Structural basis of quinone-sensing by the MarR-type repressor MhqR in Staphylococcus aureus

The MarR-family regulator MhqR of Staphylococcus aureus ( Sa MhqR) was previously characterized as quinone-sensing repressor of the mhqRED operon. Here, we resolved the crystal structures of apo- Sa MhqR and the 2-methylbenzoquinone (MBQ)-bound Sa MhqR complex. AlphaFold3 modelling was used to predict the structure of the Sa MhqR in complex with its operator DNA. In the DNA-bound Sa MhqR state, S65 and S66 of an allosteric α3-α4 loop adapted a helically wound conformation to elongate helix α4 for optimal DNA binding. Key residues for MBQ interaction were identified as F11, F39, E43, and H111, forming the MBQ-binding pocket. MBQ binding prevented the formation of the extended helix α4 in the allosteric loop, leading to steric clashes with the DNA. Molecular dynamics (MD) simulations revealed an increased intrinsic dynamics within the allosteric loop and the ß1/ß2-wing regions after MBQ binding, to prevent DNA binding. Using mutational analyses, we validated that F11, F39, and H111 are required for quinone sensing in vivo, whereas S65 and S66 of the allosteric loop and D88, K89, V91 and Y92 of the ß1/ß2-wing are essential for DNA binding in vitro and in vivo . In conclusion, our structure-guided modelling and mutational analyses identified a quinone-binding pocket of Sa MhqR and the mechanism of Sa MhqR inactivation, which involves local structural rearrangements of an allosteric loop and a high intrinsic dynamics to prevent DNA interactions. Our results provide novel insights into the redox-mechanism of the conserved Sa MhqR repressor, that functions as an important determinant of quinone and antimicrobial resistance in S. aureus .