Long Chain Fatty Acid Transporter from E. coli (EcFadL)
Created by Shane Hodson
The long-chain fatty acid (LCFA) transporter FadL from Escherichia coli (EcFadL) is a transport channel found in the bacterial outer membrane (OM) which, as the name implies, facilitates the uptake of LCFAs (1,3). EcFadL (PDB ID = 3pf1) is the most studied protein of the larger FadL family, a class of proteins involved in the transport of hydrophobic compounds across the OM of Gram-negative bacteria (1). In fact, FadL proteins are the only known OM transport proteins capable of moving hydrophobic molecules, making them vital to cell function (3). Other examples of FadL family channels are TodX from Pseudomonas putida and TbuX from Ralstonia pickettii, which both mediate the transport of aromatic hydrocarbon compounds (6).
Acquired from the extracellular environment, long-chain fatty acids provide cells with energy and carbon necessary for a variety of cell functions (7). In general, hydrophobic compounds can easily diffuse across biological membranes; as such, lipid bilayers do not typically form a permeability barrier for largely-hydrophobic LCFAs (3). However, the structure of the OM of Gram-negative bacteria is fairly unique: it consists of an inner layer of phospholipids and an outer layer of lipopolysaccharide (LPS). The structure of LPS – a hydrophobic lipid tail attached to a variety of sugars – is critical to the permeability characteristics of the OM. Due to the thick, polar sugar layer, the OM acts as a barrier for hydrophobic molecules such as LCFAs. As a result, FadL transport proteins such as EcFadL are absolutely essential for the uptake of hydrophobic compounds across the OM of Gram-negative bacteria. Once past the OM, LCFAs and other hydrophobic molecules can diffuse across the periplasm and through the inner membrane to enter the cell (the periplasm is the aqueous region between the inner and outer membranes) (3).
EcFadL is a monomeric protein of 424 residues with a molecular weight of 46273.89 Da and an isoelectric point (pI) of 5.25. The secondary structure of EcFadL is 13% helical (10 helices, both α-helices and 3/10-helices) and 54% β-sheet (24 strands), intermixed with sections of random coil (1). Similar to most OM proteins, EcFadL contains a distinct “barrel” domain constructed from 14 antiparallel β-strands. This β-barrel is approximately 50 Å in length, long enough to span the OM. Nevertheless, the barrel does not constitute an open channel between the external environment and the periplasm (5). The 42 residues at the N-terminal of the protein pack to form a "plug" domain that fills the barrel near its opening to the periplasmic space; this plug contains two 3/10-helices and one α-helix (1,5). One of these 3/10-helices contains the sequence NPA, which is conserved in all FadL transport proteins. The side chain of Asn-33 in this sequence forms an important hydrogen bond with the backbone carbonyl of Gly-21, helping to fold the compact plug domain (4).
Another important structural feature of EcFadL is an inward-pointing kink in one of the strands (S3) of the β-barrel from Thr-99 to Ala-105. Although the barrel’s β-sheet hydrogen bonding is disrupted, this kink is stabilized by interactions with an antiparallel β-strand from Asn-101 to Gly-103 and with residues Phe-3 to Leu-5. There is a hydrogen bond between Phe-3 and Gly-103, which may be consequential given that Gly-103 is conserved in all FadL channels (5). The significance of this kink is that it forms a lateral hole or gap in the side of the barrel, which is important to the channel’s transport mechanism (3,5).
