Dissection of an ABC transporter LolCDE function analyzed by photo-crosslinking.

The envelope of Escherichia coli contains approximately 100 different species of lipoproteins, most of which are localized to the inner leaflet of the outer membrane. The localization of lipoprotein (Lol) system, consisting of five Lol proteins, is responsible for the trafficking of lipoproteins to the outer membrane. LolCDE binds to lipoproteins destined for the outer membrane and transfers them to the periplasmic chaperone LolA. Although the cryo-EM structures of E. coli LolCDE have been reported, the mechanisms by which outer membrane lipoproteins are transferred to LolA remain elusive. In this study, we investigated the interaction between LolCDE and lipoproteins using site-specific photo-crosslinking. We introduced a photo-crosslinkable amino acid into different locations across the four helices which form the central lipoprotein-binding cavity, and identified domains that crosslink with peptidoglycan-associated lipoprotein (Pal) in vivo. Using one of the derivatives containing the photo-crosslinkable amino acid, we developed an in vitro system to analyze the binding of lipoproteins to LolCDE. Our results indicate that compound 2, a LolCDE inhibitor, does not inhibit the binding of lipoproteins to LolCDE, but rather promotes the dissociation of bound lipoproteins from LolCDE.

Crystal structures of multidrug efflux transporters from Burkholderia pseudomallei suggest details of transport mechanism.

BpeB and BpeF are multidrug efflux transporters from Burkholderia pseudomallei that enable multidrug resistance. Here, we report the crystal structures of BpeB and BpeF at 2.94 Å and 3.0 Å resolution, respectively. BpeB was found as an asymmetric trimer, consistent with the widely-accepted functional rotation mechanism for this type of transporter. One of the monomers has a distinct structure that we interpret as an intermediate along this functional cycle. Additionally, a detergent molecule bound in a previously undescribed binding site provides insights into substrate translocation through the pathway. BpeF shares structural similarities with the crystal structure of OqxB from Klebsiella pneumoniae, where both are symmetric trimers composed of three "binding"-state monomers. The structures of BpeB and BpeF further our understanding of the functional mechanisms of transporters belonging to the HAE1-RND superfamily.

Structural and functional characteristics of the tripartite ABC transporter.

ATP-binding cassette (ABC) transporters are one of the largest protein superfamilies and are found in all living organisms. These transporters use the energy from ATP binding and hydrolysis to transport various substrates. In this review, we focus on the structural and functional aspects of ABC transporters, with special emphasis on type VII ABC transporters, a newly defined class possessing characteristic structures. A notable feature of type VII ABC transporters is that they assemble into tripartite complexes that span both the inner and outer membranes of Gram-negative bacteria. One of the original type VII ABC transporters, which possesses all characteristic features of this class, is the macrolide efflux transporter MacB. Recent structural analyses of MacB and homologue proteins revealed the unique mechanisms of substrate translocation by type VII ABC transporters.

Structure and function relationship of OqxB efflux pump from Klebsiella pneumoniae.

OqxB is an RND (Resistance-Nodulation-Division) efflux pump that has emerged as a factor contributing to the antibiotic resistance in Klebsiella pneumoniae. OqxB underwent horizontal gene transfer and is now seen in other Gram-negative bacterial pathogens including Escherichia coli, Enterobacter cloacae and Salmonella spp., further disseminating multi-drug resistance. In this study, we describe crystal structure of OqxB with n-dodecyl-ß-D-maltoside (DDM) molecules bound in its substrate-binding pocket, at 1.85 Å resolution. We utilize this structure in computational studies to predict the key amino acids contributing to the efflux of fluoroquinolones by OqxB, distinct from analogous residues in related transporters AcrB and MexB. Finally, our complementation assays with mutated OqxB and minimum inhibitory concentration (MIC) experiments with clinical isolates of E. coli provide further evidence that the predicted structural features are indeed involved in ciprofloxacin efflux.

Tripartite transporters as mechanotransmitters in periplasmic alternating-access mechanisms.

