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Affinities of mAbs to Tet repressor complexed with operator or tetracycline suggest conformational changes associated
1Lehrstuhl für Mikrobiologie, Friedrich-Alexander Universität Erlangen-Nürnberg, Germany.
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.
Insights
Monoclonal antibodies targeting tetracycline repressor (TetR) reveal conformational changes in TetR when bound to tetracycline (Tc) or tet operator (tetO). These antibody binding differences highlight TetR
Area of Science:
- Molecular Biology
- Immunology
- Biochemistry
Background:
- The tetracycline repressor (TetR) protein regulates gene expression by binding to tetracycline (Tc) and the tet operator (tetO) DNA sequence.
- Understanding TetR's conformational changes upon ligand binding is crucial for its applications in gene regulation systems.
Purpose of the Study:
- To characterize the epitopes of monoclonal antibodies (mAbs) against TetR in different functional states (free TetR, TetR-Tc complex, TetR-tetO complex).
- To investigate conformational differences of TetR induced by tetracycline and tet operator binding using antibody-based assays.
Main Methods:
- Isolation of eight monoclonal antibodies against TetR, TetR-Tc, and TetR-tetO complexes.
- Epitope mapping of anti-TetR mAbs to the alpha-helix-turn-alpha-helix (HTH) motif, Tc binding pocket vicinity, and dimerization interface.
- Quantitative immunoprecipitation and competitive ELISA to assess mAb affinities for TetR in different functional states.
Main Results:
- Antibodies targeting the HTH motif showed similar binding to TetR and TetR-Tc, suggesting conserved conformation.
- An epitope at the dimerization interface was more strongly bound by mAbs in free TetR than in TetR-Tc, indicating conformational changes.
- Significant affinity variations were observed for epitopes near the Tc binding pocket, with distinct binding patterns for free TetR, TetR-tetO, and TetR-Tc.
Conclusions:
- Monoclonal antibodies can effectively probe conformational states of TetR.
- TetR undergoes distinct conformational changes upon binding to tetracycline and the tet operator, affecting antibody binding sites.
- These findings provide insights into TetR's allosteric regulation and have implications for designing TetR-based genetic tools.