Structural mechanisms of heavy-metal extrusion by the Cus efflux system

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.     

Crystal Structure of the Membrane Fusion Protein CusB from Escherichia coli

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.     

Substrate-linked conformational change in the periplasmic component of a Cu(I)/Ag(I) efflux system

Gram-negative bacteria utilize dual membrane resistance nodulation division-type efflux systems to export a variety of substrates. These systems contain an essential periplasmic component that is important for assembly of the protein complex. We show here that the periplasmic protein CusB from the Cus copper/silver efflux system has a critical role in Cu(I) and Ag(I) binding. Isothermal titration calorimetry experiments demonstrate that one Ag(I) ion is bound per CusB molecule with high affinity. X-ray absorption spectroscopy data indicate that the metal environment is an all-sulfur 3-coordinate environment. Candidates for the metal-coordinating residues were identified from sequence analysis, which showed four conserved methionine residues. Mutations of three of these methionine residues to isoleucine resulted in significant effects on CusB metal binding in vitro. Cells containing these CusB variants also show a decrease in their ability to grow on copper-containing plates, indicating an important functional role for metal binding by CusB. Gel filtration chromatography demonstrates that upon binding metal, CusB undergoes a conformational change to a more compact structure. Based on these structural and functional effects of metal binding, we propose that the periplasmic component of resistance nodulation division-type efflux systems plays an active role in export through substrate-linked conformational changes.     

Charged Amino Acids (R83, E567, D617, E625, R669, and K678) of CusA Are Required for Metal Ion Transport in the Cus Efflux System

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 CusB(6)-CusA(3). 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.     

Structure and mechanism of the tripartite CusCBA heavy-metal efflux complex

Gram-negative bacteria frequently expel toxic chemicals through tripartite efflux pumps that span both the inner and outer membranes. The three parts are the inner membrane, substrate-binding transporter (or pump); a periplasmic membrane fusion protein (MFP, or adaptor); and an outer membrane-anchored channel. The fusion protein connects the transporter to the channel within the periplasmic space. One such efflux system CusCBA is responsible for extruding biocidal Cu(I) and Ag(I) ions. We previously described the crystal structures of both the inner membrane transporter CusA and the MFP CusB of Escherichia coli. We also determined the co-crystal structure of the CusBA adaptor-transporter efflux complex, showing that the transporter CusA, which is present as a trimer, interacts with six CusB protomers and that the periplasmic domain of CusA is involved in these interactions. Here, we summarize the structural information of these efflux proteins, and present the accumulated evidence that this efflux system uses methionine residues to bind and export Cu(I) and Ag(I). Genetic and structural analyses suggest that the CusA pump is capable of picking up the metal ions from both the periplasm and the cytoplasm. We propose a stepwise shuttle mechanism for this pump to export metal ions from the cell.     

Interactions between CusF and CusB identified by NMR spectroscopy and chemical cross-linking coupled to mass spectrometry

The Escherichia coli periplasmic proteins CusF and CusB, as part of the CusCFBA efflux system, aid in the resistance of elevated levels of copper and silver by direct metal transfer between the metallochaperone CusF and the membrane fusion protein CusB before metal extrusion from the periplasm to the extracellular space. Although previous in vitro experiments have demonstrated highly specific interactions between CusF and CusB that are crucial for metal transfer to occur, the structural details of the interaction have not been determined. Here, the interactions between CusF and CusB are mapped through nuclear magnetic resonance (NMR) spectroscopy and chemical cross-linking coupled with high-resolution mass spectrometry to better understand how recognition and metal transfer occur between these proteins. The NMR (1)H-(15)N correlation spectra reveal that CusB interacts with the metal-binding face of CusF. In vitro chemical cross-linking with a 7.7 ? homobifunctional amine-reactive cross-linker, BS(2)G, was used to capture the CusF/CusB interaction site, and mass spectral data acquired on an LTQ-Orbitrap confirm the following two cross-links: CusF K31 to CusB K29 and CusF K58 to CusB K32, thus revealing that the N-terminal region of CusB interacts with the metal-binding face of CusF. The proteins transiently interact in a metal-dependent fashion, and contacts between CusF and CusB are localized to regions near their respective metal-binding sites.     

