A potent and highly specific FN3 monobody inhibitor of the Abl SH2 domain

Interactions between SH2 domains and phosphotyrosine sites regulate tyrosine kinase signaling networks. Selective perturbation of these interactions is challenging due to the high homology among the 120 human SH2 domains. Using an improved phage-display selection system, we generated a small antibody-mimic or ‘monobody’, termed HA4, that bound to the Abl kinase SH2 domain with low nanomolar affinity. SH2 protein microarray analysis and mass spectrometry of intracellular HA4 interactors demonstrated HA4's exquisite specificity, and a crystal structure revealed how this specificity is achieved. HA4 disrupted intramolecular interactions of Abl involving the SH2 domain and potently activated the kinase in vitro. Within cells, HA4 inhibited processive phosphorylation activity of Abl and also STAT5 activation. This work provides a design guideline for highly specific and potent inhibitors of a protein interaction domain and demonstrates their utility in mechanistic and cellular investigations.