Crystal Structure of Escherichia coli CusC, the Outer Membrane Component of a Heavy Metal Efflux Pump

Background: While copper has essential functions as an enzymatic co-factor, excess copper ions are toxic for cells, necessitating mechanisms for regulating its levels. The cusCBFA operon of E. coli encodes a four-component efflux pump dedicated to the extrusion of Cu(I) and Ag(I) ions. Methodology/Principal Findings: We have solved the X-ray crystal structure of CusC, the outer membrane component of the Cus heavy metal efflux pump, to 2.3 A ˚ resolution. The structure has the largest extracellular opening of any outer membrane factor (OMF) protein and suggests, for the first time, the presence of a tri-acylated N-terminal lipid anchor. Conclusions/Significance: The CusC protein does not have any obvious features that would make it specific for metal ions, suggesting that the narrow substrate specificity of the pump is provided by other components of the pump, most likely by the inner membrane component CusA

Functional Rotation of the Transporter AcrB: Insights into Drug Extrusion from Simulations

The tripartite complex AcrAB-TolC is the major efflux system in Escherichia coli. It extrudes a wide spectrum of noxious compounds out of the bacterium, including many antibiotics. Its active part, the homotrimeric transporter AcrB, is responsible for the selective binding of substrates and energy transduction. Based on available crystal structures and biochemical data, the transport of substrates by AcrB has been proposed to take place via a functional rotation, in which each monomer assumes a particular conformation. However, there is no molecular-level description of the conformational changes associated with the rotation and their connection to drug extrusion. To obtain insights thereon, we have performed extensive targeted molecular dynamics simulations mimicking the functional rotation of AcrB containing doxorubicin, one of the two substrates that were co-crystallized so far. The simulations, including almost half a million atoms, have been used to test several hypotheses concerning the structure-dynamics-function relationship of this transporter. Our results indicate that, upon induction of conformational changes, the substrate detaches from the binding pocket and approaches the gate to the central funnel. Furthermore, we provide strong evidence for the proposed peristaltic transport involving a zipper-like closure of the binding pocket, responsible for the displacement of the drug. A concerted opening of the channel between the binding pocket and the gate further favors the displacement of the drug. This microscopically well-funded information allows one to identify the role of specific amino acids during the transitions and to shed light on the functioning of AcrB

Crystal structure of the CusBA heavy-metal efflux complex of Escherichia coli

Gram-negative bacteria, such as Escherichia coli, expel toxic chemicals via tripartite efflux pumps spanning both the inner and outer membranes. The three parts are: 1) a membrane fusion protein connecting 2) a substrate-binding inner membrane transporter to 3) an outer membrane-anchored channel in the periplasmic space. A crystallographic model of this tripartite efflux complex has been unavailable simply because co-crystallization of different components of the system has proven to be extremely difficult. We previously described the crystal structures of both the inner membrane transporter CusA1 and membrane fusion protein CusB2 of the CusCBA efflux system3,4 from E. coli. We here report the co-crystal structure of the CusBA efflux complex, revealing the trimeric CusA efflux pump interacts with six CusB protomers at the upper half of the periplasmic domain. These six CusB molecules form a channel extending contiguously from the top of the pump. The affinity of the CusA and CusB interaction was found to be in the micromolar range. Finally, we predicted a three-dimensional structure of the trimeric CusC outer membrane channel, and develop a model of the tripartite efflux assemblage. This CusC3-CusB6-CusA3 model presents a 750 kDa efflux complex spanning the entire bacterial cell envelope to export Cu(I)/Ag(I) ions

Crystal structures of the CusA efflux pump suggest methionine-mediated metal transport

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.1,2 The efflux system CusCBA is responsible for extruding biocidal Cu(I) and Ag(I) ions.3,4 No prior structural information was available for the heavy-metal efflux (HME) subfamily of the RND efflux pumps. Here we describe the crystal structures of the inner membrane transporter CusA in the absence and presence of bound Cu(I) or Ag(I). These CusA structures provide important new structural information about the HME sub-family of RND efflux pumps. The structures suggest that the metal binding sites, formed by a three-methionine cluster, are located within the cleft region of the periplasmic domain. Intriguingly, this cleft is closed in the apo-CusA form but open in the CusA-Cu(I) and CusA-Ag(I) structures, which directly suggests a plausible pathway for ion export. Binding of Cu(I) and Ag(I) triggers significant conformational changes in both the periplasmic and transmembrane domains. The crystal structure indicates that CusA has, in addition to the three-methionine metal binding site, four methionine pairs - three located in the transmembrane region and one in the periplasmic domain. Genetic analysis and transport assays suggest that CusA is capable of actively picking up metal ions from the cytosol, utilizing these methionine pairs/clusters to bind and export metal ions. These structures suggest a stepwise shuttle mechanism for transport between these sites

Drug export and allosteric coupling in a multidrug transporter revealed by molecular simulations

Multidrug resistance is a serious problem in current chemotherapy. The efflux system largely responsible for resistance in Escherichia coli contains the drug transporter, AcrB. The structures of AcrB were solved in 2002 as the symmetric homo-trimer, and then in 2006 as the asymmetric homo-trimer. The latter suggested a functionally rotating mechanism. Here, by molecular simulations of the AcrB porter domain, we uncovered allosteric coupling and the drug export mechanism in the AcrB trimer. Allosteric coupling stabilized the asymmetric structure with one drug molecule bound, which validated the modelling. Drug dissociation caused a conformational change and stabilized the symmetric structure, providing a unified view of the structures reported in 2002 and 2006. A dynamic study suggested that, among the three potential driving processes, only protonation of the drug-bound protomer can drive the functional rotation and simultaneously export the drug

Metal-induced conformational changes in ZneB suggest an active role of membrane fusion proteins in efflux resistance systems

Resistance nodulation cell division (RND)-based efflux complexes mediate multidrug and heavy-metal resistance in many Gram-negative bacteria. Efflux of toxic compounds is driven by membrane proton/substrate antiporters (RND protein) in the plasma membrane, linked by a membrane fusion protein (MFP) to an outer-membrane protein. The three-component complex forms an efflux system that spans the entire cell envelope. The MFP is required for the assembly of this complex and is proposed to play an important active role in substrate efflux. To better understand the role of MFPs in RND-driven efflux systems, we chose ZneB, the MFP component of the ZneCAB heavy-metal efflux system from Cupriavidus metallidurans CH34. ZneB is shown to be highly specific for Zn2+ alone. The crystal structure of ZneB to 2.8 Å resolution defines the basis for metal ion binding in the coordination site at a flexible interface between the β-barrel and membrane proximal domains. The conformational differences observed between the crystal structures of metal-bound and apo forms are monitored in solution by spectroscopy and chromatography. The structural rearrangements between the two states suggest an active role in substrate efflux through metal binding and release