Periplasmic domain of CusA in an <Emphasis Type="Italic">Escherichia coli</Emphasis> Cu<Superscript>+</Superscript>/Ag<Superscript>+</Superscript> transporter has metal binding sites

The resistance nodulation division (RND)-type efflux systems are utilized in Gram-negative bacteria to export a variety of substrates. The CusCFBA system is the Cu⁺ and Ag⁺ efflux system in Escherichia coli, conferring resistance to lethal concentrations of Cu⁺ and Ag⁺. The periplasmic component, CusB, which is essential for the assembly of the protein complex, has Cu⁺ or Ag⁺ binding sites. The twelve-span membrane protein CusA is a homotrimeric transporter, and has a relatively large periplasmic domain. Here, we constructed the periplasmic domain of CusA by joining two DNA segments and then successfully expressed and purified the protein. Isothermal titration calorimetry experiments revealed Ag⁺ binding sites with Kds of 10⁻⁶–10⁻⁵ M. Our findings suggest that the metal binding in the periplasmic domain of CusA might play an important role in the function of the efflux pump.     

Direct metal transfer between periplasmic proteins identifies a bacterial copper chaperone

Transition metals require exquisite handling within cells to ensure that cells are not harmed by an excess of free metal species. In gram-negative bacteria, copper is required in only small amounts in the periplasm, not in the cytoplasm, so a key aspect of protection under excess metal conditions is to export copper from the periplasm. Additional protection could be conferred by a periplasmic chaperone to limit the free metal species prior to export. Using isothermal titration calorimetry, we have demonstrated that two periplasmic proteins, CusF and CusB, of the Escherichia coli Cu(I)/Ag(I) efflux system undergo a metal-dependent interaction. Through the development of a novel X-ray absorption spectroscopy approach using selenomethionine labeling to distinguish the metal sites of the two proteins, we have demonstrated transfer of Cu(I) occurs between CusF and CusB. The interaction between these proteins is highly specific, as a homologue of CusF with a 51% identical sequence and a similar affinity for metal, did not function in metal transfer. These experiments establish a metallochaperone activity for CusF in the periplasm of gram-negative bacteria, serving to protect the periplasm from metal-mediated damage.     

N-Terminal Region of CusB Is Sufficient for Metal Binding and Metal Transfer with the Metallochaperone CusF

Gram-negative bacteria, such as Escherichia coli, utilize efflux resistance systems in order to expel toxins from their cells. Heavy-metal resistance is mediated by resistance nodulation cell division (RND)-based efflux pumps composed of a tripartite complex that includes an RND-transporter, an outer-membrane factor (OMF), and a membrane fusion protein (MFP) that spans the periplasmic space. MFPs are necessary for complex assembly and have been hypothesized to play an active role in substrate efflux. Crystal structures of MFPs are available, however incomplete, as large portions of the apparently disordered N- and C-termini are unresolved. Such is the case for CusB, the MFP of the E. coli Cu(I)/Ag(I) efflux pump CusCFBA. In this work, we have investigated the structure and function of the N-terminal region of CusB, which includes the metal-binding site and is missing from previously determined crystal structures. Results from mass spectrometry and X-ray absorption spectroscopy show that the isolated N-terminal 61 residues (CusB-NT) bind metal in a 1:1 stoichiometry with a coordination site composed of M21, M36, and M38, consistent with full-length CusB. NMR spectra show that CusB-NT is mostly disordered in the apo state; however, some slight structure is adopted upon metal binding. Much of the intact protein's function is maintained in this fragment as CusB-NT binds metal in vivo and in vitro, and metal is transferred between the metallochaperone CusF and CusB-NT in vitro. Functional analysis in vivo shows that full-length CusB is necessary in an intact polypeptide for full metal resistance, though CusB-NT alone can contribute partial metal resistance. These findings reinforce the theory that the role of CusB is not only to bind metal but also to play an active role in efflux.     

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 Å 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.     

Periplasmic metal-resistance protein CusF exhibits high affinity and specificity for both CuI and AgI

The periplasmic protein CusF, as a part of the CusCFBA efflux complex, plays a role in resistance to elevated levels of copper and silver in Escherichia coli. Although homologues have been identified in other Gram-negative bacteria, the substrate of CusF and its precise role in metal resistance have not been described. Here, isothermal titration calorimetry (ITC) was used to demonstrate that CusF binds with high affinity to both Cu(I) and Ag(I) but not Cu(II). The affinity of CusF for Ag(I) was higher than that for Cu(I), which could reflect more efficient detoxification of Ag(I) given the lack of a cellular need for Ag(I). The chemical shifts in the nuclear magnetic resonance (NMR) spectra of CusF-Ag(I) as compared to apo-CusF show that the region of CusF most affected by Ag(I) binding encompasses three absolutely conserved residues: H36, M47, and M49. This suggests that these residues may play a role in Ag(I) coordination. The NMR spectra of CusF in the presence of Cu(II) do not indicate specific binding, which is in agreement with the ITC data. We conclude that Cu(I) and Ag(I) are the likely physiological substrates.     

