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.

Figure 1

Structural representation of MexR MDR mutation sites and overview of mexAB-oprM operon. A: MexR crystal structure 1LNW, represented by the chain A (black) + B (gray) dimer, which is likely to correspond to the unbound state,¹⁰ in stereo view. Mutation sites studied in this work are highlighted in chain A by displaying native sidechains in orange (L13M), red (R21W), green (G58E), blue (R70W), and magenta (T69I, R83H, R91H, L95F). Identified mutation sites in MexR leading to deficient DNA binding and/or multidrug resistance^{23–25,27,30,35} are indicated with Cα spheres in chain B. The figure was drawn in PyMOL.⁵⁸ B: Overview of the multidrug efflux operon mexAB-oprM and mexR gene in Pseudomonas aeruginosa. The suggested MexR binding sites PI and PII in mexO are indicated.³⁰

Figure 2

SPR sensorgrams for MexR-wt binding to mexO. Sensorgrams (solid lines) and fitted curves (broken lines) for MexR-wt binding to immobilised (A) PI-DNA using 4, 1, and 0.5 μM analyte, (B) PII-DNA using 4, 2, and 1 μM analyte. A two-state reaction conformational change model was used for MexR binding to PI and PII.

Figure 3

Oligomerization of free MexR as judged by AUC. Representative plots of sedimentation equilibrium experiments of MexR R21W at 20°C and 30,000 rpm. A: Residuals for dimer-octamer equilibrium (empty circles) and dimer-tetramer equilibrium (filled circles). B: R21W experimental values (empty circles), calculated fit of a dimer-octamer equilibrium (solid line) and calculated data points for a fitted equilibrium between dimer and tetramer (filled circles).

Figure 4

MexR structural effects of mutations and DNA binding. Far UV CD spectra of (A) MexR-wt (filled circle, black), MexR-L13M (empty rhomb, orange), MexR-R21W (filled rhomb, red), MexR-G58E (filled triangle, green), MexR-R70W (empty circle, blue) and (B) MexR-wt (solid black line) in complex with DNA binding boxes PI (solid line, dark grey) and PII (solid line, light grey) show that the structural effects of the studied MDR mutations are negligible. C: Thermal denaturation measurements at 222 nm for MexR-wt (filled circle, black), MexR-wt with PI-DNA (filled rhomb, dark grey), and MexR-wt with PII-DNA (filled square, light grey) indicate a less stable DNA-binding domain that is stabilized on DNA binding. The corresponding fits for two- and three-state denaturation (Table 4) are shown as solid lines. D: Gdn-HCl induced unfolding of MexR R21W (filled rombs, red) and R70W (empty circles, blue) indicate that the most stable MexR region is located in the dimer interface, while the DNA-binding region is unstable. Two-state (black) and three-state (red) fits (Table 4) to R21W data are shown. The R70W data were not sufficient for a full thermodynamic fit, thus the blue line only represents a graphic best fit.

Figure 5

Stability of MexR and mutants as detected by thermal stability and ANS binding measurements. A: ANS binding at 480 nm of MexR wt and mutants labeled as above, analyzed from fluorescence emission as a function of GuHCl concentration. The fluorescence of native MexR-wt (0 M GuHCl) incubated with ANS in the presence of PI- or PII-DNA was slightly higher than without DNA (+7.5 and +7.9%, respectively, data not shown in figure). B: Thermal stability measurements recording the CD ellipticity at 222 nm of MexR-wt (filled circle, black), MexR-L13M (empty rhomb, orange), MexR-R21W (filled rhomb, red), MexR-G58E (filled triangle, green), and MexR-R70W (empty circle, blue).