title: Caught in the act: covalent cross-linking captures activator-coactivator interactions in vivo.
abstract: Currently there are few methods suitable for the discovery and characterization of transient, moderate affinity protein-protein interactions in their native environment, despite their prominent role in a host of cellular functions including protein folding, signal transduction, and transcriptional activation. Here we demonstrate that a genetically encoded photoactivatable amino acid, p-benzoyl-l-phenylalanine, can be used to capture transient and/or low affinity binding partners in an in vivo setting. In this study, we focused on ensnaring the coactivator binding partners of the transcriptional activator VP16 in S. cerevisiae. The interactions between transcriptional activators and coactivators in eukaryotes are moderate in affinity and short-lived, and due in part to these characteristics, identification of the direct binding partners of activators in vivo has met with only limited success. We find through in vivo photo-cross-linking that VP16 contacts the Swi/Snf chromatin-remodeling complex through the ATPase Snf2(BRG1/BRM) and the subunit Snf5 with two distinct regions of the activation domain. An analogous experiment with Gal4 reveals that Snf2 is also a target of this activator. These results suggest that Snf2 may be a valuable target for small molecule probe discovery given the prominent role the Swi/Snf complex family plays in development and in disease. More significantly, the successful implementation of the in vivo cross-linking methodology in this setting demonstrates that it can be applied to the discovery and characterization of a broad range of transient and/or modest affinity protein-protein interactions.
authors: Malathy
authors: M
authors: Krishnamurthy
authors: Amanda
authors: A
authors: Dugan
authors: Adaora
authors: A
authors: Nwokoye
authors: Yik-Hong
authors: YH
authors: Fung
authors: Jody K
authors: JK
authors: Lancia
authors: Chinmay Y
authors: CY
authors: Majmudar
authors: Anna K
authors: AK
authors: Mapp
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D001577:Benzophenones / Q000737:chemistry; D003432:Cross-Linking Reagents / Q000737:chemistry; D017405:Herpes Simplex Virus Protein Vmw65 / Q000378:metabolism*; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000737:chemistry; D060066:Protein Interaction Maps; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D015533:Transcriptional Activation
chemical_list: C488060:4-benzoylphenylalanine; D001577:Benzophenones; D003432:Cross-Linking Reagents; D017405:Herpes Simplex Virus Protein Vmw65; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D010649:Phenylalanine; D000251:Adenosine Triphosphatases; C499333:SNF2 protein, S cerevisiae