluated for kinase activity in the presence of 50 μM ATP and the indicated concentrations of an optimal Abl substrate peptide. The K_m and v_max values are given below the graph.