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How hydrophobic molecules traverse the outer membranes of Gram-negative bacteria

June 21, 2011
108 (27) 10929-10930
Research Article
Ligand-gated diffusion across the bacterial outer membrane
Bryan W. Lepore, Mridhu Indic [...] Bert van den Berg
The outer membrane (OM) of Gram-negative bacteria provides an effective barrier to their often-harsh extracellular milieu. In particular, the outer leaflet of the OM is not a canonical monolayer of phospholipids. Rather, it is composed of lipopolysaccharide (LPS), a molecule generally consisting of a core of Lipid A decorated with inner and outer core oligosaccharides. The oligosaccharides extend ∼30 Å above the plane of the lipid headgroups of the outer leaflet. As such, it is an effective permeability barrier against potentially harmful compounds (1). However, obviously, permeability is required for bacterial survival; no bacterium is an island, as it were. For example, uptake of nutrients is essential, and OM transport proteins are required to conduct this function. The recent paper of Lepore et al. (2) has significantly extended our understanding of how hydrophobic molecules are transported across the OM. With rare exception (e.g., ref. 3), virtually all OM proteins are β-barrels, consisting of an even number of eight to twenty-four of β-strands forming a pore-like structure. Many of these OM pore-like β-barrels are classified as porins, and most nutrient uptake is accomplished by them. The effective aperture of the porin is dictated by the number of β-strands, and the aperture size then dictates the size (and shape) of the solutes that can diffuse through them. Porins function passively, permitting the energy-independent diffusion of solute molecules with a molecular mass of 600 Da or less downhill across a concentration gradient, through the porin's β-barrel, and into the periplasm. Another class of energy-independent OM transporters uses low-affinity binding sites that effectively serve to amplify small concentration gradients at the site of the transporter (4).
One can envisage situations in which porins would be insufficient for nutrient uptake. One possibility is if the nutrient (or micronutrient) is too large to traverse the lumen of a porin and/or too scarce to generate a suitable concentration gradient for diffusion. Gram-negative bacteria handle this task, primarily necessary for iron uptake, by use of the TonB-dependent OM active transport system (5). The OM transporter of this system is a 22-stranded β-barrel, with a 100- to 150-aa region of the protein at or near its amino terminus. This amino-terminal luminal domain, sometimes referred to as the plug or hatch domain, occludes the β-barrel. The substrates for different transporters are typically iron siderophores, heme, and corrinoids (e.g., vitamin B12). Remarkably, other transporters of this family bind iron-binding proteins (e.g., transferrin) and “crack” the protein open to obtain the iron (6). Additionally, substrates other than organometallics have been identified (7). The OM does not possess an equilibrium proton motive force (pmf), nor do these transporters directly use ATP (or other molecules) to drive conformational change for active transport. Instead, a multiprotein complex (ExbB-ExbD-TonB) in the inner membrane couples the inner membrane pmf to OM active transport (810), via a coupling of the periplasm-spanning TonB protein to the transporter (11, 12). Although a mechanical force transduction model seems plausible (13), the molecular mechanism of this system remains unknown.
The other possibility, addressed by the recent paper of Lepore et al. (2) in PNAS, concerns uptake of hydrophobic molecules. The oligosaccharide moieties of LPS in the OM outer leaflet can extend out from the membrane surface by as much as 30 Å, providing a very effective barrier to hydrophobic molecules. The most frequently studied exemplar of OM transport of hydrophobic molecules is FadL (14), which is responsible for the uptake of long-chain fatty acids. The crystal structure of FadL revealed the protein to be a 14-stranded β-barrel with a luminal domain occluding the barrel (15). Although the presence of this domain is reminiscent of TonB-dependent transporters, FadL alone is sufficient for transport. A significant feature of the first FadL structures (Fig. 1A) is an inward-facing kink in the third β-strand (S3) of the barrel (15). Our inspection of other protein structures suggests that this “staved β-barrel” structural motif is present in a variety of bacterial OM proteins. TonB-dependent transporters, usher proteins involved in pilus secretion, and some porins contain staved β-barrel motifs. These proteins also share a marked asymmetry of barrel height (approximately normal to the surface of the OM). This asymmetry arises from variation in β-strand length and extracellular loop size. In FadL (1T1L), the maximum height is ∼84 Å. HasR, a TonB-dependent transporter, has a similar maximum height. When the long loops of FadL and HasR are aligned structurally, the staved β-barrel motifs align as well (Fig. 1B). Moreover, there are ample data on the role of residues in this region affecting the function of each of these different protein families. This suggests, highly speculatively at this juncture, the possibility of mechanistic similarity between these disparate protein families.
