Abstract
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.
Highlights
Bruton’s tyrosine kinase (Btk) is critical for B-cell maturation and activation
We show that, based on the analysis of X-linked agammaglobulinemia (XLA) mutations in the SH2 domain, Btk activation critically depends on the formation of an allosteric interface between its SH2 and the N-lobe of kinase domain, which we mapped using a combination of enhanced sampling molecular dynamics (MD) simulations and small-angle X-ray scattering (SAXS)
Sequencing data indicate that approximately 20% of missense mutations in XLA patients are located within the Btk SH2 domain, but how these mutations result in Btk loss-offunction is poorly understood[23]
Summary
Bruton’s tyrosine kinase (Btk) is critical for B-cell maturation and activation. Btk loss-offunction mutations cause human X-linked agammaglobulinemia (XLA). 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. The SH2-kinase interface is critical for Btk activation and a targetable site for allosteric inhibition. We show that, based on the analysis of XLA mutations in the SH2 domain, Btk activation critically depends on the formation of an allosteric interface between its SH2 and the N-lobe of kinase domain, which we mapped using a combination of enhanced sampling molecular dynamics (MD) simulations and SAXS. Development of a high-affinity engineered protein antagonist to the Btk SH2 domain targeting its interface with the kinase domain prevents Btk activation in cells, inhibits proliferation and Btk-dependent signaling in malignant B-cells. We demonstrate the Btk SH2 domain as alternative allosteric site for therapeutic inhibition of Btk and its most common drug-resistant mutant
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