Structural Investigation of the Interaction between LolA and LolB Using NMR

Lipoproteins that play critical roles in various cellular functions of Gram-negative bacteria are localized in the cells inner and outer membranes. Lol proteins (LolA, LolB, LolC, LolD, and LolE) are involved in the transportation of outer membrane-directed lipoproteins from the inner to the outer membrane. LolA is a periplasmic chaperone that transports lipoproteins, and LolB is an outer membrane receptor that accepts lipoproteins. To clarify the structural basis for the lipoprotein transfer from LolA to LolB, we examined the interaction between LolA and mLolB, a soluble mutant of LolB, using solution NMR spectroscopy. We determined the interaction mode between LolA and mLolB with conformational changes of LolA. Based upon the observations, we propose that the LolA·LolB complex forms a tunnel-like structure, where the hydrophobic insides of LolA and LolB are connected, which enables lipoproteins to transfer from LolA to LolB.Gram-negative bacteria express lipid-modified proteins, lipoproteins, which are anchored to the cellular membrane via acyl chains attached to N-terminal cysteine residues of the lipoproteins. Putative lipoproteins have been found in various bacteria. For example, Escherichia coli has at least 90 types of lipoproteins (1), and the Lyme disease spirochete Borrelia burgdorferi has 105 putative lipoproteins (2). Although little is known about the functions of the majority of lipoproteins, some of the lipoproteins play essential roles in various cellular functions of Gram-negative bacteria, such as cell surface structure stabilization, cell shape maintenance, substrate transport, cell growth, and cell signaling (3).Lipoproteins are located at three cellular membrane sites; they are the periplasmic side of the inner membrane, the periplasmic side of the outer membrane, and the outside of the outer membrane (4). In E. coli most of the lipoproteins are anchored to the periplasmic side of the outer membrane, whereas others are anchored to that of the inner membrane (1). Therefore, the transportation of the lipoproteins to the outer membrane is essential for E. coli.Five Lol proteins, LolA, LolB, LolC, LolD, and LolE, play central roles in the outer membrane-directed lipoprotein localization. The Lol·CDE complex, which is anchored to the inner membrane, transfers the lipoproteins from the membrane to a soluble monomer periplasmic protein, LolA (182 amino acids) in an ATP-dependent manner (5–7). LolA transports the lipoproteins from the inner membrane through the periplasmic space to the outer membrane and transfers them to an outer membrane lipoprotein, LolB (186 amino acids). LolB is anchored to the membrane by acyl chains attached to its N-terminal cysteine, and it finally inserts the lipoproteins into the outer membrane (8–10).Among the Lol proteins the crystal structures of LolA and LolB have been solved. As for LolB, the soluble mutant of LolB, mLolB, in which the N-terminal cysteine residue was replaced with an alanine residue, was used for the structural analysis. Although LolA and mLolB share only 8% primary sequence identity, their tertiary structures are similar to each other (11). The structures of both LolA and mLolB resemble an open β-barrel with a lid. The convex side of the β-barrel is fully solvent-exposed, whereas the concave side is partly exposed (supplemental Fig. S1).The open β-barrels of LolA and LolB comprise 11 antiparallel β-strands (β1–β11) and an extra β-strand, β12 for LolA and β11′ for LolB. The lid is composed of three α-helices (α1–α3) and is embedded in the concave side of the β-barrel. The concave sides of LolA and LolB contain many hydrophobic residues. Therefore, this concave side of the proteins is speculated to be the binding site for the hydrophobic acyl chains of lipoproteins. Interestingly, one of the crystal structures of LolB accommodated a molecule of polyethylene glycol 2000 monomethyl ether, PEGMME2000, on the hydrophobic surface of the concave side (supplemental Fig. S1).The specific interaction between LolA and LolB is a decisive step in correctly sorting lipoproteins from LolA via LolB to the outer membrane. However, the structural aspects of the interaction, which would clarify how LolA transfers lipoproteins to LolB, remain unknown. To address this issue, we focused on the interaction between LolA and LolB.Here we investigated the interaction of LolA with LolB by NMR spectroscopy. We used LolA with a His₆ tag and mLolB, which retain the biological activities similar to those of the wild type protein (8, 12). By exploiting the cross-saturation and paramagnetic relaxation enhancement (PRE)² techniques, we successfully determined the interfacial residues of LolA and mLolB and the relative orientation of the two molecules in the complex. In addition, we identified the binding sites of an acyl chain analogue, decanoate, on LolA and mLolB. The results obtained from the present study not only explain how LolA might achieve lipoprotein transfer to LolB but also may provide new insights into the structural and functional aspects of other fatty acid-binding proteins.     

