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

Abstract

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.

Two Amino Acid Residues Confer Different Binding Affinities of Abelson Family Kinase Src Homology 2 Domains for Phosphorylated Cortactin

“…5, B, D, E, and F), confirming that these residues are important for cell edge protrusion in fibroblasts. We do not see a significant difference in the (22,(31)(32)(33)(34). The superposition was conducted using Topp (25).…”

“…3A, Table 1). This allowed us to directly compare the Arg SH2 domain to the Abl SH2 domain (22,(31)(32)(33)(34) and other previously determined type IA SH2 domain structures (35)(36)(37)(38)(39).…”

“…First, we compared the structure of the Arg SH2 domain with previously determined Abl crystal structures as well as an NMR structure of Arg SH2 domain (PDB codes 1OPK, 1OPL, 1AB2, 2ABL, 2ECD, 2FO0, and 3K2M) (22,(31)(32)(33)(34). The crystal structure of the Arg SH2 domain shows root mean square deviations for C␣ atoms with these structures ranging from 0.75 to 1.55 Å (Fig.…”

Allosteric Inhibition of Bcr-Abl Kinase by High Affinity Monobody Inhibitors Directed to the Src Homology 2 (SH2)-Kinase Interface

“…Although the tandem monobody construct was an effective allosteric Bcr-Abl inhibitor, it remained unclear whether targeting the SH2-kinase interface alone was sufficient for Bcr-Abl inhibition because the HA4 monobody in isolation also affected downstream signaling (13). HA4 inhibits the interaction of Abl SH2 with its phospho-Tyr-containing ligands and consequently processive phosphorylation of Bcr-Abl substrates (13).…”

“…We successfully generated a monobody termed "7c12" directed to the kinase-binding surface of the Abl SH2 domain (9). Because the 7c12 monobody had only moderate affinity and hence moderate biological effects in vitro and in cells, we needed to fuse it with another monobody, termed HA4, binding to a different surface of the SH2 domain, namely the binding site for phospho-Tyr-containing ligands (13). This tandem fusion approach enhanced the effective affinity so that the HA4 -7c12 fusion could successfully compete with the intramolecular interaction between the SH2 and kinase domains (Fig.…”