To remove xenobiotics from the periplasmic space, Gram-negative bacteria utilise unique tripartite efflux systems in which a molecular engine in the plasma membrane connects to periplasmic and outer membrane subunits. Substrates bind to periplasmic sections of the engine or sometimes to the periplasmic subunits. Then, the tripartite machines undergo conformational changes that allow the movement of the substrates down the substrate translocation pathway to the outside of the cell. The transmembrane (TM) domains of the tripartite resistance-nodulation-drug-resistance (RND) transporters drive these conformational changes by converting proton motive force into mechanical motion. Similarly, the TM domains of tripartite ATP-binding cassette (ABC) transporters transmit mechanical movement associated with nucleotide binding and hydrolysis at the nucleotide-binding domains to the relevant subunits in the periplasm. In this way, metabolic energy is coupled to periplasmic alternating-access mechanisms to achieve substrate transport across the outer membrane.

BpeB, a major resistance-nodulation-cell division transporter from Burkholderia cenocepacia: construct design, crystallization and preliminary structural analysis.

Burkholderia cenocepacia is an opportunistic pathogen that infects cystic fibrosis patients, causing pneumonia and septicemia. B. cenocepacia has intrinsic antibiotic resistance against monobactams, aminoglycosides, chloramphenicol and fluoroquinolones that is contributed by a homologue of BpeB, which is a member of the resistance-nodulation-cell division (RND)-type multidrug-efflux transporters. Here, the cloning, overexpression, purification, construct design for crystallization and preliminary X-ray diffraction analysis of this BpeB homologue from B. cenocepacia are reported. Two truncation variants were designed to remove possible disordered regions based on comparative sequence and structural analysis to salvage the wild-type protein, which failed to crystallize. The 17-residue carboxyl-terminal truncation yielded crystals that diffracted to 3.6 Šresolution. The efflux function measured using minimal inhibitory concentration assays indicated that the truncation decreased, but did not eliminate, the efflux activity of the transporter.

Crystal structure of tripartite-type ABC transporter MacB from Acinetobacter baumannii.

The MacA-MacB-TolC tripartite complex is a transmembrane machine that spans both plasma membrane and outer membrane and actively extrudes substrates, including macrolide antibiotics, virulence factors, peptides and cell envelope precursors. These transport activities are driven by the ATPase MacB, a member of the ATP-binding cassette (ABC) superfamily. Here, we present the crystal structure of MacB at 3.4-Å resolution. MacB forms a dimer in which each protomer contains a nucleotide-binding domain and four transmembrane helices that protrude in the periplasm into a binding domain for interaction with the membrane fusion protein MacA. MacB represents an ABC transporter in pathogenic microorganisms with unique structural features.

Structure of the MacAB-TolC ABC-type tripartite multidrug efflux pump.

The MacA-MacB-TolC assembly of Escherichia coli is a transmembrane machine that spans the cell envelope and actively extrudes substrates, including macrolide antibiotics and polypeptide virulence factors. These transport processes are energized by the ATPase MacB, a member of the ATP-binding cassette (ABC) superfamily. We present an electron cryo-microscopy structure of the ABC-type tripartite assembly at near-atomic resolution. A hexamer of the periplasmic protein MacA bridges between a TolC trimer in the outer membrane and a MacB dimer in the inner membrane, generating a quaternary structure with a central channel for substrate translocation. A gating ring found in MacA is proposed to act as a one-way valve in substrate transport. The MacB structure features an atypical transmembrane domain with a closely packed dimer interface and a periplasmic opening that is the likely portal for substrate entry from the periplasm, with subsequent displacement through an allosteric transport mechanism.

Structural and genomic DNA analysis of the putative TetR transcriptional repressor SCO7518 from Streptomyces coelicolor A3(2).

SCO7518 is a protein of unknown function from Streptomyces coelicolor A3(2) that has been classified into the TetR transcriptional regulator family. In this study, a crystal structure of SCO7518 was determined at 2.29Å resolution. The structure is a homodimer of protomers that comprise an N-terminal DNA-binding domain and a C-terminal dimerization and regulatory domain, and possess a putative ligand-binding cavity. Genomic systematic evolution of ligands by exponential enrichment and electrophoretic mobility shift assays revealed that SCO7518 specifically binds to an operator sequence located upstream of the sco7519 gene, which encodes a maltose O-acetyltransferase. These results suggest that SCO7518 is a transcriptional repressor of sco7519 expression.