Sorting of lipoproteins to the outer membrane in E. coli

Escherichia coli lipoproteins are anchored to the periplasmic surface of the inner or outer membrane depending on the sorting signal. An ATP-binding cassette (ABC) transporter, LolCDE, releases outer membrane-specific lipoproteins from the inner membrane, causing the formation of a complex between the released lipoproteins and the periplasmic molecular chaperone LolA. When this complex interacts with outer membrane receptor LolB, the lipoproteins are transferred from LolA to LolB and then localized to the outer membrane. The structures of LolA and LolB are remarkably similar to each other. Both have a hydrophobic cavity consisting of an unclosed beta-barrel and an alpha-helical lid. Structural differences between the two proteins reveal the molecular mechanisms underlying the energy-independent transfer of lipoproteins from LolA to LolB. Strong inner membrane retention of lipoproteins occurs with Asp at position 2 and a few limited residues at position 3. The inner membrane retention signal functions as a Lol avoidance signal and inhibits the recognition of lipoproteins by LolCDE, thereby causing their retention in the inner membrane. The positive charge of phosphatidylethanolamine and the negative charge of Asp at position 2 are essential for Lol avoidance. The Lol avoidance signal is speculated to cause the formation of a tight lipoprotein-phosphatidylethanolamine complex that has five acyl chains and therefore cannot be recognized by LolCDE.     

Mechanisms underlying energy-independent transfer of lipoproteins from LolA to LolB, which have similar unclosed {beta}-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.     

Sorting of lipoproteins to the outer membrane in E. coli

Escherichia coli lipoproteins are anchored to the periplasmic surface of the inner or outer membrane depending on the sorting signal. An ATP-binding cassette (ABC) transporter, LolCDE, releases outer membrane-specific lipoproteins from the inner membrane, causing the formation of a complex between the released lipoproteins and the periplasmic molecular chaperone LolA. When this complex interacts with outer membrane receptor LolB, the lipoproteins are transferred from LolA to LolB and then localized to the outer membrane. The structures of LolA and LolB are remarkably similar to each other. Both have a hydrophobic cavity consisting of an unclosed beta-barrel and an alpha-helical lid. Structural differences between the two proteins reveal the molecular mechanisms underlying the energy-independent transfer of lipoproteins from LolA to LolB. Strong inner membrane retention of lipoproteins occurs with Asp at position 2 and a few limited residues at position 3. The inner membrane retention signal functions as a Lol avoidance signal and inhibits the recognition of lipoproteins by LolCDE, thereby causing their retention in the inner membrane. The positive charge of phosphatidylethanolamine and the negative charge of Asp at position 2 are essential for Lol avoidance. The Lol avoidance signal is speculated to cause the formation of a tight lipoprotein-phosphatidylethanolamine complex that has five acyl chains and therefore cannot be recognized by LolCDE.     

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.     