Fig. 1.
Possible conservation of a “staved β-barrel” structural motif in bacterial OM proteins. (A) FadL (1T1L) is a 14-stranded β-barrel, with extracellular loops of varying lengths that give rise to a pronounced asymmetry in barrel height. (B) Alignment of the longest loops of FadL (blue, 1T1L) with the longest loops of the TonB-dependent heme transporter HasR (red, 3CSL). With this loop alignment, the “staved β-barrel” motifs of the two proteins are in similar locations. Mutations in this region affect function in essentially all the proteins that we have found to contain this motif, possibly suggestive of some cross-family conservation of conformational change in disparate functions. (Note: The luminal domain of HasR has been erased for ease of viewing.)
Also, structures of FadL contain ordered and bound detergent molecules. The presence of such ordered detergent molecules in membrane protein crystal structures is not unusual (16). However, what is unusual (and fortuitous) is that the bound detergent molecules in FadL provide suggestions of, and evidence for, a transport pathway. Different crystal forms of FadL display different conformations of the luminal domain amino terminus and different positions of bound detergent. In one structure, a molecule of lauryldimethylamine-N-oxide (LDAO) occupies a putative high-affinity substrate binding site and has a significant interaction with residue F3 of the luminal domain. In another structure, from a different crystal form, the LDAO molecule is “displaced” from the high-affinity site and is emergent from the side of the barrel, through the S3 kink.
It is axiomatic that structural observations of crystal packing, and of binding of nonphysiological substrates, should not be overinterpreted. However, it is equal (or greater) folly to consign, a priori, any such structural results to functional irrelevance. Subsequent publications by the van den Berg laboratory (2, 17) are superlative examples of how additional experiments using other techniques and approaches can critically assess the applicability of in crystal results to in vivo function. In the study by Hearn et al. (17), mutations that reduce the size of the aperture caused by the kinked S3 strand abrogate function; these, and other results in this paper, provided rather compelling evidence for an “escape through kink” substrate pathway. This lateral diffusion paradigm also describes multidrug ATP-binding cassette (ABC) transporters, where highly chemically diverse hydrophobic molecules (e.g., drugs) all serve as efflux substrates. Lepore et al. (2) continue this mechanistic exploration, with the discovery and characterization of gating behavior in FadL. Through a clever and comprehensive series of experiments using in vivo transport assays, fluorescence spectroscopy, and crystallography, Lepore et al. (2) demonstrate that, in the absence of occupancy of the high-affinity (sub-μM Kd) substrate binding site, the membrane-facing exit channel is not formed (2). Substrate binding at this site drives a conformational change in the portion of the luminal domain that includes residues comprising the high-affinity binding site. This conformational change drives both reduction of binding affinity for substrate release and formation of the exit channel. The manner in which the conformational changes are coupled is not yet completely clear. A previous paper in PNAS by Ferguson et al. (18) used statistical coupling analysis to map out the allosteric pathway in FecA, a TonB-dependent transporter; a similar approach may be productive for FadL (or other membrane transport proteins). This elegant transport mechanism likely applies to other members of the FadL family and indicates a degree of mechanistic complexity not typically associated with OM β-barrel proteins. Future structure-function studies of other β-barrels may reveal additional subtle and intricate mechanisms, which may be able to be exploited for novel antimicrobial lead compound discovery, bioremediation, and other societally beneficial applications.


OM transport research in the M.C.W. laboratory is funded by National Institutes of Health Grant NIGMS 079800.


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Published in

Go to Proceedings of the National Academy of Sciences
Proceedings of the National Academy of Sciences
Vol. 108 | No. 27
July 5, 2011
PubMed: 21693645


    Submission history

    Published online: June 21, 2011
    Published in issue: July 5, 2011


    OM transport research in the M.C.W. laboratory is funded by National Institutes of Health Grant NIGMS 079800.


    See companion article on page 10121 of issue 25 in volume 108.



    Michael C. Wiener1 [email protected]
    Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA 22908-0866
    Peter S. Horanyi
    Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA 22908-0866


    To whom correspondence should be addressed. E-mail: [email protected].
    Author contributions: M.C.W. and P.S.H. wrote the paper.

    Competing Interests

    The authors declare no conflict of interest.

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      How hydrophobic molecules traverse the outer membranes of Gram-negative bacteria
      Proceedings of the National Academy of Sciences
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