Lol System Research Articles

Roles of the Protruding Loop of Factor B Essential for the Localization of Lipoproteins (LolB) in the Anchoring of Bacterial Triacylated Proteins to the Outer Membrane

The Lol system comprising five Lol proteins, LolA through LolE, sorts Escherichia coli lipoproteins to outer membranes. The LolCDE complex, an ATP binding cassette transporter in inner membranes, releases outer membrane-specific lipoproteins in an ATP-dependent manner, causing formation of the LolA-lipoprotein complex in the periplasm. LolA transports lipoproteins through the periplasm to LolB on outer membranes. LolB is itself a lipoprotein anchored to outer membranes, although the membrane anchor is functionally dispensable. LolB then localizes lipoproteins to outer membranes through largely unknown mechanisms. The crystal structure of LolB is similar to that of LolA, and it possesses a hydrophobic cavity that accommodates acyl chains of lipoproteins. To elucidate the molecular function of LolB, a periplasmic version of LolB, mLolB, was mutagenized at various conserved residues. Despite the lack of acyl chains, most defective mutants were insoluble. However, a derivative with glutamate in place of leucine 68 was soluble and unable to localize lipoproteins to outer membranes. This leucine is present in a loop protruding from mLolB into an aqueous environment, and no analogous loop is present in LolA. Thus, leucine 68 was replaced with other residues. Replacement by acidic, but not hydrophobic, residues generated for the first time mLolB derivatives that can accept but cannot localize lipoproteins to outer membranes. Moreover, deletion of the leucine with neighboring residues impaired the lipoprotein receptor activity. Based on these observations, the roles of the protruding loop of LolB in the last step of lipoprotein sorting are discussed.

Folding and Lipid Membrane Interactions of BamD, An Essential Component of the β-Barrel Assembly Machine from Escherichia Coli

BamD (YfiO) [1] is an essential component of the β-barrel assembly machine of the outer membrane of Escherichia coli. BamD is synthesized in the cytosol and translocated across the cytopasmic membrane in unfolded form for processing and transport to the outer membrane by the LOL system [2].To examine the interactions of BamD with lipid membranes and BamD folding, we have over-expressed BamD into the periplasm and isolated it from membrane fractions in unfolded form in 8 M urea. Circular dichorism spectroscopy indicated secondary structure formation in BamD upon rapid dilution of the denaturant urea in aqeuous buffer. In the absence of lipid bilayers, BamD displayed a lower content of α-helix structure. The CD-data indicated that native-like folding of BamD required the presence of lipid-membranes of phosphatidylglycerol and phosphatidylcholine. An increased content of phosphatidylglycerol facilitated BamD folding. Fluorescence spectroscopy demonstrated BamD binding to both zwitterionic and negatively charged lipid. The lipid/BamD stoichiometry was estimated to 17 phosphatidylglycerol/BamD and to 31 phosphatidylcholine/BamD.[1] Malinverni JC, Werner J, Kim S, Sklar JG, Kahne D, Misra R, Silhavy TJ (2006) YfiO stabilizes the YaeT complex and is essential for outer membrane protein assembly in Escherichia coli. Mol Microbiol 61:151-164.[2] Narita S, Tokuda H (2010) Sorting of bacterial lipoproteins to the outer membrane by the Lol system. Methods Mol Biol 619:117-129.

Understanding the lid movements of LolA in Escherichia coli using molecular dynamics simulation and in silico point mutation

The Lol system in Escherichia coli is involved in localization of lipoproteins and hence is essential for growth of the organism. LolA is a periplasmic chaperone that binds to outer-membrane specific lipoproteins and transports them from inner membrane to outer membrane through LolB. The hydrophobic lipid-binding cavity of LolA consists of α-helices which act as a lid in regulating the transfer of lipoproteins from LolA to LolB. The current study aims to investigate the structural changes observed in LolA during the transition from open to closed conformation in the absence of lipoprotein. Molecular dynamics (MD) simulations were carried out for two LolA crystal structures; LolA(R43L), and in silico mutated MsL43R for a simulation time of 50ns in water environment. We have performed an in silico point mutation of leucine to arginine in MsL43R to evaluate the importance of arginine to induce structural changes and impact the stability of protein structure. A complete dynamic analysis of open to closed conformation reveals the existence of two distinct levels; closing of lid and closing of entrance of hydrophobic cavity. Our analysis reveals that the structural flexibility of LolA is an important factor for its role as a periplasmic chaperone.

