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

TL;DR

This study reveals how the SaMhqR protein in Staphylococcus aureus senses quinones and regulates antimicrobial resistance through structural changes.

Contribution

The paper identifies a quinone-binding pocket and an allosteric loop mechanism in SaMhqR that controls DNA binding and resistance.

Findings

MBQ binding disrupts the DNA-binding helical conformation of the allosteric loop in SaMhqR.

Key residues F11, F39, and H111 are essential for quinone sensing, while S65, S66, D88, K89, V91, and Y92 are critical for DNA binding.

MD simulations show increased dynamics in the allosteric loop and β1/β2-wing regions after MBQ binding, preventing DNA interactions.

Abstract

The MarR-family regulator MhqR of Staphylococcus aureus (SaMhqR) was previously characterized as a quinone-sensing repressor of the mhqRED operon. Here, we solved the crystal structures of apo-SaMhqR and the 2-methylbenzoquinone (MBQ)-bound SaMhqR complex. AlphaFold3 modeling was used to predict the structure of SaMhqR in complex with its operator DNA. In the DNA-bound SaMhqR state, S65 and S66 of an allosteric α3–α4 loop adopted 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 β1/β2-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 β1/β2-wing are essential for DNA binding in vitro and in vivo. In conclusion, our structure-guided modeling and mutational analyses identified a quinone-binding pocket in SaMhqR and the mechanism of SaMhqR inactivation, which involves local structural rearrangements of an allosteric loop and high intrinsic dynamics to prevent DNA interactions. Our results provide novel insights into the redox mechanism of the conserved SaMhqR repressor, which functions as an important determinant of quinone and antimicrobial resistance in S. aureus.

Staphylococcus aureus is a major human pathogen that can cause life-threatening infections in humans. However, treatment options are limited due to the prevalence of antimicrobial-resistant isolates in the hospital and the community. The MarR-type repressor SaMhqR was described to control resistance toward quinones and quinone-like antimicrobials. However, the redox-regulatory mechanism of SaMhqR by quinones was unknown. In this work, we explored the DNA-binding and quinone-sensing mechanism of SaMhqR and identified a quinone-binding pocket and an allosteric loop, which facilitates DNA binding activity via a helical wound conformation and adapts an unstructured coiled conformation upon quinone binding to inhibit DNA binding. A similar mechanism has been recently discovered for the regulation of uric acid resistance by UrtR family repressors (W. S. Song, D. U. Ki , H. Y. Cho, O. H. Kwon, H. Cho, S. I. Yoon, Nucleic Acids Res 52:13192–13205, 2024, https://doi.org/10.1093/nar/gkae922). Our results contribute to a better understanding of antimicrobial resistance regulation, which may be exploited for future drug design to combat multidrug-resistant S. aureus.

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