Divalent metal is required for ligand binding to the E. faecalis citrate metabolism regulator

BLANCATO, VS; MAGNI, C; LORCA, G

Rosario

Congreso; L Reunión Anual de SAIB; 2014

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

In Enterococcus faecalis, the citrate metabolism regulator (CitO) belongs to the GntR superfamily. Within the FadR family CitO and its homologs constitute a subgroup associated in Gram+ organisms to citrate utilization. In presence of citrate, CitO binds to cis-acting sequences near the cit promoters inducing citrate metabolism. CitO binds specifically citrate with high affinity (KD=1.2 µM) as determined by isothermal titration calorimetry and differential scanning fluorometry. Structure-based site directed mutagenesis was used to identify residues involved in citrate binding. Mutations in CitO Q64A, H74A and L214A did not affect citrate binding. In contrast, changes to Ala in R97, F143 and H191 modified the binding affinities for citrate indicating that these residues mediate ligand binding. These results were confirmed in vivo using fusions to lacZ in E. faecalis. In CitO structure model, residues N147, H191 and H213 that may be involved in metal binding were found. Accordingly, it was observed that binding of citrate in CitO was dependent on the presence of metal evidenced as decreased DNA binding upon preincubation with EDTA. The effect could be reverted by addition of Ca2+, Zn2+or Mn2+. The metal-binding site is buried within CitO structure, which suggests that citrate carboxylic group could interact directly with the metal at the bottom of the ligand-binding cavity.