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

Substrate Specificity within a Family of Outer Membrane Carboxylate Channels

Characterization of a large family of outer membrane channels from gram-negative bacteria suggest how they can thrive in nutrient-poor environments and how channel inactivation can contribute to antibiotic resistance

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

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

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

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 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

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

Transmembrane passage of hydrophobic compounds through a protein channel wall

Membrane proteins that transport hydrophobic compounds have important roles in multi-drug resistance and can cause a number of diseases, underscoring the importance of protein-mediated transport of hydrophobic compounds. Hydrophobic compounds readily partition into regular membrane lipid bilayers, and their transport through an aqueous protein channel is energetically unfavourable. Alternative transport models involving acquisition from the lipid bilayer by lateral diffusion have been proposed for hydrophobic substrates. So far, all transport proteins for which a lateral diffusion mechanism has been proposed function as efflux pumps. Here we present the first example of a lateral diffusion mechanism for the uptake of hydrophobic substrates by the Escherichia coli outer membrane long-chain fatty acid transporter FadL. A FadL mutant in which a lateral opening in the barrel wall is constricted, but which is otherwise structurally identical to wild-type FadL, does not transport substrates. A crystal structure of FadL from Pseudomonas aeruginosa shows that the opening in the wall of the beta-barrel is conserved and delineates a long, hydrophobic tunnel that could mediate substrate passage from the extracellular environment, through the polar lipopolysaccharide layer and, by means of the lateral opening in the barrel wall, into the lipid bilayer from where the substrate can diffuse into the periplasm. Because FadL homologues are found in pathogenic and biodegrading bacteria, our results have implications for combating bacterial infections and bioremediating xenobiotics in the environment