Sjoberg, B.-M. | Search bioRxiv

bioRxiv2024

Bacterial transcriptional repressor NrdR - a flexiblemultifactorial nucleotide sensor

NrdR is a bacterial transcriptional repressor consisting of a Zn-ribbon domain followed by an ATP-cone domain. Understanding its mechanism of action could aid the design of novel antibacterials. NrdR binds specifically to two "NrdR boxes" upstream of ribonucleotide reductase operons, of which Escherichia coli has three: nrdHIEF, nrdDG and nrdAB, where we identified a new box. We show that E. coli NrdR (EcoNrdR) has similar binding strength to all three sites when loaded with ATP plus dATP or equivalent diphosphate combinations. No other combination of nucleotides promotes binding to DNA. We present crystal structures of EcoNrdR-ATP-dATP and EcoNrdR-ADP-dATP, which are the first high-resolution crystal structures of an NrdR. We have also determined cryo-EM structures of DNA-bound EcoNrdR-ATP-dATP and novel filaments of EcoNrdR-ATP. Tetrameric forms of EcoNrdR involve alternating interactions between pairs of Zn-ribbon domains and ATP-cones. The structures reveal considerable flexibility in relative orientation of ATP-cones vs Zn-ribbon domains. The structure of DNA-bound EcoNrdR-ATP-dATP shows that significant conformational rearrangements between ATP-cones and Zn-ribbons accompany DNA binding while the ATP-cones retain the same relative orientation. In contrast, ATP-loaded EcoNrdR filaments show rearrangements of the ATP-cone pairs and sequester the DNA-binding residues of NrdR such that they are unable to bind to DNA. Our results, in combination with a previous structural and biochemical study, point to highly flexible EcoNrdR structures that when loaded with the correct nucleotides adapt to an optimal promoter binding conformation.

biophysics↗

bioRxiv2024

Strong conservation of spacer lengths in NrdR repressor DNA binding sites

The ribonucleotide reductase-specific repressor NrdR, from the human pathogens Listeria monocytogenes and Streptococcus pneumoniae, form tetramers that bind to DNA when loaded with dATP and ATP. If loaded with only ATP they form different oligomeric complexes that cannot bind to DNA. The DNA binding site in L. monocytogenes is a pair of NrdR boxes separated by 15-16 bp, whereas in Streptococcus pneumoniae the NrdR boxes are separated by 25-26 bp. However, Streptococcus pneumoniae NrdR binds stronger to the related Streptococcus thermophilus binding sites with NrdR boxes separated by 15-16 bp. This observation triggered a comprehensive binding study of four NrdRs from L. monocytogenes, Streptococcus pneumoniae, Escherichia coli and Streptomyces coelicolor to a series of synthetic dsDNA fragments where the NrdR boxes were separated by 12-27 bp. All four NrdRs bound well to NrdR boxes separated by 14-17 bp, and also to NrdR boxes separated by 24-27 bp. The worst binding occurred when NrdR boxes were separated by 20 bp. The in vitro results were confirmed in vivo in E. coli for spacer distances 12-27 bp. We conclude that NrdR repressors bind most efficiently when there is an integer number of DNA turns between the center of the two NrdR boxes.

biochemistry↗