Crystal structures of bacterial lipoprotein localization factors,LolA and LolB

Lipoproteins having a lipid-modified cysteine at the N-terminus are localized on either the inner or the outer membrane of Escherichia coli depending on the residue at position 2. Five Lol proteins involved in the sorting and membrane localization of lipoprotein are highly conserved in Gram-negative bacteria. We determined the crystal structures of a periplasmic chaperone, LolA, and an outer membrane lipoprotein receptor, LolB. Despite their dissimilar amino acid sequences, the structures of LolA and LolB are strikingly similar to each other. Both have a hydrophobic cavity consisting of an unclosed beta barrel and an alpha-helical lid. The cavity represents a possible binding site for the lipid moiety of lipoproteins. Detailed structural differences between the two proteins provide significant insights into the molecular mechanisms underlying the energy-independent transfer of lipoproteins from LolA to LolB and from LolB to the outer membrane. Furthermore, the structures of both LolA and LolB determined from different crystal forms revealed the distinct structural dynamics regarding the association and dissociation of lipoproteins. The results are discussed in the context of the current model for the lipoprotein transfer from the inner to the outer membrane through a hydrophilic environment.     

Large-scale preparation of the homogeneous LolA lipoprotein complex and efficient in vitro transfer of lipoproteins to the outer membrane in a LolB-dependent manner

An ATP-binding cassette transporter LolCDE complex of Escherichia coli releases lipoproteins destined to the outer membrane from the inner membrane as a complex with a periplasmic chaperone, LolA. Interaction of the LolA-lipoprotein complex with an outer membrane receptor, LolB, then causes localization of lipoproteins to the outer membrane. As far as examined, formation of the LolA-lipoprotein complex strictly depends on ATP hydrolysis by the LolCDE complex in the presence of LolA. It has been speculated, based on crystallographic and biochemical observations, that LolA undergoes an ATP-dependent conformational change upon lipoprotein binding. Thus, preparation of a large amount of the LolA-lipoprotein complex is difficult. Moreover, lipoproteins bound to LolA are heterogeneous. We report here that the coexpression of LolA and outer membrane-specific lipoprotein Pal from a very efficient plasmid causes the unusual accumulation of the LolA-Pal complex in the periplasm. The complex was purified to homogeneity and shown to be a functional intermediate of the lipoprotein localization pathway. In vitro incorporation of Pal into outer membranes revealed that a single molecule of LolB catalyzes the incorporation of more than 100 molecules of Pal into outer membranes. Moreover, the LolB-dependent incorporation of Pal was not affected by excess-free LolA, indicating that LolB specifically interacts with liganded LolA. Finally, the LolB depletion caused the accumulation of a significant amount of Pal in the periplasm, thereby establishing the conditions for preparation of the homogeneous LolA-lipoprotein complex.     

Roles of the hydrophobic cavity and lid of LolA in the lipoprotein transfer reaction in Escherichia coli

LolA, a periplasmic chaperone, binds to outer membrane-specific lipoproteins released from the inner membrane through the action of an ATP-binding cassette transporter, LolCDE and then transfers them to the outer membrane receptor LolB, thereby mediating the inner to outer membrane transport of lipoproteins. The crystal structure of free LolA revealed that it has an internal hydrophobic cavity, which is surrounded by hydrophobic residues and closed by a lid comprising alpha-helices. The hydrophobic cavity most likely represents the binding site for the lipid moiety of a lipoprotein. It is speculated that the lid undergoes opening and closing upon the binding and transfer of lipoproteins, respectively. To determine the functions of the hydrophobic cavity and lid in detail, 14 residues involved in the formation of these structures were subjected to random mutagenesis. Among the obtained 21 LolA derivatives that did not support growth, 14 were active as to the binding of lipoproteins but defective in the transfer of lipoproteins to LolB, causing the periplasmic accumulation of a lipoprotein as a complex with a LolA derivative. A LolA derivative, I93G, bound lipoproteins faster than wild-type LolA did, whereas it did not transfer associated lipoproteins to LolB. When I93G and wild type LolA co-existed, lipoproteins were bound only to I93G; which therefore exhibited a dominant negative property. Another derivative, L59R, was also defective in the transfer of lipoproteins to LolB but did not exhibit a dominant negative property. Taken together, these results indicate that both the hydrophobic cavity and the lid are critically important for not only the binding of lipoproteins but also their transfer.     

