No abstract

Multidrug efflux pumps cause serious problems in cancer chemotherapy and treatment of bacterial infections. Yet high-resolution structures of ligand transporter complexes have previously been unavailable. We obtained x-ray crystallographic structures of the trimeric AcrB pump from Escherichia coli with four structurally diverse ligands. The structures show that three molecules of ligands bind simultaneously to the extremely large central cavity of 5000 cubic angstroms, primarily by hydrophobic, aromatic stacking and van der Waals interactions. Each ligand uses a slightly different subset of AcrB residues for binding. The bound ligand molecules often interact with each other, stabilizing the binding.

The structure of the antenna complex not only shows Nature at its most aesthetic but also illustrates how clever and efficient the energy transfer mechanism has become, with singlet-singlet excitation being passed smoothly down the spectral gradient to the reaction centre.

The Escherichia coli AcrB multidrug efflux pump is a membrane protein that recognizes many structurally dissimilar toxic compounds. We previously reported the X-ray structures of four AcrB-ligand complexes in which the ligands were bound to the wall of the extremely large central cavity in the transmembrane domain of the pump. Genetic studies, however, suggested that discrimination between the substrates occurs mainly in the periplasmic domain rather than the transmembrane domain of the pump. We here describe the crystal structures of the AcrB mutant in which Asn109 was replaced by Ala, with five structurally diverse ligands, ethidium, rhodamine 6G, ciprofloxacin, nafcillin, and Phe-Arg-␤-naphthylamide. The ligands bind not only to the wall of central cavity but also to a new periplasmic site within the deep external depression formed by the C-terminal periplasmic loop. This depression also includes residues identified earlier as being important in the specificity. We show here that conversion into alanine of the Phe664, Phe666, or Glu673 residue in the periplasmic binding site produced significant decreases in the MIC of most agents in the N109A background. Furthermore, decreased MICs were also observed when these residues were mutated in the wild-type AcrB background, although the effects were more modest. The MIC data were also confirmed by assays of ethidium influx rates in intact cells, and our results suggest that the periplasmic binding site plays a role in the physiological process of drug efflux.Multidrug transporters cause serious problems in the chemotherapy of cancer as well as in the antibiotic treatment of bacterial infections. These membrane proteins recognize many structurally dissimilar toxic compounds and actively extrude them from cells. The Escherichia coli AcrB transporter (17, 18), which is a member of the resistance-nodulation-division (RND) family transporters (42) and is responsible for most of the intrinsic drug resistance of this organism (28,29,37), is one of the best-studied multidrug pumps. It occurs as a multiprotein complex (40,41,50) with the outer membrane channel TolC (9, 15) and a periplasmic linker protein, AcrA (17) (the structure of an AcrA homolog, MexA, was solved recently [2, 11]), and this complex structure allows the direct export of drugs to the external medium (28). The structural work of Murakami et al. (23) revealed that the unliganded AcrB is a homotrimer, where each subunit contains 12 transmembrane (TM) helices and two large periplasmic domains (each exceeding 300 residues) between TM helices TM1 and TM2 and between TM7 and TM8. The periplasmic domains of AcrB form a funnel-like structure at the top which is thought (23) to associate with the end of the periplasmic tunnel of TolC (15) and a connected "pore" at the center (Fig. 1A). The pore leads down to the large central cavity, with a diameter of 35 Å, formed by the TM domains of the three protomers. The central cavity is connected also to the periplasm through three vestibules located at subunit interfaces (...

Soft x-ray tomography (SXT) is ideally suited to imaging sub-cellular architecture and organization, particularly in eukaryotic cells. SXT is similar in concept to the well-established medical diagnostic technique computed axial tomography (CAT), except SXT is capable of imaging with a spatial resolution of 50 nm, or better. In soft x-ray tomography (SXT) cells are imaged using photons from a region of the spectrum known as the ‘water window’. This results in quantitative, high-contrast images of intact, fully hydrated cells without the need to use contrast-enhancing agents. Cells are therefore visualized very close to their native, fully functional state. The utility of SXT has recently been enhanced by the development of high numerical aperture cryogenic light microscopy for correlated imaging. Taking this multi-modal approach now allows labeled molecules to be localized in the context of a high-resolution 3-dimensional tomographic reconstruction of the cell.