Functional Mapping of PilF and PilQ in the Pseudomonas aeruginosa Type IV Pilus System

Pseudomonas aeruginosa uses type IV pili (T4P) to interact with the environment and as key virulence factors when acting as an opportunistic pathogen. Assembly of the outer membrane PilQ secretin channel through which the pili are extruded is essential for pilus biogenesis. The P. aeruginosa T4P pilotin, PilF, is required for PilQ outer membrane localization and assembly into secretins and contains six tetratricopeptide (TPR) protein-protein interaction motifs, suggesting that the two proteins interact. In this study, we found that the first four TPR motifs of PilF are sufficient for PilQ outer membrane targeting, oligomerization, and function. Guided by our structure of PilF, site-directed mutagenesis of the protein surface revealed that a hydrophobic groove on the first TPR is required for PilF-mediated PilQ assembly. Deletion of individual domains within PilQ suggests that the N0, KH-like, or secretin domain, but not the C-terminus, interacts with PilF. Purified PilQ was found to pull down PilF from Pseudomonas cell lysates. Together, these data allow us to propose a model for PilF function in the T4P system. PilF interacts directly or indirectly with the PilQ monomer after translocation of both proteins through the inner membrane and acts as a co-chaperone with the Lol system to facilitate transit across the periplasm to the outer membrane. The mechanism of PilQ insertion and assembly, which appears to be independent of the Bam system, remains to be determined.

Lipoprotein Sorting in Bacteria

Bacterial lipoproteins are synthesized as precursors in the cytoplasm and processed into mature forms on the cytoplasmic membrane. A lipid moiety attached to the N terminus anchors these proteins to the membrane surface. Many bacteria are predicted to express more than 100 lipoproteins, which play diverse functions on the cell surface. The Lol system, composed of five proteins, catalyzes the localization of Escherichia coli lipoproteins to the outer membrane. Some lipoproteins play vital roles in the sorting of other lipoproteins, lipopolysaccharides, and β-barrel proteins to the outer membrane. On the basis of results from biochemical, genetic, and structural studies, we discuss the biogenesis of lipoproteins in bacteria, their importance in cellular functions, and the molecular mechanisms underlying efficient sorting of hydrophobic lipoproteins to the outer membrane through the hydrophilic periplasm.

ABC Transporters Involved in the Biogenesis of the Outer Membrane in Gram-Negative Bacteria

The outer membrane of gram-negative bacteria is an asymmetric lipid bilayer with phospholipids and lipopolysaccharides (LPSs). β-Barreled outer membrane proteins and lipoproteins are embedded in the outer membrane. All of these constituents are essential to the function of the outer membrane. The transport systems for lipoproteins have been characterized in detail. An ATP-binding cassette (ABC) transporter, LolCDE, initiates sorting by mediating the detachment of lipoproteins from the inner membrane to form a water-soluble lipoprotein-LolA complex in the periplasm. Lipoproteins are then transferred to LolB at the outer membrane and are incorporated into the lipid bilayer. A model analogous to the Lol system has been suggested for the transport of LPS, where an ABC transporter, LptBFG, mediates the detachment of LPS from the inner membrane. Recent developments in the functional characterization of ABC transporters involved in the biogenesis of the outer membrane in gram-negative bacteria are discussed.