The extracellular end of EcFadL has both a low- and high-affinity long-chain fatty acid binding site, designated the hydrophobic groove and hydrophobic pocket, respectively. The hydrophobic groove is found between two extracellular loops (L3 and L4) extending out from the β-barrel domain. Adjacent to this site and also between these loops is the hydrophobic pocket, which is situated down within the β-barrel. (Note: while EcFadL normally binds LCFAs, the ligands shown in these slides are (hydroxyethyloxy)tri(ethyloxy)octane (C8E4), a detergent used in protein purification.) Given the largely hydrophobic nature of LCFAs, this high-affinity binding site is comprised of primarily hydrophobic amino acid residues; contributed by several strands of the β-barrel, these 15 hydrophobic residues are largely conserved among FadL transport channel proteins. The only charged residues within the high-affinity binding site are Arg-157, Lys-317, and Glu-319 (in this image, blue spheres represent positive charges and red spheres represent negative charges). The positive charges carried by the arginine and lysine allow them to interact with the negatively charged carboxylic acid head of an LCFA substrate and so better bind the molecule (5).
One final structural feature to note is an important linkage between the plug domain and the high-affinity binding site. Although the majority of the plug domain is located towards the periplasmic end of the protein, the N-terminus stretches up towards the hydrophobic pocket such that Phe-3 is actually part of the high-affinity binding site (5). This extension is stabilized by an ionic interaction between the α-amino group of Ala-1 (positively charged) and the carboxyl group of Asp-348 (negatively charged). The importance of this interaction, and thus of Asp-348, is indicated by the fact that this residue is conserved in all FadL channel proteins involved in LCFA transport (1).
The proposed mechanism for LCFA transport in EcFadL is a product of the various important structural features that have been described. The process begins with a LCFA substrate being captured by the low-affinity binding site, soon followed by the diffusion of the LCFA into the neighboring high-affinity binding site (2). The binding of the substrate in the hydrophobic pocket causes Phe-3 to be perturbed, which in turn induces a conformational change in the N-terminus. This conformational change involves breaking the ionic interaction between Ala-1 and Asp-348 and the displacement of Phe-3 from the high-affinity binding site. The removal of Phe-3 (as well as its neighbors Ala-1 and Gly-2) is key, as this action opens the channel and allows the LCFA to diffuse out through the lateral hole in the β-barrel (1). The LCFA exits the protein into the outer LPS layer of the OM, positioned such that the negatively charged carboxylic acid head will interact with the polar LPS sugars and the hydrophobic acyl chain will interact with the hydrophobic LPS lipid tails. After being flipped to the inner layer of the OM, the LCFA is free to diffuse into the periplasmic space and eventually through the inner membrane into the cell (3).
EcFadL is somewhat unique among ligand-gated channels in that the ligand that opens the channel is also the substrate that is transported by the channel. In the transport mechanism just described, an LCFA (acting as a ligand) causes the channel to open by binding to the high-affinity binding site, and is subsequently able to pass through the channel (then acting as a transported substrate). Comparison of the closed (PDB ID = 1t16) and open (PDB ID = 1t1l) conformations of EcFadL indicates that the structures are approximately the same except for the positioning of the N-terminus, which in the open conformation is shifted away from the high-affinity binding site. This observation agrees with the proposed transportation mechanism. The protein named at the beginning of this paper (PDB ID = 3pf1) is actually a mutant form of EcFadL in which Asp-348 is changed to Ala-348 (i.e., the D348A mutant). The crystal structure for this mutant is nearly indistinguishable from that of the closed wild-type EcFadL, yet the mutant demonstrates greatly impaired LCFA transport (1).
One bioinformatics search used to further investigate EcFadl is the Position-Specific Iterated Basic Local Alignment Search Tool (PSI-BLAST). PSI-BLAST is designed to find proteins with similar primary structure to a given protein (the query protein). Subject proteins are given an “E value” based on their sequence homology to the query protein, with a value under 0.05 typically indicating that the homology is significant. The protein chosen for comparison with EcFadL was TodX from Pseudomonas putida (PDB ID = 3brz), which has an E value of 0.05 and a percent identity of 20%. Although ideally a protein with a somewhat smaller E value would be chosen, options were extremely limited by the small number of subject proteins with PDB entries.
