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The AbgT family of transporters was thought to contribute to bacterial folate biosynthesis by importing the catabolite p-aminobenzoyl-glutamate for producing this essential vitamin. Approximately 13,000 putative transporters of the family have been identified. However, before our work, no structural information was available and even functional data were minimal for this family of membrane proteins. To elucidate the structure and function of the AbgT family of transporters, we recently determined the X-ray structures of the full-length Alcanivorax borkumensis YdaH and Neisseria gonorrhoeae MtrF membrane proteins. The structures reveal that these two transporters assemble as dimers with architectures distinct from all other families of transporters. Both YdaH and MtrF are bowl-shaped dimers with a solvent-filled basin extending from the cytoplasm halfway across the membrane bilayer. The protomers of YdaH and MtrF contain nine transmembrane helices and two hairpins. These structures directly suggest a plausible pathway for substrate transport. A combination of the crystal structure, genetic analysis and substrate accumulation assay indicates that both YdaH and MtrF behave as exporters, capable of removing the folate metabolite p-aminobenzoic acid from bacterial cells. Further experimental data based on drug susceptibility and radioactive transport assay suggest that both YdaH and MtrF participate as antibiotic efflux pumps, importantly mediating bacterial resistance to sulfonamide antimetabolite drugs. It is possible that many of these AbgT-family transporters act as exporters, thereby conferring bacterial resistance to sulfonamides. The AbgT-family transporters may be important targets for the rational design of novel antibiotics to combat bacterial infections., (© 2015 The Protein Society.)

The chloroplast division machinery is composed of numerous proteins that assemble as a large complex to divide double-membraned chloroplasts through binary fission. A key mediator of division-complex formation is ARC6, a chloroplast inner envelope protein and evolutionary descendant of the cyanobacterial cell division protein Ftn2. ARC6 connects stromal and cytosolic contractile rings across the two membranes through interaction with an outer envelope protein within the intermembrane space (IMS). The ARC6 IMS region bears a structurally uncharacterized domain of unknown function, DUF4101, that is highly conserved among ARC6 and Ftn2 proteins. Here we report the crystal structure of this domain from Arabidopsis thaliana ARC6. The domain forms an α/β barrel open towards the outer envelope membrane but closed towards the inner envelope membrane. These findings provide new clues into how ARC6 and its homologs contribute to chloroplast and cyanobacterial cell division., (© 2015 The Protein Society.)

Mycobacterium tuberculosis is a pathogenic bacterial species, which is neither Gram positive nor Gram negative. It has a unique cell wall, making it difficult to kill and conferring resistance to antibiotics that disrupt cell wall biosynthesis. Thus, the mycobacterial cell wall is critical to the virulence of these pathogens. Recent work shows that the mycobacterial membrane protein large (MmpL) family of transporters contributes to cell wall biosynthesis by exporting fatty acids and lipidic elements of the cell wall. The expression of the Mycobacterium tuberculosis MmpL proteins is controlled by a complicated regulatory network system. Here we report crystallographic structures of two forms of the TetR-family transcriptional regulator Rv0302, which participates in regulating the expression of MmpL proteins. The structures reveal a dimeric, two-domain molecule with architecture consistent with the TetR family of regulators. Comparison of the two Rv0302 crystal structures suggests that the conformational changes leading to derepression may be due to a rigid body rotational motion within the dimer interface of the regulator. Using fluorescence polarization and electrophoretic mobility shift assays, we demonstrate the recognition of promoter and intragenic regions of multiple mmpL genes by this protein. In addition, our isothermal titration calorimetry and electrophoretic mobility shift experiments indicate that fatty acids may be the natural ligand of this regulator. Taken together, these experiments provide new perspectives on the regulation of the MmpL family of transporters., (© 2015 The Protein Society.)

It is widely accepted that the increased use of antibiotics has resulted in bacteria with developed resistance to such treatments. These organisms are capable of forming multi-protein structures that bridge both the inner and outer membrane to expel diverse toxic compounds directly from the cell. Proteins of the resistance nodulation cell division (RND) superfamily typically assemble as tripartite efflux pumps, composed of an inner membrane transporter, a periplasmic membrane fusion protein, and an outer membrane factor channel protein. These machines are the most powerful antimicrobial efflux machinery available to bacteria. In Escherichia coli, the CusCFBA complex is the only known RND transporter with a specificity for heavy metals, detoxifying both Cu(+) and Ag(+) ions. In this review, we discuss the known structural information for the CusCFBA proteins, with an emphasis on their assembly, interaction, and the relationship between structure and function., (© 2015 The Protein Society.)