Hydrophobic Surface Patches on LolA of Pseudomonas aeruginosa Are Essential for Lipoprotein Binding

Many lipoproteins reside in the outer membrane (OM) of Gram-negative bacteria, and their biogenesis is dependent on the Lol (localization of lipoproteins) system. The periplasmic chaperone LolA accepts OM-destined lipoproteins that are released from the inner membrane by the LolCDE complex and transfers them to the OM receptor LolB. The exact nature of the LolA–lipoprotein complex is still unknown. The crystal structure of Escherichia coli LolA features an open β-barrel covered by α helices that together constitute a hydrophobic cavity, which would allow the binding of one acyl chain. However, OM lipoproteins contain three acyl chains, and the stoichiometry of the LolA–lipoprotein complex is 1:1. Here we present the crystal structure of Pseudomonas aeruginosa LolA that projects clear hydrophobic surface patches. Since these patches are large enough to accommodate acyl chains, their role in lipoprotein binding was investigated. Several LolA mutant proteins were created, and their functionality was assessed by studying their capacity to release lipoproteins produced in sphaeroplasts. Interruption of the largest hydrophobic patch completely destroyed the lipoprotein-releasing capacity of LolA, while interruption of smaller patches apparently reduced efficiency. Thus, the results show a new lipoprotein transport model that places (some of) the acyl chains on the hydrophobic surface patches.

Biogenesis of Outer Membranes in Gram-Negative Bacteria

The outer membrane, an essential organelle of Gram-negative bacteria, is composed of four major components: lipopolysaccharide, phospholipids, beta-barrel proteins, and lipoproteins. The mechanisms underlying the transport of these components to outer membranes are currently under extensive examination. Among them, the sorting of lipoproteins to the outer membrane of Escherichia coli has been clarified in detail. The Lol system, composed of five proteins, catalyzes outer membrane sorting of lipoproteins. Various Lpt proteins have recently been identified as factors involved in the transport of lipopolysaccharide to the outer membrane, although the mechanism remains largely unknown. Proteins with alpha-helical membrane spanning segments are found in the inner membrane, whereas amphipathic beta-barrel proteins span the outer membrane. These beta-barrel proteins are inserted into the outer membranes through a central core protein BamA (YaeT) with the help of four outer membrane lipoproteins. In contrast, little is known about how phospholipids are transported to the outer membrane.

2SA2-02 細菌型リポ蛋白質の選別・輸送・膜局在化を司るLolシステムの構造と機能(2SA2 タンパク質の膜透過輸送の最前線,第47回日本生物物理学会年会)

2SA2-02 細菌型リポ蛋白質の選別・輸送・膜局在化を司るLolシステムの構造と機能(2SA2 タンパク質の膜透過輸送の最前線,第47回日本生物物理学会年会)

Amino Acids at Positions 3 and 4 Determine the Membrane Specificity of Pseudomonas aeruginosa Lipoproteins

Escherichia coli lipoproteins with Asp at position 2 remain in the inner membrane, whereas those having other amino acids are targeted to the outer membrane by the Lol system. However, inner membrane lipoproteins without Asp at position 2 are found in other Gram-negative bacteria. MexA of Pseudomonas aeruginosa, an inner membrane-specific lipoprotein involved in multidrug efflux, has Gly at position 2. To identify the residue or region of MexA that functions as an inner membrane retention signal, we constructed chimeric lipoproteins comprising various regions of MexA and an outer membrane lipoprotein, OprM, and analyzed their membrane localization. Lys and Ser at positions 3 and 4, respectively, were found to be critical for the inner membrane localization of MexA in P. aeruginosa. Substitution of these residues with Leu and Ile, which are present in OprM, was sufficient to target the chimeric lipoprotein to the outer membrane and to abolish the ability of MexA to confer drug resistance. The membrane specificity of a model lipoprotein, lipoMalE, a lipidated variant of the periplasmic maltose-binding protein of E. coli, was also determined by the residues at positions 3 and 4 in P. aeruginosa. In contrast to the widely accepted "+2 rule" for E. coli lipoproteins, these results suggest a new "+3, +4 rule" for lipoprotein sorting in P. aeruginosa, namely, the final destination of lipoproteins is determined by the residues at positions 3 and 4.

