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 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.
authors.affiliation: Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, École polytechnique fédérale de Lausanne (EPFL), Station 19, 1015 Lausanne, Switzerland.
authors.forename: Tim
authors.initials: T
authors.lastname: Kükenshöner
authors.affiliation: Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, École polytechnique fédérale de Lausanne (EPFL), Station 19, 1015 Lausanne, Switzerland.
authors.forename: Nadine Eliane
authors.initials: NE
authors.lastname: Schmit
authors.affiliation: Department of Pharmacological Sciences, Stony Brook University, BST 8-140, Stony Brook, NY 11794-8651, USA.
authors.forename: Emilie
authors.initials: E
authors.lastname: Bouda
authors.affiliation: Department of Biochemistry and Molecular Biology, The University of Chicago, 929 East 57th Street, Chicago, IL 60637, USA.
authors.forename: Fern
authors.initials: F
authors.lastname: Sha
authors.affiliation: Protein Crystallography Core Facility, School of Life Sciences, École polytechnique fédérale de Lausanne, Station 19, 1015 Lausanne, Switzerland.
authors.forename: Florence
authors.initials: F
authors.lastname: Pojer
authors.affiliation: Department of Biochemistry and Molecular Biology, The University of Chicago, 929 East 57th Street, Chicago, IL 60637, USA; Laura and Isaac Perlmutter Cancer Center, New York University Langone Medical Center, 430 East 29th Street, New York, NY 10016, USA; Department of Medicine, New York University School of Medicine, 430 East 29th Street, New York, NY 10016, USA; Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, 430 East 29th Street, New York, NY 10016, USA.
authors.forename: Akiko
authors.initials: A
authors.lastname: Koide
authors.affiliation: Department of Pharmacological Sciences, Stony Brook University, BST 8-140, Stony Brook, NY 11794-8651, USA.
authors.forename: Markus
authors.initials: M
authors.lastname: Seeliger
authors.affiliation: Department of Biochemistry and Molecular Biology, The University of Chicago, 929 East 57th Street, Chicago, IL 60637, USA; Laura and Isaac Perlmutter Cancer Center, New York University Langone Medical Center, 430 East 29th Street, New York, NY 10016, USA; Department of Medicine, New York University School of Medicine, 430 East 29th Street, New York, NY 10016, USA; Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, 430 East 29th Street, New York, NY 10016, USA. Electronic address: Shohei.Koide@nyumc.org.
authors.forename: Shohei
authors.initials: S
authors.lastname: Koide
authors.affiliation: Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, École polytechnique fédérale de Lausanne (EPFL), Station 19, 1015 Lausanne, Switzerland. Electronic address: oliver.hantschel@epfl.ch.
authors.forename: Oliver
authors.initials: O
authors.lastname: Hantschel
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology*; D018360:Crystallography, X-Ray; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D019000:Phosphotyrosine / Q000276:immunology*; D011485:Protein Binding; D011487:Protein Conformation; D018909:src Homology Domains / Q000276:immunology*; D019061:src-Family Kinases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D019000:Phosphotyrosine; D019061:src-Family Kinases
keywords: crystal structure; oncogene; protein engineering; protein–protein interactions; signaling