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

Multidrug resistance (MDR) poses a major challenge to medicine. A principle cause of MDR is through active efflux by MDR transporters situated in the bacterial membrane. Here we present the crystal structure of the major facilitator superfamily (MFS) drug/H antiporter MdfA from Escherichia coli in an outward open conformation.

Generation of Conformation-Specific Antibody Fragments for Crystallization of the Multidrug Resistance Transporter MdfA.

A major hurdle in membrane protein crystallography is generating crystals diffracting sufficiently for structure determination. This is often attributed not only to the difficulty of obtaining functionally active protein in mg amounts but also to the intrinsic flexibility of its multiple conformations. The cocrystallization of membrane proteins with antibody fragments has been reported as an effective approach to improve the diffraction quality of membrane protein crystals by limiting the intrinsic flexibility.

The multidrug-resistance transporter MdfA from Escherichia coli: crystallization and X-ray diffraction analysis.

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.

Membrane composition influences the activity of in vitro refolded human vitamin K epoxide reductase.

Human vitamin K epoxide reductase (hVKOR) is an integral membrane protein responsible for the maintenance of reduced vitamin K pools, a prerequisite for the action of γ-glutamyl carboxylase and hence for hemostasis. Here we describe the recombinant expression of hVKOR as an insoluble fusion protein in Escherichia coli, followed by purification and chemical cleavage under denaturing conditions. In vitro renaturation and reconstitution of purified solubilized hVKOR in phospholipids could be established to yield active protein.

Identification of a cation transport pathway in Neisseria meningitidis PorB.

Neisseria meningitidis is the main causative agent of bacterial meningitis. In its outer membrane, the trimeric Neisserial porin PorB is responsible for the diffusive transport of essential hydrophilic solutes across the bilayer. Previous molecular dynamics simulations based on the recent crystal structure of PorB have suggested the presence of distinct solute translocation pathways through this channel.