Characterization of the Pseudomonas aeruginosa Lol System as a Lipoprotein Sorting Mechanism

Escherichia coli lipoproteins are localized to either the inner or the outer membrane depending on the residue that is present next to the N-terminal acylated Cys. Asp at position 2 causes the retention of lipoproteins in the inner membrane. In contrast, the accompanying study (9) revealed that the residues at positions 3 and 4 determine the membrane specificity of lipoproteins in Pseudomonas aeruginosa. Since the five Lol proteins involved in the sorting of E. coli lipoproteins are conserved in P. aeruginosa, we examined whether or not the Lol proteins of P. aeruginosa are also involved in lipoprotein sorting but utilize different signals. The genes encoding LolCDE, LolA, and LolB homologues were cloned and expressed. The LolCDE homologue thus purified was reconstituted into proteoliposomes with lipoproteins. When incubated in the presence of ATP and a LolA homologue, the reconstituted LolCDE homologue released lipoproteins, leading to the formation of a LolA-lipoprotein complex. Lipoproteins were then incorporated into the outer membrane depending on a LolB homologue. As revealed in vivo, lipoproteins with Lys and Ser at positions 3 and 4, respectively, remained in proteoliposomes. On the other hand, E. coli LolCDE released lipoproteins with this signal and transferred them to LolA of not only E. coli but also P. aeruginosa. These results indicate that Lol proteins are responsible for the sorting of lipoproteins to the outer membrane of P. aeruginosa, as in the case of E. coli, but respond differently to inner membrane retention signals.

A New Screening Method to Identify Inhibitors of the Lol (Localization of lipoproteins) System, a Novel Antibacterial Target

As the Lol system, which is involved in localization of lipoproteins, is essential for Escherichia coli growth and widely conserved among gram-negative bacteria, it is considered to be a promising target for the development of anti-gram-negative bacterial agents. However, no high-throughput screening method has so far been developed to screen for Lol system inhibitors. By combining three assay systems (anucleate cell blue assay, Lpp assay, and LolA-dependent release inhibition assay) and a drug susceptibility test, we have successfully developed a new screening method for identification of compounds that inhibit the Lol system. Using this new screening method, we screened 23,600 in-house chemical compounds and found 2 Lol system inhibitors. We therefore conclude that our new screening method can efficiently identify new antibacterial agents that target the Lol system.

S2c2-2 Structural basis of bacterial lipoprotein transport by Lol system(S2-c2: "Structural biology reveals macromolecular interaction",Symposia,Abstract,Meeting Program of EABS & BSJ 2006)

S2c2-2 Structural basis of bacterial lipoprotein transport by Lol system(S2-c2: "Structural biology reveals macromolecular interaction",Symposia,Abstract,Meeting Program of EABS & BSJ 2006)

An ABC transporter mediating the membrane detachment of bacterial lipoproteins depending on their sorting signals

Bacterial lipoproteins are anchored to membranes through a lipid moiety attached to the N-terminal Cys. Escherichia coli possesses more than 90 species of lipoproteins, most of which are localized in the outer membrane and others in the inner membrane. Sorting of lipoproteins to the outer membrane requires the Lol system comprising five Lol proteins. An ATP-binding cassette transporter, LolCDE, initiates the lipoprotein sorting by mediating the detachment of outer membrane-specific lipoproteins from the inner membrane. LolCDE does not recognize lipoproteins possessing Asp at position 2, which therefore remain anchored to the inner membrane. We will discuss the mechanism of LolCDE based on data obtained through in vitro experiments.

