Mechanism of NanR transcriptional activation of sialic acid metabolism in Streptococcus pneumoniae.

In Streptococcus pneumoniae, the RpiR transcriptional regulator NanR (SpNanR) senses sialic acid in the environment and upregulates transcription of the nan and sia A operons to increase uptake and metabolism of sialic acid. The molecular basis of this activation is unknown. Here, we demonstrate that SpNanR binds N-acetylmannosamine-6-phosphate, a metabolite of sialic acid catabolism. SpNanR exists in a dimer-tetramer equilibrium, and N-acetylmannosamine-6-phosphate binding strongly stabilizes the tetramer. Crystal structures and site-specific substitutions demonstrate that N-acetylmannosamine-6-phosphate bridges and stabilizes the SpNanR tetramer. SpNanR binds its DNA recognition sequence with nanomolar affinity. Notably, the effector N-acetylmannosamine-6-phosphate does not affect the affinity of SpNanR for DNA. The DNA-binding domains are not structurally coupled to the sugar isomerase domains, explaining why N-acetylmannosamine-6-phosphate binding does not affect DNA binding. Structural analysis reveals that sequence specificity arises through distortion of B-DNA and an unusual π-stack formed by two arginine residues in the minor groove, while affinity is driven by backbone contacts. We propose a mechanism by which S. pneumoniae regulates sialic acid metabolism, consistent with our biophysical experiments and in vivo regulatory behavior. These findings define a unique activation mechanism for an RpiR regulator and provide new insights into carbohydrate-responsive gene regulation in pneumococci.