Structural Characterization of Closely Related O-antigen Lipopolysaccharide (LPS) Chain Length Regulators

Kalynych, Sergei; Yao, Deqiang; Magee, James E.; Cygler, Mirosław

doi:10.1074/jbc.m112.354837

Cited by 24 publications

(68 citation statements)

References 42 publications

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“…This similarity might provide a clue to substrate specificity, explaining the targeted inhibition of OSA production in serotype O5. Recently, high-resolution X-ray crystal structures of the periplasmic domain of Wzz from Shigella flexneri and from other bacterial species have been obtained (53,54). These structures revealed a conserved structural motif, and functional investigation through site-directed mutagenesis identified residues proposed to be involved in substrate binding and oligomerization (49,50,55).…”

Section: Discussionmentioning

confidence: 99%

The D3 Bacteriophage α-Polymerase Inhibitor (Iap) Peptide Disrupts O-Antigen Biosynthesis through Mimicry of the Chain Length Regulator Wzz in Pseudomonas aeruginosa

et al. 2013

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L ipopolysaccharide (LPS) is an integral structural component of the outer membrane of Gram-negative bacteria and is important for the survival of these bacteria in the environment or in a host. In Pseudomonas aeruginosa and many other opportunistic pathogens, LPS is a major virulence factor and is composed of a lipid A membrane anchor, a core oligosaccharide linker, and a distal polysaccharide termed O antigen (O-Ag) (1). P. aeruginosa simultaneously produces two forms of O-Ag, a homopolymeric common antigen (CPA) and an immunodominant heteropolymeric O-specific antigen (OSA) composed of repeating trisaccharide units (2).In P. aeruginosa PAO1, OSA is synthesized via the Wzx/Wzydependent pathway (3), which requires the activity of several integral inner membrane (IM) proteins (4). Synthesis begins at the cytoplasmic leaflet of the IM on the lipid carrier undecaprenyl pyrophosphate (UndPP), with the formation of an OSA trisaccharide repeat constructed from newly synthesized nucleotide sugar precursors (5). The UndPP-linked OSA repeat is then transported through the cationic interior of the OSA flippase Wzx to the periplasmic leaflet of the IM (6, 7) and polymerized at the reducing terminus of the growing chain (8) by Wzy via a putative "catch-and-release" mechanism in an ␣-1-4 linkage (9, 10). The OSA chain length is regulated by the polysaccharide copolymerase (PCP) proteins Wzz 1 and Wzz 2 , which interact with the nascent polysaccharides and confer long (12 to 16 and 22 to 30 repeats) and very long (40 to 50 repeats) modal lengths, respectively (11,12). Full-length OSA is then ligated to the lipid A-core moiety by the O-Ag ligase WaaL (13,14), forming the mature LPS molecule.Variability in the OSA repeat sugar constituents, intra-and interglycosidic linkages of the OSA repeat residues, and the presence of side branch modifications are used to classify P. aeruginosa into 20 distinct serotypes according to the International Antigenic Typing Scheme (1). Similar OSA backbone sugar structures result in certain individual serotypes, for example, O2, O5, O16, O18, and O20, being classified into a single serogroup (serogroup O2). Immunochemical cross-reactivity of LPS of these serotypes with specific typing antisera or monoclonal antibodies (MAbs) substantiates their relatedness. In particular, the only difference between the OSA chemical structures of serotypes O5 and O16 is the ␣ or ␤ configuration of the interglycosidic bond at the reducing end, respectively (15,16). Interestingly, the OSA biosynthesis cluster spanning the pa3160 to pa3145 (wzz 1 to wbpL) genes of O5 is identical to that in serogroup O2. This suggests that genes located outside the wbp cluster are responsible for the chemical differences in the OSA structures. Therefore, genes responsible for the chemical differences in OSA structures were likely to have come from external sources such as lysogenic bacteriophage (17) or other ancestral microbial species.Serotype conversion following bacteriophage infection has long been observed in diverse . Upon i...

