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

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

The copper centers of tyramine β-monooxygenase and its catalytic-site methionine variants: an X-ray absorption study

Tyramine β-monooxygenase (TBM) is a member of a family of copper monooxygenases containing two noncoupled copper centers, and includes peptidylglycine monooxygenase and dopamine β-monooxygenase. In its Cu(II) form, TBM is coordinated by two to three His residues and one to two non-His O/N ligands consistent with a [CuM(His)2(OH2)2–CuH(His)3(OH2)] formulation. Reduction to the Cu(I) state causes a change in the X-ray absorption spectroscopy (XAS) spectrum, consistent with a change to a [CuM(His)2S(Met)–CuH(His)3] environment. Lowering the pH to 4.0 results in a large increase in the intensity of the Cu(I)–S extended X-ray absorption fine structure (EXAFS) component, suggesting a tighter Cu–S bond or the coordination of an additional sulfur donor. The XAS spectra of three variants, where the CuM Met471 residue had been mutated to His, Cys, and Asp, were examined. Significant differences from the wild-type enzyme are evident in the spectra of the reduced mutants. Although the side chains of His, Cys, and Asp are expected to substitute for Met at the CuM site, the data showed identical spectra for all three reduced variants, with no evidence for coordination of residue 471. Rather, the K-edge data suggested a modest decrease in coordination number, whereas the EXAFS indicated an average of two His residues at each Cu(I) center. These data highlight the unique role of the Met residue at the CuM center, and pose interesting questions as to why replacement by the cuprophilic thiolate ligand leads to detectable activity whereas replacement by imidazole generates inactive TBM

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

P5-11-10: Reproducibility and Robustness of the FDA Approved INFORM HER2 Dual ISH DNA Probe Cocktail Assay.

Abstract Background: Amplification and/or HER2 overexpression is associated with poor clinical outcome for patients with invasive breast carcinoma. Determination of HER2 status dictates eligibility of patients for trastuzumab (Herceptin) therapy, which has been shown to improve prognosis. We present data from the inter-observer reproducibility and robustness studies of the FDA approved INFORM HER2 Dual ISH DNA Probe Cocktail (HER2 Dual ISH) assay, which is an alternative to FISH. The assay is fully automated and is scored using light microscopy providing morphological context. Methods: Ninety-five human invasive breast carcinoma specimens (a mix of amplified and non-amplified cases) were stained in a study, using the HER2 Dual ISH assay. The slides were enumerated by two qualified readers. The same cohort was stained using Path Vysion FISH and read by one qualified reader. To verify the robustness of the HER2 Dual ISH assay, customer selectable options for factors known to impact the staining quality and intensity, which can be varied for troubleshooting, were tested on human invasive breast carcinoma cases and xenografts. The slides were evaluated by one qualified reader. For both studies all assay steps were fully automated on a VENTANA BenchMark XT automated slide stainer using silver metallographic and red colorimetric detections for Dual ISH. Results: In the inter-observer study the first pass staining success rate was &amp;gt;93% for the HER2 Dual ISH assay, while the FISH first pass staining success rate was 59%. Some cases were rescued after a second attempt, which yielded a second pass staining success rate of 80% for FISH and 97% for HER2 Dual ISH. The negative, positive and overall agreement rates and 95% confidence intervals for the 61 clinical samples of this cohort that were enumerable with both FISH and the HER2 Dual ISH assay are 94.7 (75.4−99.1), 95.2 (84.2−98.7), 95.1 (86.5−98.3) and 89.5 (68.6−97.1), 97.6 (87.7−99.6), 95.1 (86.5−98.3) for reader A vs. FISH and reader B vs. FISH respectively. The positive and negative agreement rates for the inter-observer reproducibility of the HER2 Dual ISH assay were 94.7 and 97.6 %, respectively. In robustness testing, the recommended staining protocol was shown to be effective. All conditions tested resulted in appropriate staining (except for mildest cell conditioning and protease options). Conclusion: The fully automated FDA approved INFORM HER2 Dual ISH assay is robust, reproducible and concordant with the manual FISH assay in determining HER2 status in invasive breast carcinoma. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-11-10.</jats:p