Src homology 2 domains (SH2) are small modular protein–protein interaction domains found in humans in many signaling proteins such as kinases, phosphatases etc. The largest class of SH2 domain-containing proteins are the Src family kinases (SFKs) where the SH2 domain of SFKs has a dual role in regulating kinase activity and signaling and represent important therapeutic targets in solid tumors and hematological malignancies. To accelerate the studies of biologically important aspects of SFK SH2 domains in normal signaling and target them in aberrant oncogenic signaling, NCCR researchers from Oliver Hantschel’s lab at the EPFL  developed high-performance synthetic binding proteins, termed monobodies, that proved to be effectively and specifically targeted to the SH2 domains of SFKs. These monobodies can now be used as excellent high-precision tools to dissect SFK signaling in normal development and signaling and to interfere with aberrant SFK signaling network in cancer cells and in vivo. The work is published on-line in the Journal of Molecular Biology.

Abstract

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. The group of Oliver Hanstchel, affiliated to the NCCR Chemical Biology at the EPFL, 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. Their 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.

Article

Article  in the Journal of Molecular Biology: http://doi.org/10.1016/j.jmb.2017.03.023

Tim Kükenshöner, Nadine Eliane Schmit, Emilie Bouda, Fern Sha, Florence Pojer, Akiko Koide, Markus Seeliger, Shohei Koide, Oliver Hantschel, “Selective Targeting of SH2 Domain–Phosphotyrosine Interactions of Src Family Tyrosine Kinases with Monobodies“, Journal of Molecular Biology, available on line since March 25, 2017.