Mechanisms Underlying Energy-independent Transfer of Lipoproteins from LolA to LolB, Which Have Similar Unclosed β-Barrel Structures

The Lol system, comprising five Lol proteins, transfers lipoproteins from the inner to the outer membrane of Escherichia coli. Periplasmic LolA accepts lipoproteins from LolCDE in the inner membrane and immediately transfers them to LolB, a receptor anchored to the outer membrane. The unclosed beta-barrel structures of LolA and LolB are very similar to each other and form hydrophobic cavities for lipoproteins. The lipoprotein transfer between these similar structures is unidirectional and very efficient, but requires no energy input. To reveal the mechanisms underlying this lipoprotein transfer, Arg and Phe at positions 43 and 47, respectively, of LolA were systematically mutagenized. The two residues were previously found to affect abilities to accept and transfer lipoproteins. Substitution of Phe-47 with polar residues inhibited the ability to accept lipoproteins from the inner membrane. No derivatives caused periplasmic accumulation of lipoproteins. In contrast, many Arg-43 derivatives caused unusual periplasmic accumulation of lipoproteins to various extents. However, all derivatives, except one having Leu instead of Arg, supported the growth of cells. All Arg-43 derivatives retained the ability to accept lipoproteins from the inner membrane, whereas their abilities to transfer associated lipoproteins to LolB were variously reduced. Assessment of the intensity of the hydrophobic interaction between lipoproteins and Arg-43 derivatives revealed that the LolA-lipoprotein interaction should be weak, otherwise lipoprotein transfer to LolB is inhibited, causing accumulation of lipoproteins in the periplasm.

Depletion of Apolipoprotein N-Acyltransferase Causes Mislocalization of Outer Membrane Lipoproteins in Escherichia coli

Lipoproteins in Gram-negative Enterobacteriaceae carry three fatty acids on the N-terminal cysteine residue, two as a diacylglyceride and one through an N-linkage following signal peptide cleavage. Most lipoproteins are anchored in the outer membrane, facing the periplasm, but some lipoproteins remain in the plasma membrane, depending on the amino acid at position +2, immediately after the fatty-acylated cysteine. In vitro, the last step in lipoprotein maturation, N-acylation of apolipoproteins by the plasma membrane apolipoprotein N-acyltransferase (Lnt), is necessary for efficient recognition of outer membrane lipoproteins by the Lol system, which transports them from the plasma to the outer membrane (Fukuda, A., Matsuyama, S.-I., Hara, T., Nakayama, J., Nagasawa, H., and Tokuda, H. (2002) J. Biol. Chem. 277, 43512-43518). To study the role of Lnt in vivo, we constructed a conditional lnt mutant of Escherichia coli. The apo-form of peptidoglycan-anchored major lipoprotein (Lpp) and two other outer membrane lipoproteins accumulated in the plasma membrane when lnt expression was reduced. We also found that Lnt is an essential protein in E. coli and that the lethality is partially because of the retention of apoLpp in the plasma membrane. Topology mapping of Lnt with beta-galactosidase and alkaline phosphatase fusions indicated the presence of six membrane-spanning segments. The lnt gene in a mutant of Salmonella enterica displaying thermosensitive Lnt activity (Gupta, S. D., Gan, K., Schmid, M. B., and Wu, H. C. (1993) J. Biol. Chem. 268, 16551-16556) was found to carry a mutation causing a single glutamate to lysine substitution at a highly conserved position in the last predicted periplasmic loop of the protein.

Lipoprotein trafficking in Escherichia coli.

Bacterial lipoproteins comprise a subset of membrane proteins that are covalently modified with lipids at the amino-terminal Cys. Lipoproteins are involved in a wide variety of functions in bacterial envelopes. Escherichia coli has more than 90 species of lipoproteins, most of which are located on the periplasmic surface of the outer membrane, while others are located on that of the inner membrane. In order to elucidate the mechanisms by which outer-membrane-specific lipoproteins are sorted to the outer membrane, biochemical, molecular biological and crystallographic approaches have been taken. Localization of lipoproteins on the outer membrane was found to require a lipoprotein-specific sorting machinery, the Lol system, which is composed of five proteins (LolABCDE). The crystal structures of LolA and LolB, the periplasmic chaperone and outer-membrane receptor for lipoproteins, respectively, were determined. On the basis of the data, we discuss here the mechanism underlying lipoprotein transfer from the inner to the outer membrane through Lol proteins. We also discuss why inner membrane-specific lipoproteins remain on the inner membrane.

