Critical biophysical properties in the Pseudomonas aeruginosa efflux gene regulator MexR are targeted by mutations conferring multidrug resistance.


Abstract

The self-assembling MexA-MexB-OprM efflux pump system, encoded by the mexO operon, contributes to facile resistance of Pseudomonas aeruginosa by actively extruding multiple antimicrobials. MexR negatively regulates the mexO operon, comprising two adjacent MexR binding sites, and is as such highly targeted by mutations that confer multidrug resistance (MDR). To understand how MDR mutations impair MexR function, we studied MexR-wt as well as a selected set of MDR single mutants distant from the proposed DNA-binding helix. Although DNA affinity and MexA-MexB-OprM repression were both drastically impaired in the selected MexR-MDR mutants, MexR-wt bound its two binding sites in the mexO with high affinity as a dimer. In the MexR-MDR mutants, secondary structure content and oligomerization properties were very similar to MexR-wt despite their lack of DNA binding. Despite this, the MexR-MDR mutants showed highly varying stabilities compared with MexR-wt, suggesting disturbed critical interdomain contacts, because mutations in the DNA-binding domains affected the stability of the dimer region and vice versa. Furthermore, significant ANS binding to MexR-wt in both free and DNA-bound states, together with increased ANS binding in all studied mutants, suggest that a hydrophobic cavity in the dimer region already shown to be involved in regulatory binding is enlarged by MDR mutations. Taken together, we propose that the biophysical MexR properties that are targeted by MDR mutations-stability, domain interactions, and internal hydrophobic surfaces-are also critical for the regulation of MexR DNA binding. Study holds ProTherm entries: 25683, 25684, 25685, 25686, 25687, 25688, 25689, 25690, 25691, 25692, 25693, 25694, 25695 Extra Details: Transition 2 DNA-binding protein; stability; efflux gene regulator; multidrug resistance; MarR family; Biacore; analytical ultracentrifugation; circular dichroism; fluorescence; real-time PCR

Submission Details

ID: QTLwoBX93

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:56 p.m.

Version: 1

Publication Details
Andrésen C;Jalal S;Aili D;Wang Y;Islam S;Jarl A;Liedberg B;Wretlind B;Mårtensson LG;Sunnerhagen M,Protein Sci. (2010) Critical biophysical properties in the Pseudomonas aeruginosa efflux gene regulator MexR are targeted by mutations conferring multidrug resistance. PMID:20095047
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
3ECH 2008-10-21 1.8 The MarR-family repressor MexR in complex with its antirepressor ArmR
3MEX 2010-07-28 2.1 Crystal structure of MexR in oxidized state
1LNW 2002-09-11 2.1 CRYSTAL STRUCTURE OF THE MEXR REPRESSOR OF THE MEXAB-OPRM MULTIDRUG EFFLUX OPERON OF PSEUDOMONAS AERUGINOSA
4ZZL 2016-07-27 2.19 MexR R21W derepressor mutant causing multidrug resistance in P. aeruginosa by mexAB-oprM efflux pump expression

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Multidrug resistance operon repressor P52003 MEXR_PSEAE