Structural basis for the transcriptional repressor NicR2 in nicotine degradation from Pseudomonas.

ABSTRACT

Nicotine is an environmental toxicant in tobacco wastes, imposing severe hazards for the health of human and other mammalians. NicR2, a TetR-like repressor from Pseudomonas putida S16, plays a critical role in regulating nicotine degradation. Here, we determined the crystal structures of NicR2 and its complex with the inducer 6-hydroxy-3-succinoyl-pyridine (HSP). The N-terminal domain of NicR2 contains a conserved helix-turn-helix (HTH) DNA-binding motif, while the C-terminal domain contains a cleft for its selective recognition for HSP. Residues R91, Y114 and Q118 of NicR2 form hydrogen bonds with HSP, their indispensable roles in NicR2's recognition with HSP were confirmed by structure-based mutagenesis combined with isothermal titration calorimetry analysis. Based on sequence alignment and structure comparison, Tyr67, Tyr68 and Lys72 of HTH motif were corroborated to take the major responsibility for DNA-binding using site-directed mutants. The 30-residue N-terminal extension of NicR2, especially residues 21-30 in the TFR arm, is required for the association with the operator DNA. Finally, we proposed that either NicR2 or the DNA would undergo a conformational change upon their association. Altogether, our structural and biochemical investigations unravel how NicR2 selectively recognizes HSP and DNA, and provide new insights into the TetR family of repressors.