LAUSR

In Staphylococcus aureus, vancomycin‐resistance‐associated response regulator (VraR) is a part of the VraSR two‐component system, which is responsible for activating a cell wall‐stress stimulon in response to an antibiotic that inhibits cell wall formation. Two VraR‐binding sites have been identified: R1 and R2 in the vraSR operon control region. However, the binding of VraR to a promoter DNA enhancing downstream gene expression remains unclear. VraR contains a conserved N‐terminal receiver domain (VraRN) connected to a C‐terminal DNA binding domain (VraRC) with a flexible linker. Here, we present the crystal structure of VraRC alone and in complex with R1‐DNA in 1.87‐ and 2.0‐Å resolution, respectively. VraRC consisting of four α‐helices forms a dimer when interacting with R1‐DNA. In the VraRC–DNA complex structure, Mg2+ ion is bound to Asp194. Biolayer interferometry experiments revealed that the addition of Mg2+ to VraRC enhanced its DNA binding affinity by eightfold. In addition, interpretation of NMR titrations between VraRC with R1‐ and R2‐DNA revealed the essential residues that might play a crucial role in interacting with DNA of the vraSR operon. The structural information could help in designing and screening potential therapeutics/inhibitors to deal with antibiotic‐resistant S. aureus via targeting VraR.