Strains of the Burkholderia cepacia complex (Bcc) are Gram-negative opportunisitic bacteria that are capable of causing serious diseases, mainly in immunocompromised individuals. Bcc pathogens are intrinsically resistant to multiple antibiotics, including β-lactams, aminoglycosides, fluoroquinolones, and polymyxins. They are major pathogens in patients with cystic fibrosis (CF) and can cause severe necrotizing pneumonia, which is often fatal. Hopanoid biosynthesis is one of the major mechanisms involved in multiple antimicrobial resistance of Bcc pathogens. The hpnN gene of B. multivorans encodes an integral membrane protein of the HpnN family of transporters, which is responsible for shuttling hopanoids to the outer membrane. Here, we report crystal structures of B. multivorans HpnN, revealing a dimeric molecule with an overall butterfly shape. Each subunit of the transporter contains 12 transmembrane helices and two periplasmic loops that suggest a plausible pathway for substrate transport. Further analyses indicate that HpnN is capable of shuttling hopanoid virulence factors from the outer leaflet of the inner membrane to the periplasm. Taken together, our data suggest that the HpnN transporter is critical for multidrug resistance and cell wall remodeling in Burkholderia.

Resistance-nodulation-cell division (RND) superfamily efflux systems are responsible for the active transport of toxic compounds from the Gram-negative bacterial cell. These pumps typically assemble as tripartite complexes, spanning the inner and outer membranes of the cell envelope. In Escherichia coli, the CusC(F)BA complex, which exports copper(I) and silver(I) and mediates resistance to these two metal ions, is the only known RND transporter with a specificity for heavy metals. We have determined the crystal structures of both the inner membrane pump CusA and membrane fusion protein CusB, as well as the adaptor–transporter CusBA complex formed by these two efflux proteins. In addition, the crystal structures of the outer membrane channel CusC and the periplasmic metallochaperone CusF have been resolved. Based on these structures, the entire assembled model of the tripartite efflux system has been developed, and this efflux complex should be in the form of CusC3–CusB6–CusA3. It has been shown that CusA utilizes methionine clusters to bind and export Cu(I) and Ag(I). This pump is likely to undergo a conformational change, and utilize a relay network of methionine clusters as well as conserved charged residues to extrude the metal ions from the bacterial cell.

The transcriptional regulators of the TetR family act as chemical sensors to monitor the cellular environment in many bacterial species. To perform this function, members of the TetR family harbor a diverse ligand-binding domain capable of recognizing the same series of compounds as the transporters they regulate. Many of the regulators can be induced by a wide array of structurally unrelated compounds. Binding of these structurally unrelated ligands to the regulator results in a conformational change that is transmitted to the DNA-binding region, causing the repressor to lose its DNA-binding capacity and allowing for the initiation of transcription. The multi-drug binding proteins AcrR of Escherichia coli and CmeR from Campylobacter jejuni are members of the TetR family of transcriptional repressors that regulate the expression of the multidrug resistant efflux pumps AcrAB and CmeABC, respectively. To gain insights into the mechanisms of transcriptional regulation and how multiple ligands induce the same physiological response, we determined the crystal structures of the AcrR and CmeR regulatory proteins. In this review, we will summarize the new findings with AcrR and CmeR, and discuss the novel features of these two proteins in comparison with other regulators in the TetR family.

The AcrAB multidrug efflux pump, which belongs to the resistance nodulation division (RND) family, recognizes and extrudes a wide range of antibiotics and chemotherapeutic agents and causes the intrinsic antibiotic resistance in Escherichia coli. The expression of AcrAB is controlled by the transcriptional regulator AcrR, whose open reading frame is located 141 bp upstream of the acrAB operon. To understand the structural basis of AcrR regulation, we have determined the crystal structure of AcrR to 2.55-Å resolution, revealing a dimeric two-domain molecule with an entirely helical architecture similar to members of the TetR family of transcriptional regulators. Each monomer of AcrR forms a multientrance pocket of 350 Å3 in the ligand-binding domain. The ligand-binding pocket is surrounded with mostly hydrophobic residues. In addition, a completely buried negatively charged glutamate, expected to be critical for drug binding, is located at the center of the binding pocket. The crystal structure provides novel insight into the mechanisms of ligand binding and AcrR regulation.

Gram-negative bacteria, such as Escherichia coli, frequently utilize tripartite efflux complexes belonging to the resistance–nodulation–division family to expel diverse toxic compounds from the cell. These systems contain a periplasmic membrane fusion protein (MFP) that is critical for substrate transport. We here present the x-ray structures of the CusB MFP from the copper/silver efflux system of E. coli. This is the first structure of any MFPs associated with heavy-metal efflux transporters. CusB bridges the inner-membrane efflux pump CusA and outer-membrane channel CusC to mediate resistance to Cu+ and Ag+ ions. Two distinct structures of the elongated molecules of CusB were found in the asymmetric unit of a single crystal, which suggests the flexible nature of this protein. Each protomer of CusB can be divided into four different domains, whereby the first three domains are mostly β-strands and the last domain adopts an entirely helical architecture. Unlike other known structures of MFPs, the α-helical domain of CusB is folded into a three-helix bundle. This three-helix bundle presumably interacts with the periplasmic domain of CusC. The N- and C-termini of CusB form the first β-strand domain, which is found to interact with the periplasmic domain of the CusA efflux pump. Atomic details of how this efflux protein binds Cu+ and Ag+ were revealed by the crystals of the CusB–Cu(I) and CusB–Ag(I) complexes. The structures indicate that CusB consists of multiple binding sites for these metal ions. These findings reveal novel structural features of an MFP in the resistance–nodulation–division efflux system and provide direct evidence that this protein specifically interacts with transported substrates.

