title: A protein-protein interaction assay based on the functional complementation of mutant firefly luciferases.
abstract: We report a novel bioluminescent protein-protein interaction (PPI) assay, which is based on the functional complementation of two mutant firefly luciferases (Fluc). The chemical reaction catalyzed by Fluc is divided into two half reactions of ATP-driven luciferin adenylation and subsequent oxidative reactions. In the former adenylation half-reaction, a luciferyl-adenylate (LH2-AMP) intermediate is produced from LH2 and ATP. With this intermediate, the latter oxidative reactions produce oxyluciferin via proton abstraction at the C4 carbon of LH2-AMP. We created and optimized two Fluc mutants; one is named "Donor", which virtually lacks oxidative activity, while the other, named "Acceptor", is almost defective in the adenylation activity. Then, the two mutants are fused to interacting partners, and prepared as pure proteins. When the interaction between the partners is induced, higher efficiency of LH2-AMP transfer between the Donor and Acceptor enzymes resulted in increased luminescence. The assay was found to work both in vitro and in cultured cells with strong signals. This would be the first example of reconstituting two divided reactions of one enzyme to detect PPI, which will not only be utilized as a robust PPI assay, but also open a way to control the activity of similar enzymes in acyl/adenylate-forming enzyme superfamily.
authors.affiliation: Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Ohmuro-Matsuyama
authors.forename: Kota
authors.initials: K
authors.lastname: Nakano
authors.forename: Aoi
authors.initials: A
authors.lastname: Kimura
authors.forename: Keiichi
authors.initials: K
authors.lastname: Ayabe
authors.forename: Masaki
authors.initials: M
authors.lastname: Ihara
authors.forename: Takeshi
authors.initials: T
authors.lastname: Wada
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Ueda
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D001517:Coleoptera / Q000201:enzymology*; D017931:DNA Primers; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism*; D016296:Mutagenesis; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D011506:Proteins; D008156:Luciferases