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 packing of CusC.

<p>Cartoon representation of the packing of CusC within the crystal, with the molecule colored by domain (β-barrel, blue; α-barrel, red; equatorial domain, yellow).</p

Electrostatic potentials of CusC.

<p>Surface representations of the outside (A) and inside (B) of CusC colored by charge (red; negative -40 kT/e, blue; positive +40 kT/e). The views in (C) and (D) are from the extracellular side and periplasmic side, respectively. The figures were made with the ABSP plug-in within PYMOL.</p

Structural overview of CusC.

<p>(A) Rainbow representation of the CusC monomer, colored from blue (N-terminus) to red (C-terminus). Selected strands (S) and extracellular loops (L) are indicated, as well as the N- and C-terminus. Residues 21–31 are not visible in the electron density, presumably because they are disordered. (B) Ribbon representation of the CusC trimer, colored by domain (blue; β-barrel, red; α-barrel, yellow; equatorial domain). The different monomers within the trimer have been indicated with different color tints. (C) Ribbon representation of the CusC trimer, colored by B-factor value (blue, low B-factors; red, high B-factors). The average B-factors for the β-barrel, α-barrel and equatorial domain are 47, 26 and 36 Ų (all atoms). This and the following figures were made with PYMOL <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015610#pone.0015610-The1" target="_blank">[21]</a>.</p

CusC is likely to be tri-acylated at the N-terminus.

<p>(A) Stereoview of the N-terminal six residues of CusC, with 2F_o-F_c density shown as a blue mesh (contoured at 1.0 σ). The three acyl chains attached to Cys1 are labeled. (B) Stereoview from the side, showing the location of the lipid anchor relative to the belt of aromatic residues (purple stick models) that delineates the interface of the inner leaflet of the OM. β-strands are colored yellow, α-helices green and loops grey.</p

Structural comparisons between CusC, TolC and OprM.

<p>Stereoview of superimposed ribbon models of CusC (orange) and TolC (green; PDB-code 1EK9), viewed from the extracellular side (A) and from the side (B). The Cα r.m.s.d. between CusC and TolC is 1.61 Å. Panels (C) and (D) show the superposition of CusC and OprM (blue), the structures of which have a Cα r.m.s.d. of 1.35 Å.</p

Structural comparisons of the intra- and extracellular openings of OMF proteins.

<p>Surface views from the extracellular side (top row) and from the periplasmic side (bottom row) of CusC (A), TolC (B), <i>P. aeruginosa</i> OprM (C; PDB-code 1WP1) and <i>Vibrio cholerae</i> VceC (D; PDB-code 1YC9).</p

Crystallographic parameters for CusC.

1<p>Values in parentheses are for the highest resolution shell.</p>2<p>R_work = Σ|Fo−Fc|/ΣFo. R_free is the cross-validation of R-factor, with 5% of the total reflections omitted in model refinement.</p>3<p>The outliers of the Ramachandran plot (Asn100, Ser311) are located in the tips of the extracellular loops, which are poorly ordered.</p