Bacterial transcriptional repressor NrdR - a flexible multifactorial nucleotide sensor.

Area of Science:

• Structural biology
• Bacteriology
• Molecular mechanisms

Background:

• NrdR is a bacterial transcriptional repressor regulating ribonucleotide reductase operons.
• Understanding NrdR's mechanism is crucial for developing novel antibacterial agents.
• Escherichia coli possesses three NrdR-regulated operons: nrdHIEF, nrdDG, and nrdAB.

Purpose of the Study:

• To elucidate the structural basis of NrdR's DNA binding and transcriptional repression.
• To investigate the role of nucleotides in NrdR's DNA binding affinity and specificity.
• To determine high-resolution structures of NrdR in various nucleotide-bound and DNA-bound states.

Main Methods:

• X-ray crystallography to determine the structures of NrdR-nucleotide complexes.
• Cryo-electron microscopy (cryo-EM) to visualize DNA-bound NrdR and NrdR filaments.
• Biochemical assays to assess NrdR binding affinities to different DNA sites and nucleotide combinations.

Main Results:

• First high-resolution crystal structures of NrdR-ATP-dATP and NrdR-ADP-dATP complexes were obtained.
• Cryo-EM revealed structures of DNA-bound NrdR and novel NrdR filaments.
• NrdR binding strength is similar across three E. coli operons when bound to ATP/dATP or diphosphate equivalents; other adenine nucleotides do not promote DNA binding.
• Structural analysis revealed flexibility in NrdR's Zn-ribbon and ATP-cone domains, with significant conformational changes upon DNA binding.
• ATP-bound NrdR filaments sequester DNA-binding residues, preventing DNA interaction.

Conclusions:

• NrdR exhibits significant structural flexibility, adapting its conformation for optimal DNA binding upon interaction with specific nucleotides.
• The nucleotide-dependent conformational changes are critical for NrdR's function as a transcriptional repressor.
• These findings provide a structural foundation for designing NrdR-targeted antibacterials.