Selective Targeting of SH2 Domain-Phosphotyrosine Interactions of Src Family Tyrosine Kinases with Monobodies

JOURNAL OF MOLECULAR BIOLOGY

卷:429

Article

Kuekenshoener, Tim¹  Schmit, Nadine Eliane¹  Bouda, Emilie²  Sha, Fern³  Pojer, Florence⁴  Koide, Akiko^3,5,6,7  Seeliger, Markus²  Koide, Shohei^3,5,6,7  Hantschel, Oliver¹ 

[1] Ecole Polytech Fed Lausanne, Sch Life Sci, Swiss Inst Expt Canc Res ISREC, Stn 19, CH-1015 Lausanne, Switzerland

[2] SUNY Stony Brook, Dept Pharmacol Sci, BST 8-140, Stony Brook, NY 11794 USA

[3] Univ Chicago, Dept Biochem & Mol Biol, 929 East 57th St, Chicago, IL 60637 USA

[4] Ecole Polytech Fed Lausanne, Sch Life Sci, Prot Crystallog Core Facil, Stn 19, CH-1015 Lausanne, Switzerland

[5] NYU, Langone Med Ctr, Laura & Isaac Perlmutter Canc Ctr, 430 East 29th St, New York, NY 10016 USA

[6] NYU, Sch Med, Dept Med, 430 East 29th St, New York, NY 10016 USA

[7] NYU, Sch Med, Dept Biochem & Mol Pharmacol, 430 East 29th St, New York, NY 10016 USA

关键词: protein engineering;    signaling;    protein-protein interactions;    oncogene;    crystal structure;   

DOI  :  10.1016/j.jmb.2017.03.023

来源: Elsevier

【 摘 要 】

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. (C) 2017 The Authors. Published by Elsevier Ltd.

【 授权许可】

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