The multidrug-resistance transporter MdfA fromEscherichia coli: crystallization and X-ray diffraction analysis

Abstract

The active efflux of antibiotics by multidrug-resistance (MDR) transporters is a major pathway of drug resistance and complicates the clinical treatment of bacterial infections. MdfA is a member of the major facilitator superfamily (MFS) from Escherichia coli and provides resistance to a wide variety of dissimilar toxic compounds, including neutral, cationic and zwitterionic substances. The 12-transmembrane-helix MdfA was expressed as a GFP-octahistidine fusion protein with a TEV protease cleavage site. Following tag removal, MdfA was purified using two chromatographic steps, complexed with a Fab fragment and further purified using size-exclusion chromatography. MdfA and MdfA-Fab complexes were subjected to both vapour-diffusion and lipidic cubic phase (LCP) crystallization techniques. Vapour-diffusion-grown crystals were of type II, with poor diffraction behaviour and weak crystal contacts. LCP lipid screening resulted in type I crystals that diffracted to 3.4 Å resolution and belonged to the hexagonal space group P622.

Outward open conformation of a Major Facilitator Superfamily multidrug/H+ antiporter provides insights into switching mechanism

“…The C-terminal lobe is more rigid than the N-terminal lobe, and the cytoplasmic halves of the TM helices (relative to the center of the lipid bilayer) showed smaller root mean square deviation (RMSD) values than those of the periplasmic halves of the TM helices (Supplementary Table 1 )—despite the fact that the Fab binds to the cytoplasmic face of MdfA. We therefore conclude that the outward-open conformation of MdfA is little affected by Fab binding (corroborated by low-resolution data from crystals of MdfA alone 20 ) and is stably maintained in a solvated membrane environment.…”

“…Isolation of Fab fragments that recognize native conformations of MdfA in proteoliposomes has been described elsewhere 31 . Co-crystals of MdfA in complex with Fab fragments grew in the lipidic cubic phase and diffracted to 3.4 Å resolution 20 , 31 . Diffraction data were collected at 100 K at a wavelength of 1.0 Å on the SLS beamline PXI (X06SA) using the 16M Eiger detector.…”

“…Atoms for difference density corresponding to solvent molecules, lipids, and/or detergents were not added to the model in accordance with the low resolution of the data. The Fab binds to the cytoplasmic side of MdfA, where it may stabilize the outward open state and enhances crystal contacts 20 . Data collection and refinement statistics are given in Table 1 .…”

Copper and cadmium administration induce toxicity and oxidative stress in the marine flatworm Macrostomum lignano

“…The membrane transporters, which play important roles in pumping out toxicants, are also differentially regulated under stress conditions. In humans, the multidrug resistance protein is an ATPbinding cassette (ABC) transporter that confers resistance to many drugs and regulates redox homeostasis, inflammation and hormone secretion [76][77][78]. It uses ATP to transport drugs through a channel formed by the trans-membrane segments [79,80].…”

Probing the solution structure of the E. coli multidrug transporter MdfA using DEER distance measurements with nitroxide and Gd(III) spin labels

“…We used DEER to elucidate the solution conformation of MdfA solubilized in detergent, in light of the recently published crystal structures 64,66,68 . For this purpose, we have labeled a series of double cysteine MdfA mutants with both MTSSL and C2-Gd, a Gd(III) tag (Fig.…”