Mutant of LolA, a lipoprotein-specific molecular chaperone of Escherichia coli, defective in the transfer of lipoproteins to LolB

The outer membrane-specific lipoproteins of Escherichia coli are released from the inner membrane as a water-soluble complex with LolA and then transferred to the outer membrane receptor, LolB. LolA thus plays a critical role in the sorting and outer membrane localization of lipoproteins. To dissect the LolA function, the highly conserved residues were subjected to random mutagenesis, followed by selection for a growth defect. LolA(R43L), one of mutants thus constructed, possessed Leu in place of Arg at position 43 and caused accumulation of the LolA(R43L)-lipoprotein complex in the periplasm. LolA(R43L) was as active as wild-type LolA as to the release of lipoproteins from spheroplasts. In marked contrast, the transfer of lipoproteins from LolA(R43L) to LolB was completely inhibited, indicating that Arg at position 43 of LolA is involved in the lipoprotein transfer reaction.     

A short helix in the C-terminal region of LolA is important for the specific membrane localization of lipoproteins

The structures of a lipoprotein carrier, LolA, and a lipoprotein receptor, LolB, are similar except for an extra C-terminal loop containing a 3(10) helix and beta-strand 12 in LolA. Lipoprotein release was significantly reduced when beta-12 was deleted. Deletion of the 3(10) helix also inhibited the lipoprotein release. Furthermore, lipoproteins were non-specifically localized to membranes when LolA lacked the 3(10) helix. Thus, the membrane localization of lipoproteins with the LolA derivative lacking the 3(10) helix was independent of LolB whereas LolB was essential for the outer membrane localization of lipoproteins with the wild-type LolA.     

Secretion of Bacterial Lipoproteins: Through the Cytoplasmic Membrane,the Periplasm and Beyond

Bacterial lipoproteins are peripherally anchored membrane proteins that play a variety of roles in bacterial physiology and virulence in monoderm (single membrane-enveloped, e.g., gram-positive) and diderm (double membrane-enveloped, e.g., gram-negative) bacteria. After export of prolipoproteins through the cytoplasmic membrane, which occurs predominantly but not exclusively via the general secretory or Sec pathway, the proteins are lipid-modified at the cytoplasmic membrane in a multistep process that involves sequential modification of a cysteine residue and cleavage of the signal peptide by the signal II peptidase Lsp. In both monoderms and diderms, signal peptide processing is preceded by acylation with a diacylglycerol through preprolipoprotein diacylglycerol transferase (Lgt). In diderms but also some monoderms, lipoproteins are further modified with a third acyl chain through lipoprotein N-acyl transferase (Lnt). Fully modified lipoproteins that are destined to be anchored in the inner leaflet of the outer membrane (OM) are selected, transported and inserted by the Lol (lipoprotein outer membrane localization) pathway machinery, which consists of the inner-membrane (IM) ABC transporter-like LolCDE complex, the periplasmic LolA chaperone and the OM LolB lipoprotein receptor. Retention of lipoproteins in the cytoplasmic membrane results from Lol avoidance signals that were originally described as the “+ 2 rule”. Surface localization of lipoproteins in diderms is rare in most bacteria, with the exception of several spirochetal species. Type 2 (T2SS) and type 5 (T5SS) secretion systems are involved in secretion of specific surface lipoproteins of γ-proteobacteria. In the model spirochete Borrelia burgdorferi, surface lipoprotein secretion does not follow established sorting rules, but remains dependent on N-terminal peptide sequences. Secretion through the outer membrane requires maintenance of lipoproteins in a translocation-competent unfolded conformation, likely through interaction with a periplasmic holding chaperone, which delivers the proteins to an outer membrane lipoprotein flippase. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

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.     

Real time analysis of lipoprotein transfer from LolA to LolB by means of surface plasmon resonance

Lipoproteins of Escherichia coli are sorted to the outer membrane through a pathway composed of five Lol proteins. LolA transports lipoproteins released from the inner membrane by LolCDE to LolB on the outer membrane via the periplasm. Interaction between LolA and LolB was speculated to be strong when LolA binds lipoprotein. However, due to a lack of a sensitive method, the kinetics of this reaction have not been examined in detail. We report here the detection of lipoprotein transfer in real time by means of surface plasmon resonance. The kinetic parameters of lipoprotein transfer were determined with wild-type LolA and a mutant defective in it.

