Biochemical and Structural Analysis of Bacterial O-antigen Chain Length Regulator Proteins Reveals a Conserved Quaternary Structure

Larue, Kane; Kimber, Matthew S.; Ford, Robert C.; Whitfield, Chris

doi:10.1074/jbc.m809068200

Cited by 62 publications

(70 citation statements)

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“…Cross-linking analysis with formaldehyde suggests that detergent-solubilized FepE can form dimeric and tetrameric complexes that can assemble into higher molecular mass oligomer forms (Larue et al, 2009). In this study, in situ treatment of FepE and FepE mutants with APDP cross-linker showed that FepE was able to form a number of higher order oligomers (Figs 5 and S2) that did not appear to be larger than 260 kDa (Fig.…”

Section: Discussionmentioning

confidence: 79%

“…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: 89%

“…X-ray crystallographic studies of the periplasmic domain of FepE show that the protein forms a nonamer (Tocilj et al, 2008), while cryo-electron microscopy analysis on the fulllength FepE protein showed that FepE probably exists as a hexamer state (Larue et al, 2009). Cross-linking analysis with formaldehyde suggests that detergent-solubilized FepE can form dimeric and tetrameric complexes that can assemble into higher molecular mass oligomer forms (Larue et al, 2009).…”

Section: Discussionmentioning

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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Section: Discussionmentioning

confidence: 79%

Section: Introductionmentioning

confidence: 89%

Section: Discussionmentioning

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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“…Cross-linking studies indicate Wzz proteins form homo-oligomers (11), which has been confirmed via several structural analyses (9,12,13). This ability to homo-oligomerize is tied to * This work was supported in part by National Institutes of Health Grants 1 R01 AI068112 and 1 R21 AI53842 (to J.…”

mentioning

confidence: 97%

Site-directed Mutagenesis Reveals Key Residue for O Antigen Chain Length Regulation and Protein Stability in Pseudomonas aeruginosa Wzz2

Kintz¹,

Goldberg²

2011

Journal of Biological Chemistry

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Background: How Wzz proteins control O antigen chain length is not well understood. Results: Residue 321, located within the second coiled-coil region, of the Wzz2 protein influences the chain length produced. Conclusion: Position 321 affected the chain length regulating activity and oligomer stability of Wzz2. Significance: Prior studies focusing on Wzz and Wzy interactions in regulating chain length do not fully explain Wzz activity.

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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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Biochemical and Structural Analysis of Bacterial O-antigen Chain Length Regulator Proteins Reveals a Conserved Quaternary Structure

Abstract: Lipopolysaccharide (LPS) is a major component of the

Cited by 62 publications

References 61 publications

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

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

Site-directed Mutagenesis Reveals Key Residue for O Antigen Chain Length Regulation and Protein Stability in Pseudomonas aeruginosa Wzz2

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

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