Molecular properties of bacterial multidrug transporters.

One of the mechanisms that bacteria utilize to evade the toxic effects of antibiotics is the active extrusion of structurally unrelated drugs from the cell. Both intrinsic and acquired multidrug transporters play an important role in antibiotic resistance of several pathogens, including Neisseria gonorrhoeae, Mycobacterium tuberculosis, Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, and Vibrio cholerae. Detailed knowledge of the molecular basis of drug recognition and transport by multidrug transport systems is required for the development of new antibiotics that are not extruded or of inhibitors which block the multidrug transporter and allow traditional antibiotics to be effective. This review gives an extensive overview of the currently known multidrug transporters in bacteria. Based on energetics and structural characteristics, the bacterial multidrug transporters can be classified into five distinct families. Functional reconstitution in liposomes of purified multidrug transport proteins from four families revealed that these proteins are capable of mediating the export of structurally unrelated drugs independent of accessory proteins or cytoplasmic components. On the basis of (i) mutations that affect the activity or the substrate specificity of multidrug transporters and (ii) the three-dimensional structure of the drug-binding domain of the regulatory protein BmrR, the substrate-binding site for cationic drugs is predicted to consist of a hydrophobic pocket with a buried negatively charged residue that interacts electrostatically with the positively charged substrate. The aromatic and hydrophobic amino acid residues which form the drug-binding pocket impose restrictions on the shape and size of the substrates. Kinetic analysis of drug transport by multidrug transporters provided evidence that these proteins may contain multiple substrate-binding sites.     

A single acidic residue can guide binding site selection but does not govern QacR cationic-drug affinity

Structures of the multidrug-binding repressor protein QacR with monovalent and bivalent cationic drugs revealed that the carboxylate side-chains of E90 and E120 were proximal to the positively charged nitrogens of the ligands ethidium, malachite green and rhodamine 6G, and therefore may contribute to drug neutralization and binding affinity. Here, we report structural, biochemical and in vivo effects of substituting these glutamate residues. Unexpectedly, substitutions had little impact on ligand affinity or in vivo induction capabilities. Structures of QacR(E90Q) and QacR(E120Q) with ethidium or malachite green took similar global conformations that differed significantly from all previously described QacR-drug complexes but still prohibited binding to cognate DNA. Strikingly, the QacR(E90Q)-rhodamine 6G complex revealed two mutually exclusive rhodamine 6G binding sites. Despite multiple structural changes, all drug binding was essentially isoenergetic. Thus, these data strongly suggest that rather than contributing significantly to ligand binding affinity, the role of acidic residues lining the QacR multidrug-binding pocket is primarily to attract and guide cationic drugs to the "best available" positions within the pocket that elicit QacR induction.     

Role of transmembrane segment 10 in efflux mediated by the staphylococcal multidrug transport protein QacA

The staphylococcal multidrug exporter QacA confers resistance to a wide range of structurally dissimilar monovalent and bivalent cationic antimicrobial compounds. To understand the functional importance of transmembrane segment 10, which is thought to be involved in substrate binding, cysteine-scanning mutagenesis was performed in which 35 amino acid residues in the putative transmembrane helix and its flanking regions were replaced in turn with cysteine. Solvent accessibility analysis of the introduced cysteine residues using fluorescein maleimide indicated that transmembrane segment 10 of QacA contains a 20-amino-acid hydrophobic core and may extend from Pro-309 to Ala-334. Phenotypic analysis and fluorimetric transport assays of these mutants showed that Gly-313 is important for the efflux of both monovalent and bivalent cationic substrates, whereas Asp-323 is only important for the efflux of bivalent substrates and probably forms part of the bivalent substrate-binding site(s) together with Met-319. Furthermore, the effects of N-ethyl-maleimide treatment on ethidium and 4',6-diamidino-2-phenylindole export mediated by the QacA mutants suggest that the face of transmembrane segment 10 that contains Asp-323 may also be close to the monovalent substrate-binding site(s), making this helix an integral component of the QacA multidrug-binding pocket.     

Crystal structure of 2-hydroxyl-6-oxo-6-phenylhexa-2,4-dienoic acid (HPDA) hydrolase (BphD enzyme) from the Rhodococcus sp. strain RHA1 of the PCB degradation pathway

2-Hydroxyl-6-oxo-6-phenylhexa-2,4-dienoic acid (HPDA) hydrolase (the BphD enzyme) hydrolyzes a ring-cleavage product of an aromatic compound generated in a biphenyl/polychlorinated biphenyl (PCB) degradation pathway of bacteria. The crystal structure of the BphD enzyme has been determined at 2.4 A resolution by the multiple isomorphous replacement method. The final refined model of the BphD enzyme yields an R-factor of 17.5 % at 2.4 A resolution with reasonable geometry. The BphD enzyme is an octameric enzyme with a 422 point-group symmetry. The subunit can be divided into core and lid domains. The active site of the enzyme is situated in the substrate-binding pocket, which is located between the two domains. The substrate-binding pocket can be divided into hydrophobic and hydrophilic regions. This feature of the pocket seems to be necessary for substrate binding, as the substrate is composed of hydrophilic and hydrophobic parts. The proposed orientation of the substrate seems to be consistent with the general catalytic mechanism of alpha/beta-hydrolases.     

