Escherichia coli OmpW belongs to a family of small outer membrane proteins that are widespread in Gram-negative bacteria. Their functions are unknown, but recent data suggest that they may be involved in the protection of bacteria against various forms of environmental stress. To gain insight into the function of these proteins we have determined the crystal structure of E. coli OmpW to 2.7-Å resolution. The structure shows that OmpW forms an 8-stranded ␤-barrel with a long and narrow hydrophobic channel that contains a bound n-dodecyl-N,N-dimethylamine-N-oxide detergent molecule. Single channel conductance experiments show that OmpW functions as an ion channel in planar lipid bilayers. The channel activity can be blocked by the addition of n-dodecyl-N,Ndimethylamine-N-oxide. Taken together, the data suggest that members of the OmpW family could be involved in the transport of small hydrophobic molecules across the bacterial outer membrane. The outer membrane (OM)2 of Gram-negative bacteria is a protective barrier that hinders the permeability of both hydrophilic and hydrophobic compounds, because of the presence of lipopolysaccharide (LPS) within the outer leaflet of the OM (1). To obtain nutrients and other molecules that are necessary for growth and function of the cell, Gramnegative bacteria have channels within their OM that facilitate uptake of these molecules. With respect to the transport of small, hydrophilic substances, these channels can be divided in three classes, based on their mode of transport (1): general porins, substrate-specific transporters, and active transporters. A wealth of structural and functional information is available for many of these OM channel proteins, which form monomeric or trimeric barrels that are each composed of 12-22 antiparallel ␤-strands. In addition to OM proteins with established transport functions, the OM also contains a considerable number of smaller, monomeric ␤-barrels that are composed of 8 or 10 ␤-strands. These proteins have been implicated in a wide range of functions including OM lipid metabolism, cell adhesion, and structural functions. One of these small OM proteins is OmpA from Escherichia coli, which belongs to a protein family with a number of established and putative functions, the most important of which is to provide structural stability to the cell via interactions of its C-terminal domain with the periplasmic peptidoglycan layer (1). Another member of the small OM protein family is NspA from Neisseria meningitidis, which belongs to the Opa family of proteins that are thought to mediate adhesion to host cells (2).A fundamental question is whether these small barrels can function as transport channels. Arguing against this possibility are the crystal structures that have been determined for several of these proteins, and which do not show continuous channels that would be consistent with transport functions. On the other hand, it has been shown that, at least in vitro, OmpA forms both small and large ion channels (3) and is permeable to larger uncha...

OprD proteins form a large family of substrate-specific outer-membrane channels in Gram-negative bacteria. We report here the X-ray crystal structure of OprD from Pseudomonas aeruginosa, which reveals a monomeric 18-stranded beta-barrel characterized by a very narrow pore constriction, with a positively charged basic ladder on one side and an electronegative pocket on the other side. The location of highly conserved residues in OprD suggests that the structure represents the general architecture of OprD channels.

Membrane proteins that transport hydrophobic compounds play important roles in multi-drug resistance1–3 and can cause a number of diseases4,5, underscoring the importance of protein-mediated transport of hydrophobic compounds. Hydrophobic compounds readily partition into regular membrane lipid bilayers6, and their transport through an aqueous protein channel is energetically unfavourable3. Alternative transport models, involving acquisition from the lipid bilayer by lateral diffusion have been proposed for hydrophobic substrates3,4,7–12. To date, 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 (LCFA) 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 β-barrel is conserved and delineates a long, hydrophobic tunnel that could mediate substrate passage from the extracellular environment, through the polar lipopolysaccharide layer and, via the lateral opening in the barrel wall, into the lipid bilayer from where the substrate can diffuse into the periplasm. Since FadL homologues are found in pathogenic and biodegrading bacteria, our results have implications for combating bacterial infections and bioremediating xenobiotics in the environment.

BackgroundThe 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 FindingsIn 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 Å resolution. OprG forms an eight-stranded β-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/SignificanceThe 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.

Bacterial biodegradation of hydrocarbons, an important process for environmental remediation, requires the passage of hydrophobic substrates across the cell membrane. Here, we report crystal structures of two outer membrane proteins, Pseudomonas putida TodX and Ralstonia pickettii TbuX, which have been implicated in hydrocarbon transport and are part of a subfamily of the FadL fatty acid transporter family. The structures of TodX and TbuX show significant differences with those previously determined for Escherichia coli FadL, which may provide an explanation for the substrate-specific transport of TodX and TbuX observed with in vivo transport assays. The TodX and TbuX structures revealed 14-stranded ␤-barrels with an N-terminal hatch domain blocking the barrel interior. A hydrophobic channel with bound detergent molecules extends from the extracellular surface and is contiguous with a passageway through the hatch domain, lined by both hydrophobic and polar or charged residues. The TodX and TbuX structures support a mechanism for transport of hydrophobic substrates from the extracellular environment to the periplasm via a channel through the hatch domain.membrane protein ͉ x-ray structure