As one of the world's most prevalent enteric pathogens, Campylobacter jejuni is a major causative agent of human enterocolitis and is responsible for more than 400 million cases of diarrhea each year. The impact of this pathogen on children is of particular significance. Campylobacter has developed resistance to many antimicrobial agents via multidrug efflux machinery. The CmeABC tripartite multidrug efflux pump, belonging to the resistance-nodulation-cell division (RND) superfamily, plays a major role in drug resistant phenotypes of C. jejuni. This efflux complex spans the entire cell envelop of C. jejuni and mediates resistance to various antibiotics and toxic compounds. We here report the crystal structure of C. jejuni CmeC, the outer membrane component of the CmeABC tripartite multidrug efflux system. The structure reveals a possible mechanism for substrate export., (© 2014 The Protein Society.)

The Rv1217c-Rv1218c multidrug efflux system, which belongs to the ATP-binding cassette superfamily, recognizes and actively extrudes a variety of structurally unrelated toxic chemicals and mediates the intrinsic resistance to these antimicrobials in Mycobacterium tuberculosis. The expression of Rv1217c-Rv1218c is controlled by the TetR-like transcriptional regulator Rv1219c, which is encoded by a gene immediately upstream of rv1218c. To elucidate the structural basis of Rv1219c regulation, we have determined the crystal structure of Rv1219c, which reveals a dimeric two-domain molecule with an entirely helical architecture similar to members of the TetR family of transcriptional regulators. The N-terminal domains of the Rv1219c dimer are separated by a large center-to-center distance of 64 Å. The C-terminal domain of each protomer possesses a large cavity. Docking of small compounds to Rv1219c suggests that this large cavity forms a multidrug binding pocket, which can accommodate a variety of structurally unrelated antimicrobial agents. The internal wall of the multidrug binding site is surrounded by seven aromatic residues, indicating that drug binding may be governed by aromatic stacking interactions. In addition, fluorescence polarization reveals that Rv1219c binds drugs in the micromolar range., (© 2014 The Protein Society.)

The TetR family of transcription regulators are diverse proteins capable of sensing and responding to various structurally dissimilar antimicrobial agents. Upon detecting these agents, the regulators allow transcription of an appropriate array of resistance markers to counteract the deleterious compounds. Campylobacter jejuni CmeR is a pleiotropic regulator of multiple proteins, including the membrane-bound multidrug efflux transporter CmeABC. CmeR represses the expression of CmeABC and is induced by bile acids, which are substrates of the CmeABC tripartite pump. The multiligand-binding pocket of CmeR has been shown to be very extensive and consists of several positively charged and multiple aromatic amino acids. Here we describe the crystal structures of CmeR in complexes with the bile acids, taurocholate and cholate. Taurocholate and cholate are structurally related, differing by only the anionic charged group. However, these two ligands bind distinctly in the binding tunnel. Taurocholate spans the novel bile acid binding site adjacent to and without overlapping with the previously determined glycerol-binding site. The anionic aminoethanesulfonate group of taurocholate is neutralized by a charge-dipole interaction. Unlike taurocholate, cholate binds in an anti-parallel orientation but occupies the same bile acid-binding site. Its anionic pentanoate moiety makes a water-mediated hydrogen bond with a cationic residue to neutralize the formal negative charge. These structures underscore the promiscuity of the multifaceted binding pocket of CmeR. The capacity of CmeR to recognize bile acids was confirmed using isothermal titration calorimetry and fluorescence polarization. The results revealed that the regulator binds these acids with dissociation constants in the micromolar region., (Copyright © 2011 The Protein Society.)

Gram-negative bacteria, such as Escherichia coli, frequently utilize tripartite efflux complexes in the resistance-nodulation-cell division (RND) family to expel diverse toxic compounds from the cell. These efflux systems span the entire cell envelope to mediate the phenomenon of bacterial multidrug resistance. The three parts of the efflux complexes are: (1) a membrane fusion protein (MFP) connecting (2) a substrate-binding inner membrane transporter to (3) an outer membrane-anchored channel in the periplasmic space. One such efflux system CusCBA is responsible for extruding biocidal Cu(I) and Ag(I) ions. We recently determined the crystal structures of both the inner membrane transporter CusA and MFP CusB of the CusCBA tripartite efflux system from E. coli. These are the first structures of the heavy-metal efflux (HME) subfamily of the RND efflux pumps. Here, we summarize the structural information of these two efflux proteins and present the accumulated evidence that this efflux system utilizes methionine residues to bind and export Cu(I)/Ag(I). Genetic and structural analyses suggest that the CusA pump is capable of picking up the metal ions from both the periplasm and cytoplasm. We propose a stepwise shuttle mechanism for this pump to extrude metal ions from the cell.