Sequence-structure relationships in polysaccharide co-polymerase (PCP) proteins

Morona, Renato; Purins, Leanne; Tocilj, Ante; Matte, Allan; Cygler, Mirosław

doi:10.1016/j.tibs.2008.11.001

Cited by 80 publications

(75 citation statements)

References 42 publications

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“…Nonetheless, our results demonstrate that WzzB could form oligomeric complexes, most probably homodimers, in agreement with Stenberg et al (2005), who performed mass spectrometry of E. coli WzzB oligomers and concluded that they consist exclusively of WzzB. Our results indicating that the amount of Wzy is much smaller than that of both chain length regulators, together with the apparent absence of proteinprotein interactions between WzzB and Wzy, are not consistent with the recent model proposing that Wzz oligomers serve as a scaffold for multiple Wzy polymerase molecules to facilitate OAg polymerization (Morona et al, 2009;Tocilj et al, 2008). Besides protein-protein interaction, Wzz and Wzy could also interact via contacts mediated by the OAg chains.…”

Section: Discussionsupporting

confidence: 71%

The cellular level of O-antigen polymerase Wzy determines chain length regulation by WzzB and WzzpHS-2 in Shigella flexneri 2a

et al. 2009

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The lipopolysaccharide O antigen of Shigella flexneri 2a has two preferred chain lengths, a short (S-OAg) composed of an average of 17 repeated units and a very long (VL-OAg) of about 90 repeated units. These chain length distributions are controlled by the chromosomally encoded WzzB and the plasmid-encoded Wzz pHS-2 proteins, respectively. In this study, genes wzzB, wzz pHS-2 and wzy (encoding the O-antigen polymerase) were cloned under the control of arabinose-and rhamnose-inducible promoters to investigate the effect of varying their relative expression levels on O antigen polysaccharide chain length distribution. Controlled expression of the chain length regulators wzzB and wzz pHS-2 revealed a dose-dependent production of each modal length. Increase in one mode resulted in a parallel decrease in the other, indicating that chain length regulators compete to control the degree of O antigen polymerization. Also, when expression of the wzy gene is low, S-OAg but not VL-OAg is produced. Production of VL-OAg requires high induction levels of wzy. Thus, the level of expression of wzy is critical in determining O antigen modal distribution. Western blot analyses of membrane proteins showed comparable high levels of the WzzB and Wzz pHS-2 proteins, but very low levels of Wzy. In vivo cross-linking experiments and immunoprecipitation of membrane proteins did not detect any direct interaction between Wzy and WzzB, suggesting the possibility that these two proteins may not interact physically but rather by other means such as via translocated O antigen precursors.

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Section: Discussionsupporting

confidence: 71%

The cellular level of O-antigen polymerase Wzy determines chain length regulation by WzzB and WzzpHS-2 in Shigella flexneri 2a

et al. 2009

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“…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.…”

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confidence: 99%

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 Wzz proteins, which are members of the polysaccharide co-polymerase family of proteins (4), are responsible for determining the number of O antigen subunits that are linked together to form the side chain. In strains producing two different preferred chain lengths, different Wzz proteins are used to regulate each chain length (5)(6)(7).…”

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confidence: 99%

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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Sequence-structure relationships in polysaccharide co-polymerase (PCP) proteins

Cited by 80 publications

References 42 publications

The cellular level of O-antigen polymerase Wzy determines chain length regulation by WzzB and WzzpHS-2 in Shigella flexneri 2a

The cellular level of O-antigen polymerase Wzy determines chain length regulation by WzzB and WzzpHS-2 in Shigella flexneri 2a

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

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