Affinities of mAbs to Tet repressor complexed with operator or tetracycline suggest conformational changes associated with induction.
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.
Pook
E
E
Lehrstuhl für Mikrobiologie, Friedrich-Alexander Universität Erlangen-Nürnberg, Germany.
Grimm
S
S
Bonin
A
A
Winkler
T
T
Hillen
W
W
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*