Structured summary

MINT-7259948: mlolB (uniprotkb:P61320) binds (MI:0407) to pal (uniprotkb:P0A912) by surface plasmon resonance (MI:0107)     

Lipoprotein trafficking in <Emphasis Type="Italic">Escherichia coli</Emphasis>

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.     

Introduction of a lethal redox switch that controls the opening and closing of the hydrophobic cavity in LolA

LolA plays a critical role in the outer membrane sorting of Escherichia coli lipoproteins because it carries a hydrophobic lipoprotein from the inner membrane through the hydrophilic periplasm to the outer membrane receptor LolB. LolA has an incomplete beta-barrel structure composed of 11 beta-strands with an alpha-helical lid forming a hydrophobic cavity inside. The accompanying study revealed that the hydrophobic cavity opens and closes upon the binding and release of lipoproteins, respectively. Ile(93) in the alpha-helix and Phe(140) in the beta-strand are located close to each other in the hydrophobic cavity. These two residues were replaced by Cys to construct the I93C/F140C derivative. Expression of I93C/F140C immediately arrested growth whether wild-type LolA was present or not. However, this dominant negative phenotype was abolished by reducing agents, indicating that the intramolecular disulfide bonding between the two Cys residues is lethal. I93C/F140C was unstable, and its periplasmic level was lower than that of wild-type LolA or its single Cys derivative. Reduction of I93C/F140C was essential for the release of lipoproteins from the inner membrane. Moreover, treatment of I93C/F140C with divalent cross-linkers having different side chain lengths revealed that opening of the lid for a sufficient distance is required for the release activity. The binding of a fluorescent probe to the hydrophobic cavity of I93C/F140C also depended on reducing agents. Taken together, these results indicate that the two Cys residues introduced into LolA function as a redox switch, which regulates the opening and closing of the hydrophobic cavity.     

Opening and closing of the hydrophobic cavity of LolA coupled to lipoprotein binding and release

Outer membrane-specific lipoproteins of Escherichia coli are released from the inner membrane through the action of Lol-CDE, which leads to the formation of a complex between the lipoprotein and LolA, a periplasmic chaperone. LolA then transfers lipoproteins to LolB, a receptor in the outer membrane. The structures of LolA and LolB are very similar, having an incomplete beta-barrel covered with an alpha-helical lid forming a hydrophobic cavity inside. The cavity of LolA, but not that of LolB, is closed and thus inaccessible to the bulk solvent. Previous studies suggested that Arg at position 43 of LolA is critical for maintaining this closed structure. We show here, through a crystallographic study, that the cavity of the LolA(R43L) mutant, in which Leu replaces Arg-43, is indeed open to the external milieu. We then found that the binding of a fluorescence probe distinguishes the open/close state of the cavity. Furthermore, it was revealed that the hydrophobic cavity of LolA opens upon the binding of lipoproteins. Such a liganded LolA was found to be inactive in the release of lipoproteins from the inner membrane. On the other hand, the liganded LolA became fully functional when lipoproteins were removed from LolA by detergent treatment or transferred to LolB. Free LolA thus formed was inaccessible to a fluorescence probe. These results, taken together, reveal the LolA cycle, in which the hydrophobic cavity undergoes opening and closing upon the binding and release of lipoproteins, respectively.     

A Periplasmic LolA Derivative with a Lethal Disulfide Bond Activates the Cpx Stress Response System

