Substrate binding to the multidrug transporter MepA.

MepA is a multidrug transporter from Staphylococcus aureus that confers multidrug resistance through the efflux of a wide array of hydrophobic substrates. To evaluate the ability of MepA to recognize different substrates, the dissociation constants for interactions between MepA and three of its substrates (acriflavine (Acr), rhodamine 6G (R6G), and ethidium (Et)) were measured. Given that MepA is purified in the presence of detergents and that its substrates are hydrophobic, we examined the effect of the detergent concentration on the dissociation constant. We demonstrate that all three substrates interact directly with the detergent micelles. Additionally, we find the detergent effect on the KD value to be highly substrate-dependent. The KD value for R6G is greatly influenced by the detergent, whereas the KD values for Acr and Et are only modestly affected. The effect of the inactive D183A mutant on binding was also evaluated. The D183A mutant shows lower affinity toward Acr and Et.

Structural characterization of MepB from Staphylococcus aureus reveals homology to endonucleases.

The MepRAB operon in Staphylococcus aureus has been identified to play a role in drug resistance. Although the functions of MepA and MepR are known, little information is available on the function of MepB. Here we report the X-ray structure of MepB to 2.1 Å revealing its structural similarity to the PD-(D/E)XK family of endonucleases. We further show that MepB binds DNA and RNA, with a higher affinity towards RNA and single stranded DNA than towards double stranded DNA. Notably, the PD-(D/E)XK catalytic active site residues are not conserved in MepB. MepB's association with a drug resistance operon suggests that it plays a role in responding to antimicrobials. This role is likely carried out through MepB's interactions with nucleic acids.

Crystallization and preliminary X-ray crystallographic analysis of the Escherichia coli outer membrane cobalamin transporter BtuB in complex with the carboxy-terminal domain of TonB.

The energy-dependent uptake of organometallic compounds and other micronutrients across the outer membranes of Gram-negative bacteria is carried out by outer membrane active-transport proteins that utilize the proton-motive force of the inner membrane via coupling to the TonB protein. The Escherichia coli outer membrane cobalamin transporter BtuB and a carboxy-terminal domain of the TonB protein, residues 147-239 of the wild-type protein, were expressed and purified individually. A complex of BtuB and TonB(147-239) was formed in the presence of the substrate cyanocobalamin (CN-Cbl; vitamin B12) and calcium and was crystallized. BtuB was purified in the detergent LDAO (n-dodecyl-N,N-dimethylamine-N-oxide) and the complex was formed in a detergent mixture of LDAO and C8E4 (tetraethylene glycol monooctylether). Crystals were obtained by sitting-drop vapor diffusion, with the reservoir containing 30%(v/v) polyethylene glycol (PEG 300) and 100 mM sodium acetate pH 5.2. The crystals belong to space group P2(1)2(1)2(1) (unit-cell parameters a = 74.3, b = 82.4, c = 122.6 angstroms). The asymmetric unit consists of a single BtuB-TonB complex. Data sets have been collected to 2.1 angstroms resolution at a synchrotron beamline (APS SER-CAT 22-ID).

Outer membrane active transport: structure of the BtuB:TonB complex.

In Gram-negative bacteria, the import of essential micronutrients across the outer membrane requires a transporter, an electrochemical gradient of protons across the inner membrane, and an inner membrane protein complex (ExbB, ExbD, TonB) that couples the proton-motive force to the outer membrane transporter. The inner membrane protein TonB binds directly to a conserved region, called the Ton-box, of the transporter. We solved the structure of the cobalamin transporter BtuB in complex with the C-terminal domain of TonB. In contrast to its conformations in the absence of TonB, the Ton-box forms a beta strand that is recruited to the existing beta sheet of TonB, which is consistent with a mechanical pulling model of transport.