Another bioinformatics search used to investigate EcFadL is the Dali Server. The Dali Server is designed to find proteins with similar tertiary structure to a given query protein, with each subject protein being assigned a Z-score based on its similarity to the query. In general, a Z-score above 2 indicates that a subject has significant similarities to the query. Again, the protein chosen for comparison with EcFadL was TodX from Pseudomonas putida (PDB ID = 3brz), which has a Z-score of 35.4 (8). This protein was picked because it has an E value and a Z-score within the desired ranges, and because it is in the FadL family of hydrophobic compound transport channels found in the OM of Gram-negative bacteria. Furthmore, TodX provides an instructive example of how proteins with ostensibly similar structures can have differing functions (6).
As members of the same FadL family of transport proteins, EcFadL and TodX share some distinct structural similarities (also, see superimposed comparison). To begin, both proteins contain the 14 antiparallel β-strand barrel so common to OM transport proteins. Furthermore, both proteins have a compact plug domain at the N-terminus that features three helices – two 310-helices and one α-helix (3, 6). This plug domain fills the barrel near its opening to the periplasmic space; however, the exact positioning of this domain in TodX varies noticeably from its positioning in EcFadL. The result of this shift is significant, as the plug placement in TodX leaves open a small channel through the domain, a channel lined with both hydrophobic and hydrophilic residues. Therefore, unlike EcFadL, TodX contains a pathway between the extracellular environment and the periplasmic space. The existence of this passageway is also due in part to the less prominent kink in the β-barrel wall of TodX; in fact, the kink is so minimal in this protein that the stabilizing hydrogen bonding network between the β-strands involved remains unbroken (6).
Another significant difference between EcFadL and TodX involves the positioning of the extracellular loops. While in EcFadL two of these loops extend out into the extracellular space, all of the loops in TodX remain close to the barrel. The most functionally significant of these loops is L3, which is comprised of two antiparallel α-helices (like in EcFadL) and rests flat on top of the barrel domain. The inside of this loop is lined with hydrophobic residues and is called the hydrophobic cleft, producing (in conjunction with other neighboring loops and their hydrophobic residues) a hydrophobic channel that leads all the way to the N-terminal plug domain. This structure stands in contrast to the low- and high-affinity LCFA binding sites (i.e., the hydrophobic groove and the hydrophobic pocket found between loops L3 and L4) found in EcFadL (6).
The structural differences between TodX and EcFadL are manifested in the differing functions of the proteins. Foremostly, the substrates transported by the proteins are different: EcFadL transports LCFAs while TodX transports aromatic hydrocarbon compounds such as benzene, toluene, ethylbenzene, and xylene. This affinity for different compounds is a result of structural differences in the binding sites of the proteins. As discussed previously, the high-affinity binding site of EcFadL, while predominately hydrophobic, contains two positively charged residues capable of interacting with the negatively charged carboxylic acid head group of a LCFA. The hydrophobic cleft of TodX, however, contains no such residues, giving it poor affinity for LCFAs. Instead, the TodX binding cleft is ideally suited for entirely hydrophobic molecules, which is exactly what is observed with the transport of aromatic hydrocarbon substrates (6).
The structural differences between TodX and EcFadL also potentially suggest a different mechanism for substrate transportation in TodX. While lateral diffusion through the barrel wall is still a possibility (the kink/hole is still present, although less pronounced), the presence of the channel through the plug domain gives the potential for substrate passage directly into the periplasm (4,6). In this mechanism, an aromatic hydrocarbon molecule is first captured by the hydrophobic cleft and subsequently diffuses down the hydrophobic channel towards the N-terminal plug domain. An N-terminal conformational change allows the substrate to enter the plug domain channel and eventually diffuse though to the periplasmic space. It has been suggested that the presence of both hydrophobic and hydrophilic residues in the plug domain channel lowers the affinity of the hydrophobic substrate for its surroundings, and thus prevents the substrate from being held for too long in the channel. While reasonable, this transportation mechanism of TodX has not yet been experimentally verified. Nevertheless, this type of mechanism is structurally impossible with EcFadL, thus indicating how small changes in protein structure can have potentially large impacts on protein function (6).