title: Affinities of mAbs to Tet repressor complexed with operator or tetracycline suggest conformational changes associated with induction.
abstract: We isolated five monoclonal antibodies (mAbs) made against tetracycline repressor (TetR), one against the TetR tetracycline complex (Tc) and two against the TetR-tet operator (tetO) complex. The epitopes of the anti-TetR mAbs are localized in the alpha-helix-turn-alpha-helix motif (HTH), at different sites near the Tc binding pocket and at the dimerization interface. The anti-TetR-Tc and one of the anti-TetR-tetO mAbs recognize epitopes near the Tc binding pocket. The other anti-TetR-tetO mAb binds to an epitope within the HTH. Quantitative immunoprecipitation and competitive ELISA employing TetR, TetR-Tc, or TetR-tetO revealed different affinities of the mAbs for TetR in these functional states. Binding of the two mAbs to epitopes in the HTH was identical for TetR and TetR-Tc indicating the same conformation in both forms. The epitope located in the dimerization interface is bound more strongly in TetR compared to TetR-Tc, supporting the idea of different conformations of that epitope in these forms of TetR. The greatest affinity differences were found for epitopes around the Tc binding pocket. Two anti-TetR mAbs have the highest affinities for free TetR, somewhat reduced affinity for TetR-tetO and the lowest affinities for TetR-Tc. The anti-TetR-Tc mAb has a discontinuous epitope, formed in TetR-Tc, which is less well bound in TetR and not bound in the TetR-tetO complex. One anti-TetR-tetO mAb does not recognize TetR-Tc. Since the epitopes do not overlap with the respective ligand binding sites on TetR, these results are interpreted as conformational differences of the epitopes in these forms of TetR.
authors.affiliation: Lehrstuhl für Mikrobiologie, Friedrich-Alexander Universität Erlangen-Nürnberg, Germany.
authors.forename: E
authors.initials: E
authors.lastname: Pook
authors.forename: S
authors.initials: S
authors.lastname: Grimm
authors.forename: A
authors.initials: A
authors.lastname: Bonin
authors.forename: T
authors.initials: T
authors.lastname: Winkler
authors.forename: W
authors.initials: W
authors.lastname: Hillen
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D000915:Antibody Affinity; D001426:Bacterial Proteins / Q000737:chemistry / Q000276:immunology*; D001666:Binding Sites, Antibody; D002352:Carrier Proteins; D018604:Epitope Mapping; D046911:Macromolecular Substances; D051379:Mice; D008958:Models, Molecular; D009875:Operator Regions, Genetic / Q000276:immunology; D011487:Protein Conformation; D012097:Repressor Proteins / Q000737:chemistry / Q000276:immunology*; D013752:Tetracycline / Q000737:chemistry / Q000276:immunology*