Mg2+-dependent mechanism of environmental versatility in a multidrug efflux pump

Tripartite resistance nodulation and cell division multidrug efflux pumps span the periplasm and are a major driver of multidrug resistance among Gram-negative bacteria. The periplasm provides a distinct environment between the inner and outer membranes of Gram-negative bacteria. Cations, such as Mg2+, become concentrated within the periplasm and, in contrast to the cytoplasm, its pH is sensitive to conditions outside the cell. Here, we reveal an interplay between Mg2+ and pH in modulating the dynamics of the periplasmic adaptor protein, AcrA, and its function within the prototypical AcrAB-TolC multidrug efflux pump from Escherichia coli. In the absence of Mg2+, AcrA becomes increasingly plastic within acidic conditions, but when Mg2+ is bound this is ameliorated, resulting in domain specific organisation in neutral to weakly acidic regimes. We establish a unique histidine residue directs these structural dynamics and is essential for sustaining pump efflux activity across acidic, neutral, and alkaline conditions. Overall, we propose Mg2+ conserves the structural mobility of AcrA to ensure optimal AcrAB-TolC function within rapid changing environments commonly faced by the periplasm during bacterial infection and colonization. This work highlights that Mg2+ is an important mechanistic component in this pump class and possibly across other periplasmic lipoproteins.


Figure S2. E. coli proteome within the periplasmic space.
MeBiPred analysis on the entire subcellular E. coli proteome.Plotted are the theroretical total perecentage of proteins predicted to bind different metals.OMLP; outer membrane lipoprotein, IMLP; inner membrane lipoprotein, IM-peri; peripheral inner membrane protein, periplasm; periplasmic protein.2+ the reported predicted template modelling (pTM) score and the interface predicted template modelling (ipTM) score were 0.60 and 0.80, respectively and had a ranking score of 0.82.For MdtE and Mg 2+ the pTM score and the ipTM score were 0.62 and 0.83 respectively and had a ranking score of 0.85.For MdtA and Mg 2+ the pTM score and the ipTM score were 0.55 and 0.74 respectively and had a ranking score of 0.81.For MexA and Mg 2+ the pTM score and the ipTM score were 0.62 and 0.79 respectively and had a ranking score of 0.83.For CusB and Cu 2+ the pTM score and the ipTM score were 0.49 and 0.73 respectively and had a ranking score of 0.75.2+ .Native-MS characterisation of AcrA S at pH 6.0 with 100 μM MgCl2.Proteins buffer was exchanged in 100 mM ammonium acetate buffer prior to MS.Two monomeric CSD's observed for AcrA S + ± MgCl2.F. Full length scans of AcrA +/-± MgCl2 at pH 6.0 and pH 7.4.Only 190-250 nm analysed due to noisy data at the ends.Proteins loaded at 1 mg/mL and ± MgCl2 at 1 mM.Each scan was repeated in quadruplet, and the averaged scan was analysed.Delta epsilon conversion completed by BeStSel online program 1 .G. Circular dichroism thermal melts of AcrA S ± MgCl2.Scans at 222 nm were taken between 40-60 ˚C with 1 ˚C increments of AcrA ± ± MgCl2 at pH 6.0.Proteins loaded at 0.0075 mg/mL and ± MgCl2 at 1 mM.Plot shows fraction denatured vs temperature.Tm's reported are 52.7 ˚C for AcrA S and 53.6 for AcrA S + ± MgCl2.

Figure S1 .
Figure S1.Location of Asp and Glu residues on AcrA.Mapping of the Asp and Glu residues on AcrA.Asp residues labelled pink, Glu residues labelled red.

Figure S4 .
Figure S4.Biophysical characterisation of AcrA and magnesium.A. Characteristic size exclusion chromatogram (SEC) for AcrA S .Fractions 17-22 were taken and run on an SDS-PAGE to assess sample purity.Void peak is shown at 40 mL and the main peak elutes at 75 mL, suggesting a homogenous monomer.B. Characteristic SDS-PAGE showing the eluted SEC fractions.C. Isothermal titration calorimetry of AcrA and magnesium.micro-ITC (Microcal

Figure S6 .
Figure S6.Structural dynamics of AcrA in the presence of MgCl2 at pH 7.4.The differential HDX (ΔHDX) plots for ((AcrA S + Mg 2+ ) -AcrA S ), at pH 7.4 for the 10-min time point painted onto the structure of AcrA (PDB: 5066) using HDeXplosion and Chimera. 2,3White signifies areas with no significant change in HDX.Significance was defined to be ≥ 0.5 Da change with a P-value ≤ 0.01 in a Welch's t-test (n=4).Uptake plots for three peptides in different domains of AcrA.Uptake plots are the average deuterium uptake and error bars indicate the standard deviation.Due to the experiment being at pH 7.4, it was more practicably feasible to collect a longer 4-hour time point to see if Mg2+  had an effect on dynamics over a longer period of time; again, there were no observed peptides that saw a change in uptake at this time point.

Figure S7 .
Figure S7.RMSF from MD simulations of AcrA S and MgCl2 at neutral conditions.AcrA coloured according to the difference in root-mean-square fluctuations (RMSF) between simulations of the AcrA S wild-type state without MgCl2 and with MgCl2 (AcrA S WT MgCl2 -AcrA S WT), averaged over four replicas for each.Red indicates that the RMSF has increased whereas blue indicates it has decreased.RMSF was calculated over the last 70 ns of each 100-ns simulation.

Figure S8 .
Figure S8.Protein expression of AcrA mutant.AcrA can be seen for all conditions as labelled on the gel.

Figure S10 .
Figure S10.Effect of MgCl2 on the NPN efflux activity of AcrAB-TolC and its AcrA H285A variant.Steady-state accumulation levels of the fluorescent probe NPN in E. coli 9(Pore) cells carrying a AcrAB-TolC or AcrA H285A B-TolC as a function of the external NPN concentration.Experiments completed ± MgCl2 at pH 6.0 and pH 8.0.

Figure S11 .
Figure S11.Inhibition of AcrA by NSC 60339 in the presence of magnesium.A. Chiclet plot displaying the differential HDX (ΔHDX) plots for ((AcrA S + Mg 2+ ) -AcrA S ), at pH 6,0 the single time point collected.Blue signifies areas with decreased HDX between states and white signifies areas with no significant change in HDX.Significance was defined to be ≥ 0.4 Da change with a P-value ≤ 0.01 in a Welch's t-test (n=4), as previously reported. 4B. ΔHDX for ((AcrA S + Mg 2+ ) -AcrA S ) for the only time point is painted onto the AcrA structure (PDB:5O66) using HDeXplosion and Chimera. 2,3C. The m/z spectrum for peptides 75-86 and 308-323 under nondeuterating conditions and deuterating conditions with Mg 2+ and Mg 2+ + NSC 60339.The centroid is represented by the dotted line, and the mass change of the deuterated samples is written in Daltons.