Two-way interdomain signal transduction in tetracycline repressor.
The transcription of genes encoding resistance to the antibiotic, tetracycline (Tc), is repressed by tetracycline repressor (TetR), which is a homodimeric alpha-helical protein possessing a small N-terminal DNA binding domain (DNB domain) and a larger C-terminal domain (TBD domain). Binding of Tc to the TBD domain induces a subtle conformational change in the DNB domain that leads to abrogation of its DNA-binding activity, and induction of Tc resistance. While most previous studies on TetR have focused on the effects of Tc-binding on the DNB domain conformation, here we have investigated the role of the DNB domain in modulating Tc binding. We have discovered that a TBD domain construct entirely lacking the DNB domain displays a drastic reduction in Tc-binding affinity even though the DNB domain is far from the Tc-binding site. In the context of full-length TetR, highly destabilizing amino acid substitutions in the DNB domain cause reductions in Tc-binding activity. Strikingly, the DNB domains of these mutants, which are completely unfolded in the absence of Tc, are induced to fold when Tc is bound. These results demonstrate that there is a previously unrecognized two-way interdomain signaling mechanism in TetR whereby the DNB domain is required for maximal Tc-binding by the TBD domain, and Tc-binding in the TBD domain leads to stabilization of the DNB domain. Furthermore, our work suggests that detailed thermodynamic and kinetic studies on mutant forms of other allosteric proteins may also reveal surprising and previously undetected modes of interdomain communication.
Reichheld
Sean E
SE
Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada M5S 1A8.
Davidson
Alan R
AR
D019943:Amino Acid Substitution; D015964:Gene Expression Regulation, Bacterial; D007700:Kinetics; D008958:Models, Molecular; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013753:Tetracycline Resistance / Q000502:physiology*; D013754:Tetracyclines / Q000494:pharmacology*; D013816:Thermodynamics
D012097:Repressor Proteins; D013754:Tetracyclines; C034811:tetracycline resistance-encoding transposon repressor protein