Disulfide Cross-linking of a Multidrug and Toxic Compound Extrusion Transporter Impacts Multidrug Efflux

Abstract

Multidrug and toxic compound extrusion (MATE) transporters contribute to multidrug resistance by extruding different drugs across cell membranes. The MATE transporters alternate between their extracellular and intracellular facing conformations to propel drug export, but how these structural changes occur is unclear. Here we combine site-specific cross-linking and functional studies to probe the movement of transmembrane helices in NorM from Neiserria gonorrheae (NorM-NG), a MATE transporter with known extracellular facing structure. We generated an active, cysteine-less NorM-NG and conducted pairwise cysteine mutagenesis on this variant. We found that copper phenanthroline catalyzed disulfide bond formation within five cysteine pairs and increased the electrophoretic mobility of the corresponding mutants. Furthermore, copper phenanthroline abolished the activity of the five paired cysteine mutants, suggesting that these substituted amino acids come in spatial proximity during transport, and the proximity changes are functionally indispensable. Our data also implied that the substrate-binding transmembrane helices move up to 10 Å in NorM-NG during transport and afforded distance restraints for modeling the intracellular facing transporter, thereby casting new light on the underlying mechanism.

Keywords: conformational change; cysteine cross-linking; drug resistance; exchanger; membrane protein; multidrug efflux; multidrug transporter.

FIGURE 1.

Structural basis for the design of paired cysteine mutagenesis in NorM-NG. A, periplasmic view of the extracellular facing NorM-NG structure (Protein Data Bank code 4HUK), which shows the bound substrate tetraphenylphosphonium (magenta) sandwiched between the N (cyan) and C (yellow) domains of the transporter. B, the intracellular facing model of NorM-NG highlights the relative movement between the N and C domains. Relevant amino acids (sticks) and transmembrane helices are labeled. This figure was prepared using the software PyMOL (version 1.5.0.4, Schrodinger).

FIGURE 2.

Western blot analysis of the NorM-NG variants. Western blot analysis of NorM-NG variants in membrane preparations was performed by using an antibody against the His tag. This analysis suggested that all of the NorM-NG variants investigated in this work were expressed at similar levels in the E. coli membrane. The positions of two molecular weight markers are also indicated.

FIGURE 3.

Cross-linking of five NorM-NG mutants revealed by gel mobility shift assays. A and B, nonreducing SDS-PAGE analysis of detergent-purified NorM-NG variants with and without copper phenanthroline (Cu) treatment. The proteins were purified in the presence of 100 mm NaCl and incubated with and without 200 μm CuSO₄ plus 400 μm phenanthroline. The proteins were visualized by Coomassie staining (A) or UV illumination (B). Free cysteine thiol groups in the protein reacted with N-(5-fluoresceinyl)maleimide and thus gave rise to fluorescence signals (B). C, Western blot of membrane-embedded NorM-NG variants with and without copper phenanthroline treatment. Copper phenanthroline stimulated the formation of disulfide bonds and the loss of free cysteine thiol groups in five dicysteine NorM-NG mutants and markedly increased their electrophoretic mobility.

FIGURE 4.

SDS-PAGE analysis revealed no cross-linking in monocysteine mutants of NorM-NG. Nonreducing SDS-PAGE analysis of detergent-purified NorM-NG variants with and without copper phenanthroline (Cu) treatment (200 μm CuSO₄ plus 400 μm phenanthroline). 100 mm NaCl was included during this analysis. The proteins were visualized either by Coomassie staining (top panel) or UV illumination (middle panel). Western blot of membrane-embedded NorM-NG variants with and without copper phenanthroline treatment is shown (bottom panel). Notably, copper phenanthroline had no measurable effect on the electrophoretic mobility of the 10 monocysteine mutants of NorM-NG.

FIGURE 5.

Effects of substrate-binding on the cross-linking of NorM-NG mutants. Nonreducing SDS-PAGE analysis of detergent-purified NorM-NG variants with and without copper phenanthroline (Cu) treatment (50 μm CuSO₄ plus 100 μm phenanthroline), in the absence and presence of 1 mm TPP. No NaCl was added to the samples during this analysis. The proteins were visualized by Coomassie staining (top panel) and UV illumination (bottom panel). TPP stimulated the electrophoretic mobility shift in the NorM-NG mutants and enhanced their loss of free cysteine thiols.

FIGURE 6.

Functional consequences of cysteine cross-linking as measured in the drug resistance assay. A, optical density measurement of bacteria expressing NorM-NG variants in the absence and presence of copper phenanthroline (Cu) and DTT. 3 μg/ml of R6G or 0.5 μg/ml of ethidium (ET) was used. The results represent means ± S.D. (error bars) of at least three independent experiments performed in duplicate. B, nonreducing SDS-PAGE analysis of the NorM-NG variants, with and without copper phenanthroline and DTT treatment. Copper phenanthroline gave rise to gel mobility shift of five dicysteine NorM-NG mutants, which could be reversed by DTT.

FIGURE 7.

Functional impact of disulfide cross-linking as measured in the drug accumulation assay. The accumulation of R6G or ethidium (ET) in cells expressing the NorM-NG variants, in the absence or presence of copper phenanthroline (Cu) and DTT. The results represent means ± S.D. (error bars) of at least three independent experiments performed in duplicate. The data are given as percentages of the values for cells expressing the inactive D41A in the presence of both copper phenanthroline and DTT.

FIGURE 8.

Lack of functional impact of copper phenanthroline on monocysteine NorM-NG mutants. The accumulation of R6G in cells expressing the NorM-NG variants, in the absence or presence of copper phenanthroline (Cu) and DTT. The results represent means ± S.D. (error bars) of at least three independent experiments performed in duplicate. The data are given as percentages of the values for cells expressing D41A in the presence of copper phenanthroline and DTT. In stark contrast to the five dicysteine mutants of NorM-NG, copper phenanthroline or DTT had little, if any, effect on the accumulation of R6G in bacteria expressing the 10 monocysteine mutants.

FIGURE 9.

Proposed model for the interconversion between the extracellular and intracellular facing NorM-NG. Rotational movement alone between the N (cyan) and C (yellow) domains is unlikely to bring all the five cysteine pairs studied in this work (blue dots) into sufficient proximity for disulfide formation. Helix bending (highlighted by a red arrow) likely occurs as the transporter adopts the intracellular facing conformation, which enables the disulfide bond formation (red dots). Although helix bending may occur in the N and/or C domain, for simplicity the bending of helices is only shown in the C domain.