The Cus efflux system removes toxic ions via a methionine shuttle

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.     

Periplasmic oxidized-protein repair during copper stress in E. coli: A focus on the metallochaperone CusF

Methionine residues are particularly sensitive to oxidation by reactive oxygen or chlorine species (ROS/RCS), leading to the appearance of methionine sulfoxide in proteins. This post-translational oxidation can be reversed by omnipresent protein repair pathways involving methionine sulfoxide reductases (Msr). In the periplasm of Escherichia coli, the enzymatic system MsrPQ, whose expression is triggered by the RCS, controls the redox status of methionine residues. Here we report that MsrPQ synthesis is also induced by copper stress via the CusSR two-component system, and that MsrPQ plays a role in copper homeostasis by maintaining the activity of the copper efflux pump, CusCFBA. Genetic and biochemical evidence suggest the metallochaperone CusF is the substrate of MsrPQ and our study reveals that CusF methionines are redox sensitive and can be restored by MsrPQ. Thus, the evolution of a CusSR-dependent synthesis of MsrPQ allows conservation of copper homeostasis under aerobic conditions by maintenance of the reduced state of Met residues in copper-trafficking proteins.     

Structural and metal binding characterization of the C-terminal metallochaperone domain of membrane fusion protein SilB from Cupriavidus metallidurans CH34

Detoxification of heavy metal ions in Proteobacteria is tightly controlled by various systems regulating their sequestration and transport. In Cupriavidus metallidurans CH34, a model organism for heavy metal resistance studies, the sil determinant is potentially involved in the efflux of silver and copper ions. Proteins SilA, SilB, and SilC form a resistance nodulation cell division (RND)-based transport system in which SilB is the periplasmic adaptor protein belonging to the membrane fusion protein (MFP) family. In addition to the four domains typical of known MFPs, SilB has a fifth additional C-terminal domain, called SilB(440-521), which is characterized here. Structure and backbone dynamics of SilB(440-521) have been investigated using nuclear magnetic resonance, and the residues of the metal site were identified from (15)N- and (13)C-edited HSQC spectra. The solution structure and additional metal binding experiments demonstrated that this C-terminal domain folds independently of the rest of the protein and has a conformation and a Ag(+) and Cu(+) binding specificity similar to those determined for CusF from Escherichia coli. The small protein CusF plays a role in metal trafficking in the periplasm. The similarity with CusF suggests a potential metallochaperone role for SilB(440-521) that is discussed in the context of simultaneous expression of different determinants involved in copper resistance in C. metallidurans CH34.     

Mechanism of ATPase-mediated Cu+ Export and Delivery to Periplasmic Chaperones: THE INTERACTION OF ESCHERICHIA COLI CopA AND CusF*

Cellular copper homeostasis requires transmembrane transport and compartmental trafficking while maintaining the cell essentially free of uncomplexed Cu^2+/+. In bacteria, soluble cytoplasmic and periplasmic chaperones bind and deliver Cu⁺ to target transporters or metalloenzymes. Transmembrane Cu⁺-ATPases couple the hydrolysis of ATP to the efflux of cytoplasmic Cu⁺. Cytosolic Cu⁺ chaperones (CopZ) interact with a structural platform in Cu⁺-ATPases (CopA) and deliver copper into the ion permeation path. CusF is a periplasmic Cu⁺ chaperone that supplies Cu⁺ to the CusCBA system for efflux to the extracellular milieu. In this report, using Escherichia coli CopA and CusF, direct Cu⁺ transfer from the ATPase to the periplasmic chaperone was observed. This required the specific interaction of the Cu⁺-bound form of CopA with apo-CusF for subsequent metal transfer upon ATP hydrolysis. As expected, the reverse Cu⁺ transfer from CusF to CopA was not observed. Mutation of CopA extracellular loops or the electropositive surface of CusF led to a decrease in Cu⁺ transfer efficiency. On the other hand, mutation of Met and Glu residues proposed to be part of the metal exit site in the ATPase yielded enzymes with lower turnover rates, although Cu⁺ transfer was minimally affected. These results show how soluble chaperones obtain Cu⁺ from transmembrane transporters. Furthermore, by explaining the movement of Cu⁺ from the cytoplasmic pool to the extracellular milieu, these data support a mechanism by which cytoplasmic Cu⁺ can be precisely directed to periplasmic targets via specific transporter-chaperone interactions.     