A practical phasing procedure using the MAD method without the aid of XAFS measurements: successful solution in the structure determination of the outer-membrane lipoprotein carrier LolA.

A practical procedure for MAD phasing was successfully performed in the structure determination of the LolA protein, even though the XAFS data could not be measured owing to the overlap of the fluorescence spectra of the atoms that contribute significant signals for anomalous dispersion. The LolA protein, a periplasmic chaperone functioning as an outer-membrane lipoprotein carrier of the Lol system which mediates translocation of the water-insoluble outer-membrane lipoprotein across the periplasm in Gram-negative bacteria, was crystallized in two forms: orthorhombic (I222) and trigonal (P3(1)21 or P3(2)21). A multi-wavelength data set was collected from a platinum derivative of the orthorhombic crystals grown from a buffer solution containing zinc acetate and cacodylate (an arsenic compound), but XAFS measurements could not be performed because the energies of the fluorescence spectra of Zn atoms, As atoms and Pt atoms are in very close proximity. However, effective MAD data were collected with six wavelength data sets near the platinum absorption edge and the f' and f" values could subsequently be estimated from the data statistics and the peak height of the dispersive and anomalous difference Patterson maps. The subsequent MAD phasing gave a high-quality initial electron-density map which was sufficient to construct a complete molecular model of the LolA protein.

Crystallization and preliminary crystallographic study of the outer-membrane lipoprotein receptor LolB, a member of the lipoprotein localization factors.

The Lol system mediates the translocation of the water-insoluble outer-membrane lipoprotein across the periplasm of Gram-negative bacteria depending on the sorting signal. The outer-membrane lipoprotein receptor LolB (21.2 kDa) is a member of the Lol system. A soluble mutant of LolB (mLolB) from Escherichia coli was crystallized in two forms. Monoclinic crystals diffract X-rays to 1.9 A resolution and belong to space group P2(1), with unit-cell parameters a = 37.2, b = 112.4, c = 47.8 A, beta = 111.4 degrees. The V(M) value is most likely to be 2.2 A(3) Da(-1), assuming the presence of two molecules in the asymmetric unit. Hexagonal crystals diffract X-rays to 2.2 A resolution and belong to space group P6(3)22, with unit-cell parameters a = b = 71.4, c = 133.9 A. The V(M) value is determined as 2.3 A(3) Da(-1), assuming a single molecule in the asymmetric unit. A four-wavelength data set was collected from a monoclinic crystal of selenomethionylated mLolB in order to perform MAD phasing. The quality of the initial electron-density map was sufficient to build a molecular model.

Aminoacylation of the N-terminal Cysteine Is Essential for Lol-dependent Release of Lipoproteins from Membranes but Does Not Depend on Lipoprotein Sorting Signals

Lipoproteins are present in a wide variety of bacteria and are anchored to membranes through lipids attached to the N-terminal cysteine. The Lol system of Escherichia coli mediates the membrane-specific localization of lipoproteins. Aspartate at position 2 functions as a Lol avoidance signal and causes the retention of lipoproteins in the inner membrane, whereas lipoproteins having residues other than aspartate at position 2 are released from the inner membrane and localized to the outer membrane by the Lol system. Phospholipid:apolipoprotein transacylase, Lnt, catalyzes the last step of lipoprotein modification, converting apolipoprotein into mature lipoprotein. To reveal the importance of this aminoacylation for the Lol-dependent membrane localization, apolipoproteins were prepared by inhibiting lipoprotein maturation. Lnt was also purified and used to convert apolipoprotein into mature lipoprotein in vitro. The release of these lipoproteins was examined in proteoliposomes. We show here that the aminoacylation is essential for the Lol-dependent release of lipoproteins from membranes. Furthermore, lipoproteins with aspartate at position 2 were found to be aminoacylated both in vivo and in vitro, indicating that the lipoprotein-sorting signal does not affect lipid modification.