•The crystal structures of the wild-type CusC outer membrane channel protein and two of its mutant proteins are reported.•These structures suggest that the first N-terminal cysteine residue plays an important role in protein–membrane interactions and is critical for the insertion of this channel protein into the outer membrane.•These structures provide insight into the mechanisms on CusC folding and transmembrane channel formation.Gram-negative bacteria, such as Escherichia coli, frequently utilize tripartite efflux complexes in the RND (resistance–nodulation–cell division) family to expel diverse toxic compounds from the cell. These complexes span both the inner and outer membranes of the bacterium via an α-helical, inner membrane transporter; a periplasmic membrane fusion protein; and a β-barrel, outer membrane channel. One such efflux system, CusCBA, is responsible for extruding biocidal Cu(I) and Ag(I) ions. To remove these toxic ions, the CusC outer membrane channel must form a β-barrel structural domain, which creates a pore and spans the entire outer membrane. We here report the crystal structures of wild-type CusC, as well as two CusC mutants, suggesting that the first N-terminal cysteine residue plays an important role in protein–membrane interactions and is critical for the insertion of this channel protein into the outer membrane. These structures provide insight into the mechanisms on CusC folding and transmembrane channel formation. It is found that the interactions between CusC and membrane may be crucial for controlling the opening and closing of this β-barrel, outer membrane channel.Download high-res image (351KB)Download full-size image

Gram-negative bacteria expel various toxic chemicals via tripartite efflux pumps belonging to the resistance–nodulation–cell division superfamily. These pumps span both the inner and outer membranes of the cell. The three components of these tripartite systems are an inner‐membrane, substrate-binding transporter (or pump); a periplasmic membrane fusion protein (or adaptor); and an outer‐membrane-anchored channel. These three efflux proteins interact in the periplasmic space to form the three-part complexes. We previously presented the crystal structures of both the inner‐membrane transporter CusA and membrane fusion protein CusB of the CusCBA tripartite efflux system from Escherichia coli. We also described the co-crystal structure of the CusBA adaptor–transporter, revealing that the trimeric CusA efflux pump assembles with six CusB protein molecules to form the complex CusB6–CusA3. We here report three different conformers of the crystal structures of CusBA–Cu(I), suggesting a mechanism on how Cu(I) binding initiates a sequence of conformational transitions in the transport cycle. Genetic analysis and transport assays indicate that charged residues, in addition to the methionine pairs and clusters, are essential for extruding metal ions out of the cell.Download high-res image (176KB)Download full-size imageResearch Highlights► Three different conformers of the crystal structures of CusBA–Cu(I) are reported. ► These structures suggest a sequence of conformational transitions in the transport cycle. ► Conserved charged residues of CusA are essential for extruding metal ions out of the cell.

The CmeABC multidrug efflux pump, which belongs to the resistance-nodulation-division (RND) family, recognizes and extrudes a broad range of antimicrobial agents and is essential for Campylobacter jejuni colonization of the animal intestinal tract by mediating the efflux of bile acids. The expression of CmeABC is controlled by the transcriptional regulator CmeR, whose open reading frame is located immediately upstream of the cmeABC operon. To understand the structural basis of CmeR regulation, we have determined the crystal structure of CmeR to 2.2 Å resolution, revealing a dimeric two-domain molecule with an entirely helical architecture similar to members of the TetR family of transcriptional regulators. Unlike the rest of the TetR regulators, CmeR has a large center-to-center distance (54 Å) between two N termini of the dimer, and a large flexible ligand-binding pocket in the C-terminal domain. Each monomer forms a 20 Å long tunnel-like cavity in the ligand-binding domain of CmeR and is occupied by a fortuitous ligand that is identified as glycerol. The binding of glycerol to CmeR induces a conformational state that is incompatible with target DNA. As glycerol has a chemical structure similar to that of potential ligands of CmeR, the structure obtained mimics the induced form of CmeR. These findings reveal novel structural features of a TetR family regulator, and provide new insight into the mechanisms of ligand binding and CmeR regulation.

The AcrB of Escherichia coli pumps out a wide range of compounds, including most of the currently available antibiotics, and contributes significantly to the serious problem of multidrug resistance of pathogenic bacteria. Quantitative analysis of drug efflux by this pump requires the measurement of the affinity of ligands. Yet there has been no success in determining these values. We introduce here an approach of steady-state fluorescence polarization to study the interactions between four different ligands and the purified AcrB transporter in a detergent environment. Our assays indicate that the transporter binds these drugs with KD values ranging from 5.5 to 74.1 μM.