Dominant negative mutant of a lipoprotein‐specific molecular chaperone, LolA, tightly associates with LolCDE

Miyamoto, Atsuki; Matsuyama, Shin-ichi; Tokuda, Harukuni

doi:10.1016/s0014-5793(02)03305-7

Cited by 16 publications

(17 citation statements)

References 17 publications

(44 reference statements)

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“…These proteins may play many roles in oxidation or reduction pathways in the cytoplasmic membrane or periplasmis space, similar to the Dsb proteins in E. coli (31). The observation that the gene encoding LolA, a prokaryotic periplasmic lipoproteinspecific molecular chaperone (27,28), is cotranscribed with trxB in an operon regulated by oxidative and thiol redox stress supports the idea that some of these proteins are localized in the periplasm and membrane of this anaerobe. Further studies should help us to clarify how B. fragilis TrxB, the Trxs, and thiol disulfide oxidoreductase-like proteins are employed to reduce different cytoplasm and periplasm substrates in an anaerobic organism.…”

Section: Discussionmentioning

confidence: 69%

Thioredoxin Reductase Is Essential for Thiol/Disulfide Redox Control and Oxidative Stress Survival of the Anaerobe Bacteroides fragilis

2007

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Results of this study showed that the anaerobic, opportunistic pathogen Bacteroides fragilis lacks the glutathione/glutaredoxin redox system and possesses an extensive number of putative thioredoxin (Trx) orthologs. Analysis of the genome sequence revealed six Trx orthologs and an absence of genes required for synthesis of glutathione and glutaredoxins. In addition, it was shown that the thioredoxin reductase (TrxB)/Trx system is the major or sole redox system for thiol/disulfide cellular homeostasis in this anaerobic bacterium. Expression of the B. fragilis trxB gene was induced following treatment with diamide or H 2 O 2 or exposure to oxygen. This inducible trxB expression was OxyR independent. Northern blot hybridization analysis showed that the trxB mRNA was cotranscribed with lolA as a bicistronic transcript or was present as a monocistronic transcript that was also highly induced under the same conditions. The role of LolA, a prokaryotic periplasmic lipoprotein-specific molecular chaperone in the thiol/disulfide redox system, is unknown. A trxB deletion mutant was more sensitive to the effects of diamide and oxygen than the parent strain. In addition, the trxB mutant was unable to grow in culture media without addition of a reductant. Furthermore, the trxB mutant was not able to induce intraabdominal abscess formation in a mouse model, whereas the parent strain was. Taken together, these data strongly suggest that TrxB/Trx is the major, if not the sole, thiol/disulfide redox system in this anaerobe required for survival and abscess formation in a peritoneal cavity infection model.

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Section: Discussionmentioning

confidence: 69%

Thioredoxin Reductase Is Essential for Thiol/Disulfide Redox Control and Oxidative Stress Survival of the Anaerobe Bacteroides fragilis

2007

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“…Previous biochemical data (13,15) have suggested that differences in the affinity for lipoproteins drive the transport of lipoproteins to the outer membrane via LolB. The crystal structures of LolA and LolB (18), and the phenotypes of LolA mutants, R43L (19) and F47E (20), indicated the importance of opening and closing of the LolA lid for lipoprotein binding to LolA, and subsequent lipoprotein transfer from LolA to LolB, respectively (Fig. 7A).…”

Section: Discussionmentioning

confidence: 73%

“…X 1 -X 3 and X 4 -X 6 for the respective derivatives are shown above. Construction of R43L (19) and F47E (20) was reported previously. Although not reported, the F47D, F47K, and F47P mutants have previously been isolated in addition to F47E by means of random mutagenesis.…”

Section: Construction Of Lola Mutants As To Arg At Position 43 and Phmentioning

confidence: 99%

“…Because the closed form of the R43L mutant was speculated to be unstable without stabilizing hydrogen bonds, closing of the LolA lid seemed to be critically important for efficient transfer of associated lipoproteins to LolB (18,19). Another mutant, F47E, having Glu in place of Phe at position 47 in the ␤3 strand, was defective in the complex formation with lipoproteins and remained tightly associated with LolCDE, thereby inhibiting the wildtype LolA (20). The LolA structure suggested the formation of additional hydrogen bonds between the ␤-sheet and the lid helices of the F47E derivative, making opening of the lid more difficult (18).…”

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confidence: 99%

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

Taniguchi

Matsuyama

Tokuda

2005

Journal of Biological Chemistry

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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 ␤-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.Bacterial lipoproteins are synthesized as precursors and then translocated to the periplasmic side of the inner membrane, where they are processed to mature forms (1, 2). Mature lipoproteins have an N-terminal Cys that is modified by thioether-linked diacylglycerol and aminolinked acyl chains (3). Escherichia coli has at least 90 lipoproteins (4 -6), which are anchored through N-terminal lipids to the periplasmic leaflet of either the inner or outer membrane depending on the sorting signal. When lipoproteins have Asp at position 2 (7), they are retained in the inner membrane, presumably through an interaction between Asp at position 2 and phospholipids (8). Residues other than Asp at position 2 cause outer membrane localization of lipoproteins (9, 10).The Lol system, comprising five Lol proteins, is required for the sorting and outer membrane localization of lipoproteins. The LolCDE complex, an ATP binding cassette transporter, releases outer membranedirected lipoproteins from the inner membrane in an ATP-dependent manner (11, 12), leading to the formation of a water-soluble complex comprising one molecule each of a lipoprotein and LolA in the periplasm (13, 14). Interaction of the LolA-lipoprotein complex with outer membrane receptor LolB induces the energy-independent transfer of lipoproteins from LolA to LolB, and then from LolB to the outer membrane (15,16). LolB is itself a lipoprotein anchored to the outer me...

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“…The replacement of Phe at position 47 with Glu gave a dominant negative LolA derivative, which cannot accept lipoproteins and remains tightly associated with LolCDE (17). Since Phe 47 is one of the residues forming the hydrophobic cavity (Fig.…”

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confidence: 99%

Roles of the Hydrophobic Cavity and Lid of LolA in the Lipoprotein Transfer Reaction in Escherichia coli

Watanabe

Matsuyama

Tokuda

2006

Journal of Biological Chemistry

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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 ␣-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.

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Dominant negative mutant of a lipoprotein‐specific molecular chaperone, LolA, tightly associates with LolCDE

Cited by 16 publications

References 17 publications

Thioredoxin Reductase Is Essential for Thiol/Disulfide Redox Control and Oxidative Stress Survival of the Anaerobe Bacteroides fragilis

Thioredoxin Reductase Is Essential for Thiol/Disulfide Redox Control and Oxidative Stress Survival of the Anaerobe Bacteroides fragilis

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

Roles of the Hydrophobic Cavity and Lid of LolA in the Lipoprotein Transfer Reaction in Escherichia coli

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