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Section: Discussionmentioning

confidence: 99%

The D3 Bacteriophage α-Polymerase Inhibitor (Iap) Peptide Disrupts O-Antigen Biosynthesis through Mimicry of the Chain Length Regulator Wzz in Pseudomonas aeruginosa

et al. 2013

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“…Kalynych et al (2012) observed that an FepE mutant, although conferring a shortened LPS Oag modal chain length, had the same 3D structure as FepE (Kalynych et al, 2012;Tocilj et al, 2008), suggesting that Oag modal chain length regulation by FepE is probably controlled by amino acid residues along the structure of the oligomer, rather than either the oligomer size or conformational changes.…”

Section: Discussionmentioning

confidence: 99%

“…Studies on the structure of FepE show that the protein oligomerizes, but the size of this oligomer appears to be variable. Crystallization data on the periplasmic region of FepE and a FepE mutant conferring a shorter LPS Oag chain length show that both form nonamer structures (Kalynych et al, 2012;Tocilj et al, 2008). In contrast, cryo-electron microscopy studies on the full-length FepE reconstituted into proteoliposomes suggest a preference for a hexameric state (Larue et al, 2009).…”

Section: Introductionmentioning

confidence: 92%

“…1), both proteins are structurally similar (Kalynych et al, 2011(Kalynych et al, , 2012Tocilj et al, 2008) and belong to the same family of PCP1a proteins. Analysis of several WzzB SF mutants showed that mutational alterations made between amino acids 102-107, 128-131 and 219-232 consistently had an effect on Oag modal chain length determination (Daniels & Morona, 1999;Papadopoulos & Morona, 2010;R.…”

Section: Identification Of Putative Sites That May Affect Fepe Functionmentioning

confidence: 99%

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Residues located inside the Escherichia coli FepE protein oligomer are essential for lipopolysaccharide O-antigen modal chain length regulation

Tran

Morona

2013

Microbiology

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The Escherichia coli O157 : H7 FepE protein regulates lipopolysaccharide (LPS) O-antigen (Oag) chain length to confer a very long modal chain length of .80 Oag repeat units (RUs). The mechanism by which FepE regulates Oag modal chain length and the regions within it that are important for its function remain unclear. Studies on the structure of FepE show that the protein oligomerizes. However, the exact size of the oligomer is in dispute, further hampering our understanding of its mechanism. Guided by information previously obtained for regions known to be important for Oag modal chain length determination in the homologous Shigella flexneri WzzB SF protein, a set of FepE mutant constructs with single amino acid substitutions was created. Analysis of the resulting LPS conferred by these mutant His 6 -FepE proteins showed that amino acid substitutions of leucine 168 (L168) and aspartic acid 268 (D268) resulted in LPS with consistently shortened Oag chain lengths of ,80 Oag RUs. Substitution of FepE's transmembrane cysteine residues did not affect function. Chemical cross-linking experiments on mutant FepE proteins showed no consistent correlation between oligomer size and functional activity, and MS analysis of FepE oligomers indicated that the in vivo size of FepE is consistent with a maximum size of a hexamer. Our findings suggest that different FepE residues, mainly located within the internal cavity of the oligomer, contribute to Oag modal chain length determination but not the oligomeric state of the protein. INTRODUCTIONLipopolysaccharide (LPS) is an important virulence factor of many Gram-negative bacteria and generally consists of three distinct regions: the membrane-anchored lipid A domain, the core sugar region and the O-antigen (Oag) polysaccharide chains (Raetz & Whitfield, 2002). In Shigella flexneri, as well as in other members of the family Enterobacteriaceae, the genes encoding enzymes for Oag biosynthesis and polymerization are mainly found in the bacterial chromosome, but can also be plasmid-encoded (Raetz & Whitfield, 2002). Oag is a polymer of sugar repeat units (RUs) which define the Oag serotype specificity. The basic Oag RU of S. flexneri is a tetrasaccharide made up of three rhamnose sugars and one N-acetylglucosamine sugar.The following describes the Oag synthesis strategy in S. flexneri and Escherichia coli K-12 known as the Wzydependent polymerization pathway (Morona et al., 2009;Raetz & Whitfield, 2002;Samuel & Reeves, 2003;Tocilj et al., 2008). Biosynthesis of the Oag is initiated on the cytoplasmic side of the inner membrane. After a series of successive glycoyltransferase reactions, an RU is assembled on the membrane-bound carrier undecaprenyl pyrophosphate (Und-PP) and is then transferred across to the periplasmic side of the membrane by the Wzx flippase. Oag RUs then undergo polymerization by the Wzy polymerase and are attached on to the lipid A-core molecule by the WaaL ligase to form a chain of Oag RUs on a complete LPS molecule. Oag RUs can vary considerably from 0 to .100 in n...