LolA accommodates the acyl chains of lipoproteins in its hydrophobic cavity and shuttles between the inner and outer membranes through the hydrophilic periplasm to place lipoproteins in the outer membrane. The LolA(I93C/F140C) derivative, in which Cys replaces Ile at position 93 and Phe at position 140, strongly inhibited growth in the absence of a reducing agent because of the lethal intramolecular disulfide bond between the two Cys residues. Expression of I93C/F140C was found to activate the Cpx two-component system, which responds to cell envelope stress. The inhibition of growth by I93C/F140C was partly suppressed by overproduction of LolCDE, which is an ATP-binding cassette transporter and mediates the transfer of lipoproteins from the inner membrane to LolA. A substantial portion of the oxidized form, but not the reduced one, of I93C/F140C expressed on LolCDE overproduction was recovered in the membrane fraction, whereas wild-type LolA was localized in the periplasm even when LolCDE was overproduced. Moreover, LolCDE overproduction stabilized I93C/F140C and therefore caused an increase in its level. Taken together, these results indicate that oxidized I93C/F140C stably binds to LolCDE, which causes strong envelope stress.There are more than 90 different species of lipoproteins in the Escherichia coli envelope, most of which are localized on the periplasmic side of the outer membrane (29, 31) They each have an N-terminal cysteine covalently modified with three acyl chains, and are anchored to membranes via these lipid tails (25). Although some of these proteins have been shown to be involved in important cellular processes, such as biogenesis of the outer membrane (1, 14, 24, 35), drug transport (11), and signal transduction (7), the functions of the majority of them remain unknown. The Lol system, composed of five Lol proteins, is required for the sorting and targeting of outer membrane-specific lipoproteins (30).Lipoprotein precursors are sequentially processed to their mature forms on the periplasmic side of the inner membrane after their translocation across the inner membrane by Sec translocon (21). Those destined for the outer membrane then each form a complex with LolCDE (36), a member of ATP-binding cassette (ABC) transporter family, in the inner membrane. LolA, a periplasmic lipoprotein-specific carrier, receives lipoproteins from LolCDE in an ATP hydrolysis-dependent manner and forms a water-soluble complex with a lipoprotein (13). The complex then traverses the periplasmic space from the inner to the outer membrane, where lipoproteins are transferred from LolA to a lipoprotein receptor, LolB (14), in a mouth-to-mouth manner (20). Finally, lipoproteins are anchored to the outer membrane through the action of LolB (32).LolA is composed of 11 antiparallel β-sheets and 3 α-helices, which form an incomplete β-barrel structure with a lid covering the barrel (28). The cavity formed inside the barrel is hydrophobic and is considered to be the binding site for the acyl chains of lipoproteins. To elucidate the role of the opening and closing of the hydrophobic cavity in lipoprotein transfer reactions, the LolA(I93C/F140C) mutant, in which cysteine replaces Ile93 in the α2 helix and Phe140 in the β10 strand, was previously constructed (34). In I93C/F140C expressed in the periplasm, an intramolecular disulfide bond was formed between the two cysteine residues. This oxidized form of I93C/F140C was fixed in the closed conformation and was unable to release lipoproteins from the inner membrane, suggesting that opening of the cavity is crucial for the LolA function (34). Biochemical studies subsequently showed that the LolA cavity indeed undergoes opening and closing upon the binding and release of lipoproteins, respectively (19). Moreover, it was found that only the closed form of LolA is active in the lipoprotein release reaction.I93C/F140C exhibited the strongest growth inhibition among the LolA mutants so far isolated, although it was fully active in the presence of a reducing agent (34). We show here that oxidized I93C/F140C strongly activates the Cpx two-component system (23) that responds to cell envelope stress, whereas overproduction of LolCDE partly suppresses the toxic effect of I93C/F140C.     

Characterization of the LolA-LolB system as the general lipoprotein localization mechanism of Escherichia coli.