Structural and biochemical characterization of CIB1 delineates a new family of EF-hand-containing proteins

CIB1 (CIB) is an EF-hand-containing protein that binds multiple effector proteins, including the platelet alphaIIbbeta3 integrin and several serine/threonine kinases and potentially modulates their function. The crystal structure for Ca(2+)-bound CIB1 has been determined at 2.0 A resolution and reveals a compact alpha-helical protein containing four EF-hands, the last two of which bind calcium ions in the standard fashion seen in many other EF-hand proteins. CIB1 shares high structural similarity with calcineurin B and the neuronal calcium sensor (NCS) family of EF-hand-containing proteins. Most importantly, like calcineurin B and NCS proteins, which possess a large hydrophobic pocket necessary for ligand binding, CIB1 contains a hydrophobic pocket that has been implicated in ligand binding by previous mutational analysis. However, unlike several NCS proteins, Ca(2+)-bound CIB1 is largely monomeric whether bound to a relevant peptide ligand or ligand-free. Differences in structure, oligomeric state, and phylogeny define a new family of CIB1-related proteins that extends from arthropods to humans.     

Electron crystallography reveals plasticity within the drug binding site of the small multidrug transporter EmrE

EmrE is a Small Multidrug Resistance transporter (SMR) family member that mediates counter transport of protons and hydrophobic cationic drugs such as tetraphenylphosphonium (TPP⁺), ethidium, propidium and dequalinium. It is thought that the selectivity of the drug binding site in EmrE is defined by two negatively charged glutamate residues within a hydrophobic pocket formed from six of the α-helices, three from each monomer of the asymmetric EmrE homodimer. It is not apparent how such a binding pocket accommodates drugs of various sizes and shapes or whether the conformational changes that occur upon drug binding are identical for drugs of diverse chemical nature. Here, using electron cryomicroscopy of EmrE two-dimensional crystals we have determined projection structures of EmrE bound to three structurally different planar drugs, ethidium, propidium and dequalinium. Using image analysis and rigorous comparisons between these density maps and the density maps of the ligand-free and TPP⁺-bound forms of EmrE, we identify regions within the transporter that adapt differentially depending on the type of ligand bound. We show that all three planar drugs bind at the same pocket within the protein as TPP⁺. Furthermore, our analysis indicates that, while retaining the overall fold of the protein, binding of the planar drugs is accompanied by small rearrangements of the transmembrane domains that are different to those that occur when TPP⁺ binds. The regions in the EmrE dimer that are remodelled surround the drug binding site and include transmembrane domains from both monomers.     

Crystal structure of lactose synthase reveals a large conformational change in its catalytic component, the beta1,4-galactosyltransferase-I.

The lactose synthase (LS) enzyme is a 1:1 complex of a catalytic component, beta1,4-galactosyltransferse (beta4Gal-T1) and a regulatory component, alpha-lactalbumin (LA), a mammary gland-specific protein. LA promotes the binding of glucose (Glc) to beta4Gal-T1, thereby altering its sugar acceptor specificity from N-acetylglucosamine (GlcNAc) to glucose, which enables LS to synthesize lactose, the major carbohydrate component of milk. The crystal structures of LS bound with various substrates were solved at 2 A resolution. These structures reveal that upon substrate binding to beta4Gal-T1, a large conformational change occurs in the region comprising residues 345 to 365. This repositions His347 in such a way that it can participate in the coordination of a metal ion, and creates a sugar and LA-binding site. At the sugar-acceptor binding site, a hydrophobic N-acetyl group-binding pocket is found, formed by residues Arg359, Phe360 and Ile363. In the Glc-bound structure, this hydrophobic pocket is absent. For the binding of Glc to LS, a reorientation of the Arg359 side-chain occurs, which blocks the hydrophobic pocket and maximizes the interactions with the Glc molecule. Thus, the role of LA is to hold Glc by hydrogen bonding with the O-1 hydroxyl group in the acceptor-binding site on beta4Gal-T1, while the N-acetyl group-binding pocket in beta4Gal-T1 adjusts to maximize the interactions with the Glc molecule. This study provides details of a structural basis for the partially ordered kinetic mechanism proposed for lactose synthase.     

枯草杆菌耐药转运蛋白Bmr及其基因bmr的表达调控研究进展

枯草杆菌多重耐药转运蛋白Bmr是其主要的耐药外排蛋白之一,由位于基因组DNA的bmr基因编码,介导对多种抗生素、杀菌剂等药物的耐药性。bmr基因的表达受到BmrR及MtaN的转录调控,二者均属于MerR家族调节子。关于近年对多重耐药转运蛋白Bmr和调节蛋白BmrR、MtaN的结构、生理功能及作用机制等研究情况进行综述。