Alternative periplasmic copper-resistance mechanisms in Gram negative bacteria

Bacteria have evolved different systems to tightly control both cytosolic and envelope copper concentration to fulfil their requirements and at the same time, avoid copper toxicity. We have previously demonstrated that, as in Escherichia coli , the Salmonella cue system protects the cytosol from copper excess. On the other hand, and even though Salmonella lacks the CusCFBA periplasmic copper efflux system, it can support higher copper concentrations than E. coli under anaerobic conditions. Here we show that the Salmonella cue regulon is also responsible for the control of copper toxicity in anaerobiosis. We establish that resistance in this condition requires a novel CueR-controlled gene named cueP . A Δ cueP mutant is highly susceptible to copper in the absence of oxygen, but shows a faint phenotype in aerobic conditions unless other copper-resistance genes are also deleted, resembling the E. coli CusCFBA behaviour. Species that contain a cueP homologue under CueR regulation have no functional CusR/CusS-dependent Cus-coding operon. Conversely, species that carry a CusR/CusS-regulated cus operon have no cueP homologues. Even more, we show that the CueR-controlled cueP expression increases copper resistance of a Δ cus E. coli . We posit that CueP can functionally replace the Cus complex for periplasmic copper resistance, in particular under anaerobic conditions.     

A novel copper-binding fold for the periplasmic copper resistance protein CusF

We have determined the crystal structure of apo-CusF, a periplasmic protein involved in copper and silver resistance in Escherichia coli. The protein forms a five-stranded beta-barrel, classified as an OB-fold, which is a unique topology for a copper-binding protein. NMR chemical shift mapping experiments suggest that Cu(I) is bound by conserved residues H36, M47, and M49 located in beta-strands 2 and 3. These residues are clustered at one end of the beta-barrel, and their side chains are oriented toward the interior of the barrel. Cu(I) can be modeled into the apo-CusF structure with only minimal structural changes using H36, M47, and M49 as ligands. The unique structure and metal binding site of CusF are distinct from those of previously characterized copper-binding proteins.     

Proteomic identification of the Cus system as a major determinant of constitutive Escherichia coli silver resistance of chromosomal origin

Although silver is one of the most potent and rapidly acting toxic metals to bacteria, silver-resistant bacteria do exist with low incidence. A proteomic approach was employed to identify the silver resistance determinants of a silver-resistant Escherichia coli strain isolated from stepwise selection against increasing concentrations of silver (Li et al. J. Bacteriol 1997, 179, 6127-32). Two-dimensional gel electrophoresis and mass spectrometry analysis revealed that members of the CusCFBA copper/silver chemiosmotic efflux system were highly expressed in the silver-resistant strain but undetectable in the parental silver-sensitive strain. Disruption of the cus locus of the silver-resistant strain resulted in a decrease of the minimum inhibitory concentration of Ag (+) from more than 1 mM to 12 microM. These results suggest that the chromosomally encoded Cus system, which naturally controls the periplasmic copper concentrations, is selectable to confer a constitutive silver resistance phenotype.     

Crystal Structures of CusC Review Conformational Changes Accompanying 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.     

Unusual Cu(I)/Ag(I) coordination of Escherichia coli CusF as revealed by atomic resolution crystallography and X-ray absorption spectroscopy

Elevated levels of copper or silver ions in the environment are an immediate threat to many organisms. Escherichia coli is able to resist the toxic effects of these ions through strictly limiting intracellular levels of Cu(I) and Ag(I). The CusCFBA system is one system in E. coli responsible for copper/silver tolerance. A key component of this system is the periplasmic copper/silver-binding protein, CusF. Here the X-ray structure and XAS data on the CusF-Ag(I) and CusF-Cu(I) complexes, respectively, are reported. In the CusF-Ag(I) structure, Ag(I) is coordinated by two methionines and a histidine, with a nearby tryptophan capping the metal site. EXAFS measurements on the CusF-Cu(I) complex show a similar environment for Cu(I). The arrangement of ligands effectively sequesters the metal from its periplasmic environment and thus may play a role in protecting the cell from the toxic ion.