The major outer membrane lipoprotein (Lpp) of Escherichia coli requires LolA for its release from the cytoplasmic membrane, and LolB for its localization to the outer membrane. We examined the significance of the LolA-LolB system as to the outer membrane localization of other lipoproteins. All lipoproteins possessing an outer membrane-directed signal at the N-terminal second position were efficiently released from the inner membrane in the presence of LolA. Some lipoproteins were released in the absence of externally added LolA, albeit at a slower rate and to a lesser extent. This LolA-independent release was also strictly dependent on the outer membrane sorting signal. A lipoprotein-LolA complex was formed when the release took place in the presence of LolA, whereas lipoproteins released in the absence of LolA existed as heterogeneous complexes, suggesting that the release and the formation of a complex with LolA are distinct events. The release of LolB, an outer membrane lipoprotein functioning as the receptor for a lipoprotein-LolA complex, occurred with a trace amount of LolA, and therefore was extremely efficient. The LolA-dependent release of lipoproteins was found to be crucial for the specific incorporation of lipoproteins into the outer membrane, whereas lipoproteins released in the absence of LolA were nonspecifically and inefficiently incorporated into the membrane. The outer membrane incorporation of lipoproteins including LolB per se was dependent on LolB in the outer membrane. From these results, we conclude that lipoproteins in E. coli generally utilize the LolA-LolB system for efficient release from the inner membrane and specific localization to the outer membrane.     

Deletion of lolB, Encoding an Outer Membrane Lipoprotein, Is Lethal for Escherichia coli and Causes Accumulation of Lipoprotein Localization Intermediates in the Periplasm

Outer membrane lipoproteins of Escherichia coli are released from the inner membrane upon the formation of a complex with a periplasmic chaperone, LolA, followed by localization to the outer membrane. In vitro biochemical analyses revealed that the localization of lipoproteins to the outer membrane generally requires an outer membrane lipoprotein, LolB, and occurs via transient formation of a LolB-lipoprotein complex. On the other hand, a mutant carrying the chromosomal lolB gene under the control of the lac promoter-operator grew normally in the absence of LolB induction if the mutant did not possess the major outer membrane lipoprotein Lpp, suggesting that LolB is only important for the localization of Lpp in vivo. To examine the in vivo function of LolB, we constructed a chromosomal lolB null mutant harboring a temperature-sensitive helper plasmid carrying the lolB gene. At a nonpermissive temperature, depletion of the LolB protein due to loss of the lolB gene caused cessation of growth and a decrease in the number of viable cells irrespective of the presence or absence of Lpp. LolB-depleted cells accumulated the LolA-lipoprotein complex in the periplasm and the mature form of lipoproteins in the inner membrane. Taken together, these results indicate that LolB is the first example of an essential lipoprotein for E. coli and that its depletion inhibits the upstream reactions of lipoprotein trafficking.     

副溶血性弧菌脂蛋白定位系统转运蛋白结构与功能的生物信息学分析

【目的】副溶血性弧菌是一种重要的人畜共患病原菌,脂蛋白定位系统(Localization of lipoprotein system,Lol)负责该菌脂蛋白的转运与定位,与其致病力及耐药性密切相关,对Lol系统转运蛋白进行系统的生物信息学分析,有助于推动副溶血性弧菌致病与耐药机理的进一步研究。【方法】本文通过生物信息学分析技术,结合ExPASy在线工具、SignalP 4.0 Server、TMHMM-2.0、STRING、SWISS-MODEL等软件,分析了副溶血性弧菌Lol系统转运蛋白LolA-E及LolCD_2E的基本性质、蛋白互作关系及三级结构。【结果】LolA和LolB为酸性亲水蛋白,含信号肽位点,无跨膜区域。LolC和LolE为碱性疏水膜蛋白,LolCD_2E为中性疏水膜蛋白,LolC-E及LolCD_2E均无显著的信号肽位点。蛋白相互作用网络显示,LolA–E五个蛋白的编码基因均共表达,负责脂蛋白的合成与转运,并与BamA、Pal、MacB、CmeC等外膜蛋白具有密切的互作关系。三级结构同源建模发现,副溶血性弧菌与大肠杆菌拥有相似的LolA和LolB结构,LolC-E含有MacB蛋白的同源结构,赋予了该系统消耗ATP运输脂蛋白的重要功能。此外,本研究还首次发现了副溶血性弧菌LolC和LolE中存在一段保守的Hook结构,是LolCD_2E复合物与LolA结合并转运脂蛋白的关键区域。【结论】本研究为副溶血性弧菌Lol系统转运蛋白的表达纯化、结构与功能的研究提供了重要的数据基础,为后续抗菌药物的研发提供了新型作用靶点。