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Title: Selective Targeting of SH2 Domain–Phosphotyrosine Interactions of Src Family Tyrosine Kinases with Monobodies

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. 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 SH2more » 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.« less

Authors:
; ; ; ; ; ; ; ;
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
FOREIGN
OSTI Identifier:
1368269
Resource Type:
Journal Article
Journal Name:
Journal of Molecular Biology
Additional Journal Information:
Journal Volume: 429; Journal Issue: 9; Journal ID: ISSN 0022-2836
Publisher:
Elsevier
Country of Publication:
United States
Language:
ENGLISH
Subject:
60 APPLIED LIFE SCIENCES; 59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Kükenshöner, Tim, Schmit, Nadine Eliane, Bouda, Emilie, Sha, Fern, Pojer, Florence, Koide, Akiko, Seeliger, Markus, Koide, Shohei, and Hantschel, Oliver. Selective Targeting of SH2 Domain–Phosphotyrosine Interactions of Src Family Tyrosine Kinases with Monobodies. United States: N. p., 2017. Web. doi:10.1016/j.jmb.2017.03.023.
Kükenshöner, Tim, Schmit, Nadine Eliane, Bouda, Emilie, Sha, Fern, Pojer, Florence, Koide, Akiko, Seeliger, Markus, Koide, Shohei, & Hantschel, Oliver. Selective Targeting of SH2 Domain–Phosphotyrosine Interactions of Src Family Tyrosine Kinases with Monobodies. United States. https://doi.org/10.1016/j.jmb.2017.03.023
Kükenshöner, Tim, Schmit, Nadine Eliane, Bouda, Emilie, Sha, Fern, Pojer, Florence, Koide, Akiko, Seeliger, Markus, Koide, Shohei, and Hantschel, Oliver. Mon . "Selective Targeting of SH2 Domain–Phosphotyrosine Interactions of Src Family Tyrosine Kinases with Monobodies". United States. https://doi.org/10.1016/j.jmb.2017.03.023.
@article{osti_1368269,
title = {Selective Targeting of SH2 Domain–Phosphotyrosine Interactions of Src Family Tyrosine Kinases with Monobodies},
author = {Kükenshöner, Tim and Schmit, Nadine Eliane and Bouda, Emilie and Sha, Fern and Pojer, Florence and Koide, Akiko and Seeliger, Markus and Koide, Shohei and Hantschel, Oliver},
abstractNote = {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.},
doi = {10.1016/j.jmb.2017.03.023},
url = {https://www.osti.gov/biblio/1368269}, journal = {Journal of Molecular Biology},
issn = {0022-2836},
number = 9,
volume = 429,
place = {United States},
year = {2017},
month = {5}
}