SCO4008, a putative TetR transcriptional repressor from Streptomyces coelicolor A3(2), regulates transcription of sco4007 by multidrug recognition.

SCO4008 from Streptomyces coelicolor A3(2) is a member of the TetR family. However, its precise function is not yet clear. In this study, the crystal structure of SCO4008 was determined at a resolution of 2.3Å, and its DNA-binding properties were analyzed. Crystal structure analysis showed that SCO4008 forms an Ω-shaped homodimer in which the monomer is composed of an N-terminal DNA-binding domain containing a helix-turn-helix and a C-terminal dimerization and regulatory domain possessing a ligand-binding cavity. The genomic systematic evolution of ligands by exponential enrichment and electrophoretic mobility shift assay revealed that four SCO4008 dimers bind to the two operator regions located between sco4008 and sco4007, a secondary transporter belonging to the major facilitator superfamily. Ligand screening analysis showed that SCO4008 recognizes a wide range of structurally dissimilar cationic and hydrophobic compounds. These results suggested that SCO4008 is a transcriptional repressor of sco4007 responsible for the multidrug resistance system in S. coelicolor A3(2).

Structural and functional analysis of the TetR-family transcriptional regulator SCO0332 from Streptomyces coelicolor.

SCO0332 protein is a putative TetR-family transcriptional regulator from Streptomyces coelicolor A3(2). The crystal structure of SCO0332 was determined at 2.25 A resolution by single-wavelength anomalous diffraction (SAD) phasing using the S atoms of the native protein. SCO0332 contains a helix-turn-helix (HTH) DNA-binding motif in its N-terminal region and forms a homodimer. The overall structure of SCO0332 shows significant similarity to other TetR-family regulators. A systematic evolution of ligands by exponential enrichment (SELEX) analysis indicated that SCO0332 has sequence-specific DNA-binding ability and determined the position of the operator element of SCO0332 on the chromosomal DNA of S. coelicolor. An electrophoretic mobility-shift assay (EMSA) showed that SCO0332 binds to the operator sequence upstream of the sco0330 gene, which encodes a putative short-chain oxidoreductase. These results suggest that SCO0332 is a transcriptional repressor that regulates sco0330 gene expression.

The CGL2612 protein from Corynebacterium glutamicum is a drug resistance-related transcriptional repressor: structural and functional analysis of a newly identified transcription factor from genomic DNA analysis.

The emergence of antibiotic-resistant bacteria often causes serious clinical problems. The TetR family is one of the major transcription factor families that regulate expression of genes involved in bacterial antimicrobial resistance systems. CGL2612 protein is a transcription factor newly identified by genomic DNA analysis on Corynebacterium glutamicum, which belongs to the mycolic acid-containing Actinomycetales, including the well known pathogens Corynebacterium diphtheriae and Mycobacterium tuberculosis. Crystal structure analysis showed that the CGL2612 protein exhibits significant structural similarity to the multidrug resistance (MDR)-related transcription factor QacR from Staphylococcus aureus, despite poor amino acid sequence similarity between these proteins. Binding DNA sequence analysis of CGL2612 protein using the systematic evolution of ligands by the exponential enrichment (systematic evolution of ligands by exponential enrichment, or SELEX) method revealed that this protein is a new member of the TetR family, which regulates expression of the immediately upstream gene, cgl2611, probably encoding a major facilitator superfamily permease. Subsequent functional analyses confirmed a function of the CGL2612 as a transcriptional repressor responsible for the antimicrobial resistance system in C. glutamicum. The strategy used in the present study is one of the most convenient and powerful methods to analyze functionally unknown transcription factors, and the results obtained here will contribute to our understanding of the drug resistance mechanism not only in C. glutamicum but also in the related bacteria, C. diphtheriae and M. tuberculosis.