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

a Potential Channel for Transport of Hydrophobic Molecules across the Outer Membrane. PLoS ONE 5(11): e15016. doi:10.1371/journal.pone.0015016 The Crystal Structure of OprG from Pseudomonas aeruginosa , a Potential Channel for Transport of Hydrophobic Molecules across the Outer Membrane Debra S. Touw 0 Dimki R. Patel 0 Bert van den Berg 0 Raphael H. Valdivia, Duke University, United States of America 0 Program in Molecular Medicine, University of Massachusetts Medical School , Worcester, Massachusetts , United States of America 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 lateral diffusion mechanism similar to that of E. coli FadL. - Funding: This work has been supported by National Institutes of Health grant 5R01GM085785 (NIGMS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic human pathogen associated with lung infections in cystic fibrosis patients and nosocomial infections [1]. It has the ability to grow on a diverse range of carbon sources [2]. Like in other Gram-negative bacteria, the outer membrane (OM) creates an effective protective barrier to the permeation of small molecules [3]. Due to the impermeability of the OM, gramnegative bacteria have evolved three major classes of outer membrane proteins to facilitate the transport of nutrients into the cell: active transporters, general porins, and diffusion-driven specific transporters [2]. The TonB-dependent active transporters (e.g. FhuA and FepA in E. coli) are used for the uptake of large molecules such as iron-siderophore complexes [4,5]. General porins (e.g. E coli OmpF) occur in many Gram-negative bacteria and form water-filled channels that facilitate the non-specific diffusion of small hydrophilic compounds across the outer membrane [6]. P. aeruginosa and other pseudomonads lack general porins and instead have a large number of substrate-specific channels for nutrient transport [2]. Due to the lack of porins, the OM of P. aeruginosa is highly impermeable, making it resistant to many antibiotics [1]. Besides small water-soluble compounds, the OM also forms an effective barrier against the permeation of hydrophobic molecules due to the presence of lipopolysaccharide (LPS) on the outside of the cell. The diffusion of many hydrophobic compounds across the OM is mediated by proteins belonging to the FadL family of OM channels [7]. FadL-mediated transport occurs via a mechanism involving lateral diffusion of the substrate from the barrel lumen, via an opening in the barrel wall, into the OM [8]. The OmpW family of small OM proteins is widespread among Gram-negative bacteria, with orthologs found in a, b, c, and d proteobacteria. Recent research in Vibrio cholerae has shown that OmpW is very immunogenic and present in all V. cholerae strains analyzed to date [9,10,11]. E. coli OmpW has been shown to be a receptor for colicin S4, which is part of the E. coli bacteriocin defense system [9,12]. In addition, recent proteomic profiling studies have suggested a role for OmpW in osmoregulation [13]. However, none of these studies provid11/29/2010es any direct evidence for OmpW protein function. Perhaps the best clue for a putative function for OmpW family members is provided by sequence similarity to OM proteins present in operons dedicated to the biodegradation of small, hydrophobic molecules such as medium-chain alkanes (AlkL) and naphthalene (NahQ and DoxH) [14,15,16]. The crystal structure of E. coli OmpW showed an eight-stranded b-barrel with a hydrophobic channel containing a LDAO detergent molecule, supporting the notion that OmpW could mediate diffusion of hydrophobic molecules [17]. The (...truncated)