The Crystal Structure of OprG from Pseudomonas aeruginosa, a Potential Channel for Transport of Hydrophobic Molecules across the Outer Membrane

Background: The outer membrane (OM) of Gram-negative bacteria provides a barrier to the passage of hydrophobic and hydrophilic compounds into the cell. The OM has embedded proteins that serve important functions in signal transduction and in the transport of molecules into the periplasm. The OmpW family of OM proteins, of which P. aeruginosa OprG is a member, is widespread in Gram-negative bacteria. The biological functions of OprG and other OmpW family members are still unclear. Methodology/Principal Findings: In order to obtain more information about possible functions of OmpW family members we have solved the X-ray crystal structure of P. aeruginosa OprG at 2.4 A ˚ resolution. OprG forms an eightstranded b-barrel with a hydrophobic channel that leads from the extracellular surface to a lateral opening in the barrel wall. The OprG barrel is closed off from the periplasm by interacting polar and charged residues on opposite sides of the barrel wall. Conclusions/Significance: The crystal structure, together with recent biochemical data, suggests that OprG and other OmpW family members form channels that mediate the diffusion of small hydrophobic molecules across the OM by a latera

Structural overview of OprG.

<p>Views from the side (a) and from the extracellular side (b; top panel) and periplasmic side (b; bottom panel). β-strands are colored blue, α-helices red and loops green. Selected extracellular loops are indicated. The approximate positions of the outer membrane interface regions are indicated by horizontal lines. (c) Structural comparison between OprG (blue) and <i>E. coli</i> OmpW (red). Loops have been smoothed for clarity. This and the following figures were made with PYMOL <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015016#pone.0015016-DeLano1" target="_blank">[32]</a>.</p

ClustalW alignment of OprG and other OmpW family members.

<p>The observed secondary structure of OprG is shown above the alignment, with β-strands (S) in blue and the α-helix in loop l3 in red. OprG residues are colored as follows: red; hydrophobic with sidechains pointing inwards, purple; polar/charged with sidechains pointing inwards and green; absolutely conserved prolines lining the lateral opening. The following orthologs have been aligned: Pa, <i>Pseudomonas aeruginosa</i> OprG; Pp, <i>Pseudomonas putida</i> OprG; Ec, <i>E. coli</i> OmpW; Ah, <i>Aeromonas hydrophila</i> OmpW; Vc, <i>Vibrio cholerae</i> OmpW; AlkL, <i>Pseudomonas oleovorans</i> AlkL; DoxH, <i>Pseudomonas</i> sp. (strain C18) DoxH.</p

Data collection and refinement statistics for OprG.

1<p>Values in parentheses are for the highest resolution shell.</p

Proposed transport mechanism for OmpW family members.

<p>(a) Cartoon of <i>Pseudomonas aeruginosa</i> FadL (PDB ID: 3DWO) viewed from the side <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015016#pone.0015016-Hearn1" target="_blank">[8]</a>. The hatch domain, closing off the barrel on the periplasmic side, is colored red. Bound detergent molecules delineating the hydrophobic transport channel are shown as space-filling models in blue. An arrow marks the lateral opening into the membrane. (b) Surface slab through the center of OprG, showing the hydrophobic channel as a dark tube. Residues Trp170 and Val65, forming the bottom of the channel, are shown in red. An arrow marks the lateral opening into the membrane. (c) Schematic model for transport of small hydrophobic substrates (depicted as octane in green) by members of the OmpW family. The bottom of the channel is shown in red.</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