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“…The periplasmic domain structures of a collection of PCP proteins (that also include WzzB SF and WzzB ST ) have been solved (Chang et al, 2015;Kalynych et al, 2012;Larue et al, 2009;Tocilj et al, 2008;Kalynych et al, 2015) and shown that the protomers self-assemble into 3-8 protein oligomers. As Wzz oligomerization has been previously established, the aim of this study is to explore the underlying basis of this interaction.…”

Section: Introductionmentioning

confidence: 99%

Conserved transmembrane glycine residues in the Shigella flexneri polysaccharide co-polymerase protein WzzB influence protein–protein interactions

et al. 2016

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The O antigen (Oag) component of lipopolysaccharides (LPS) is crucial for virulence and Oag chain-length regulation is controlled by the polysaccharide co-polymerase class 1 (PCP1) proteins. Crystal structure analyses indicate that structural conservation among PCP1 proteins is highly maintained, however the mechanism of Oag modal-chain-length control remains to be fully elucidated. Shigella flexneri PCP1 protein WzzB SF confers a modal-chain length of 10-17 Oag repeat units (RUs), whereas the Salmonella enterica Typhimurium PCP1 protein WzzB ST confers a modal-chain length of~16-28 Oag RUs. Both proteins share >70 % overall sequence identity and contain two transmembrane (TM1 and TM2) regions, whereby a conserved prolineglycine-rich motif overlapping the TM2 region is identical in both proteins. Conserved glycine residues within TM2 are functionally important, as glycine to alanine substitutions at positions 305 and 311 confer very short Oag modal-chain length (~2-6 Oag RUs). In this study, WzzB SF was co-expressed with WzzB ST in S. flexneri and a single intermediate modal-chain length of 11-21 Oag RUs was observed, suggesting the presence of Wzz:Wzz interactions. Interestingly, co-expression of WzzB SF with WzzB G305A/G311A conferred a bimodal LPS Oag chain length (despite over 99 % protein sequence identity), and we hypothesized that the proteins fail to interact. Co-purification assays detected His 6 -WzzB SF co-purifying with FLAGtagged WzzB ST but not with FLAG-tagged WzzB G305A/G311A , supporting our hypothesis. These data indicate that the conserved glycine residues in TM2 are involved in Wzz:Wzz interactions, and provide insight into key interactions that drive Oag modal length control.

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Structural Characterization of Closely Related O-antigen Lipopolysaccharide (LPS) Chain Length Regulators

Cited by 24 publications

References 42 publications

The D3 Bacteriophage α-Polymerase Inhibitor (Iap) Peptide Disrupts O-Antigen Biosynthesis through Mimicry of the Chain Length Regulator Wzz in Pseudomonas aeruginosa

The D3 Bacteriophage α-Polymerase Inhibitor (Iap) Peptide Disrupts O-Antigen Biosynthesis through Mimicry of the Chain Length Regulator Wzz in Pseudomonas aeruginosa

Residues located inside the Escherichia coli FepE protein oligomer are essential for lipopolysaccharide O-antigen modal chain length regulation

Conserved transmembrane glycine residues in the Shigella flexneri polysaccharide co-polymerase protein WzzB influence protein–protein interactions

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