title: Large-Scale Screening of Preferred Interactions of Human Src Homology-3 (SH3) Domains Using Native Target Proteins as Affinity Ligands.
abstract: The Src Homology-3 (SH3) domains are ubiquitous protein modules that mediate important intracellular protein interactions via binding to short proline-rich consensus motifs in their target proteins. The affinity and specificity of such core SH3 - ligand contacts are typically modest, but additional binding interfaces can give rise to stronger and more specific SH3-mediated interactions. To understand how commonly such robust SH3 interactions occur in the human protein interactome, and to identify these in an unbiased manner we have expressed 324 predicted human SH3 ligands as full-length proteins in mammalian cells, and screened for their preferred SH3 partners using a phage display-based approach. This discovery platform contains an essentially complete repertoire of the ∼300 human SH3 domains, and involves an inherent binding threshold that ensures selective identification of only SH3 interactions with relatively high affinity. Such strong and selective SH3 partners could be identified for only 19 of these 324 predicted ligand proteins, suggesting that the majority of human SH3 interactions are relatively weak, and thereby have capacity for only modest inherent selectivity. The panel of exceptionally robust SH3 interactions identified here provides a rich source of leads and hypotheses for further studies. However, a truly comprehensive characterization of the human SH3 interactome will require novel high-throughput methods based on function instead of absolute binding affinity.
authors.affiliation: From the ‡Department of Virology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland.
authors.forename: Arunas
authors.initials: A
authors.lastname: Kazlauskas
authors.affiliation: From the ‡Department of Virology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland.
authors.forename: Constanze
authors.initials: C
authors.lastname: Schmotz
authors.affiliation: From the ‡Department of Virology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland.
authors.forename: Tapio
authors.initials: T
authors.lastname: Kesti
authors.affiliation: From the ‡Department of Virology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland.
authors.forename: Jussi
authors.initials: J
authors.lastname: Hepojoki
authors.affiliation: From the ‡Department of Virology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland.
authors.forename: Iivari
authors.initials: I
authors.lastname: Kleino
authors.affiliation: §Department of Biochemistry and the Siebens-Drake Research Institute, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario N6A 5C1, Canada.
authors.forename: Tomonori
authors.initials: T
authors.lastname: Kaneko
authors.affiliation: §Department of Biochemistry and the Siebens-Drake Research Institute, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario N6A 5C1, Canada.
authors.forename: Shawn S C
authors.initials: SS
authors.lastname: Li
authors.affiliation: From the ‡Department of Virology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland; kalle.saksela@helsinki.fi.
authors.forename: Kalle
authors.initials: K
authors.lastname: Saksela
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D006801:Humans; D008024:Ligands; D019151:Peptide Library; D011485:Protein Binding; D060066:Protein Interaction Maps; D020543:Proteome / Q000032:analysis* / Q000737:chemistry; D018909:src Homology Domains; D019061:src-Family Kinases / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D008024:Ligands; D019151:Peptide Library; D020543:Proteome; D019061:src-Family Kinases