The binding of Src-homology 2 (SH2) domains to phosphotyrosine (pY) sites is critical for the autoinhibition and substrate recognition of the eight Src family kinases (SFKs). The high sequence conservation of the 120 human SH2 domains poses a significant challenge to selectively perturb the interactions of even the SFK SH2 family against the rest of the SH2 domains. We have developed synthetic binding proteins, termed monobodies, for six of the SFK SH2 domains with nanomolar affinity. Most of these monobodies competed with pY ligand binding and showed strong selectivity for either the SrcA (Yes, Src, Fyn, Fgr) or SrcB subgroup (Lck, Lyn, Blk, Hck). Interactome analysis of intracellularly expressed monobodies revealed that they bind SFKs but no other SH2-containing proteins. Three crystal structures of monobody–SH2 complexes unveiled different and only partly overlapping binding modes, which rationalized the observed selectivity and enabled structure-based mutagenesis to modulate inhibition mode and selectivity. In line with the critical roles of SFK SH2 domains in kinase autoinhibition and T-cell receptor signaling, monobodies binding the Src and Hck SH2 domains selectively activated respective recombinant kinases, whereas an Lck SH2-binding monobody inhibited proximal signaling events downstream of the T-cell receptor complex. Our results show that SFK SH2 domains can be targeted with unprecedented potency and selectivity using monobodies. They are excellent tools for dissecting SFK functions in normal development and signaling and to interfere with aberrant SFK signaling networks in cancer cells.

The two isoforms of the Bcr-Abl tyrosine kinase, p210 and p190, are associated with different leukemias and have a dramatically different signaling network, despite similar kinase activity. To provide a molecular rationale for these observations, we study the Dbl-homology (DH) and Pleckstrin-homology (PH) domains of Bcr-Abl p210, which constitute the only structural differences to p190. Here we report high-resolution structures of the DH and PH domains and characterize conformations of the DH–PH unit in solution. Our structural and functional analyses show no evidence that the DH domain acts as a guanine nucleotide exchange factor, whereas the PH domain binds to various phosphatidylinositol-phosphates. PH-domain mutants alter subcellular localization and result in decreased interactions with p210-selective interaction partners. Hence, the PH domain, but not the DH domain, plays an important role in the formation of the differential p210 and p190 Bcr-Abl signaling networks.

The transcription factor STAT3 is frequently activated in human solid and hematological malignancies and remains a challenging therapeutic target with no approved drugs to date. Here, we develop synthetic antibody mimetics, termed monobodies, to interfere with STAT3 signaling. These monobodies are highly selective for STAT3 and bind with nanomolar affinity to the N-terminal and coiled-coil domains. Interactome analysis detects no significant binding to other STATs or additional off-target proteins, confirming their exquisite specificity. Intracellular expression of monobodies fused to VHL, an E3 ubiquitin ligase substrate receptor, results in degradation of endogenous STAT3. The crystal structure of STAT3 in complex with monobody MS3-6 reveals bending of the coiled-coil domain, resulting in diminished DNA binding and nuclear translocation. MS3-6 expression strongly inhibits STAT3-dependent transcriptional activation and disrupts STAT3 interaction with the IL-22 receptor. Therefore, our study establishes innovative tools to interfere with STAT3 signaling by different molecular mechanisms.

Bruton’s tyrosine kinase (Btk) is critical for B-cell maturation and activation. Btk loss-of-function mutations cause human X-linked agammaglobulinemia (XLA). In contrast, Btk signaling sustains growth of several B-cell neoplasms which may be treated with tyrosine kinase inhibitors (TKIs). Here, we uncovered the structural mechanism by which certain XLA mutations in the SH2 domain strongly perturb Btk activation. Using a combination of molecular dynamics (MD) simulations and small-angle X-ray scattering (SAXS), we discovered an allosteric interface between the SH2 and kinase domain required for Btk activation and to which multiple XLA mutations map. As allosteric interactions provide unique targeting opportunities, we developed an engineered repebody protein binding to the SH2 domain and able to disrupt the SH2-kinase interaction. The repebody prevents activation of wild-type and TKI-resistant Btk, inhibiting Btk-dependent signaling and proliferation of malignant B-cells. Therefore, the SH2-kinase interface is critical for Btk activation and a targetable site for allosteric inhibition.

Exclusive of membrane-proximal mutations seen commonly in chronic neutrophilic leukemia (e.g., T618I), functionally defective mutations in the extracellular domain of the G-CSF receptor (CSF3R) have been reported only in severe congenital and idiopathic neutropenia patients. Here, we describe the first activating mutation in the fibronectin-like type III domain of the extracellular region of CSF3R (W341C) in a leukemia patient. This mutation transformed cells via cysteine-mediated intermolecular disulfide bonds, leading to receptor dimerization. Interestingly, a CSF3R cytoplasmic truncation mutation (W791X) found on the same allele as the extracellular mutation and the expansion of the compound mutation was associated with increased leukocytosis and disease progression of the patient. Notably, the primary patient sample and cells transformed by W341C and W341C/W791X exhibited sensitivity to JAK inhibitors. We further showed that disruption of original cysteine pairs in the CSF3R extracellular domain resulted in either gain- or loss-of-function changes, part of which was attributable to cysteine-mediated dimer formation. This, therefore, represents the first characterization of unpaired cysteines that mediate both gain- and loss-of-function phenotypes. Overall, our results show the structural and functional importance of conserved extracellular cysteine pairs in CSF3R and suggest the necessity for broader screening of CSF3R extracellular domain in leukemia patients.