1887

Abstract

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. PCP1 protein WzzB confers a modal-chain length of 10–17 Oag repeat units (RUs), whereas the PCP1 protein WzzB 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 proline-glycine-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 was co-expressed with WzzB in 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 with WzzB 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-WzzB co-purifying with FLAG-tagged WzzB but not with FLAG-tagged WzzB, 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.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.000282
2016-06-01
2019-12-06
Loading full text...

Full text loading...

/deliver/fulltext/micro/162/6/921.html?itemId=/content/journal/micro/10.1099/mic.0.000282&mimeType=html&fmt=ahah

References

  1. Arselin G., Giraud M. F., Dautant A., Vaillier J., Brèthes D., Coulary-Salin B., Schaeffer J., Velours J.. 2003; The GxxxG motif of the transmembrane domain of subunit e is involved in the dimerization/oligomerization of the yeast ATP synthase complex in the mitochondrial membrane. Eur J Biochem270:1875–1884 [CrossRef][PubMed]
    [Google Scholar]
  2. Becker A., Niehaus K., Puhler A.. 1995; Low-molecular-weight succinoglycan is predominantly produced by Rhizobium meliloti strains carrying a mutated ExoP protein characterized by a periplasmic N-terminal domain and a missing C-terminal domain. Mol Microbiol16:191–203 [CrossRef][PubMed]
    [Google Scholar]
  3. Becker A., Puhler A.. 1998; Specific amino acid substitutions in the proline-rich motif of the Rhizobium meliloti ExoP protein result in enhanced production of low-molecular-weight succinoglycan at the expense of high-molecular-weight succinoglycan. J Bacteriol180:395–399
    [Google Scholar]
  4. Chang C. W., Tran E. N., Ericsson D. J., Casey L. W., Lonhienne T., Benning F., Morona R., Kobe B.. 2015; Structural and biochemical analysis of a single amino-acid mutant of WzzBSF that alters lipopolysaccharide O-antigen chain length in Shigella flexneri . PLoS One10:e0138266 [CrossRef][PubMed]
    [Google Scholar]
  5. Daniels C., Morona R.. 1999; Analysis of Shigella flexneri wzz (Rol) function by mutagenesis and cross-linking: wzz is able to oligomerize. Mol Microbiol34:181–194 [CrossRef][PubMed]
    [Google Scholar]
  6. Elbaz Y., Salomon T., Schuldiner S.. 2008; Identification of a glycine motif required for packing in EmrE, a multidrug transporter from Escherichia coli . J Biol Chem283:12276–12283 [CrossRef][PubMed]
    [Google Scholar]
  7. Guzman L. M., Belin D., Carson M. J., Beckwith J.. 1995; Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol177:4121–4130[PubMed]
    [Google Scholar]
  8. Hong M., Payne S. M.. 1997; Effect of mutations in Shigella flexneri chromosomal and plasmid-encoded lipopolysaccharide genes on invasion and serum resistance. Mol Microbiol24:779–791 [CrossRef][PubMed]
    [Google Scholar]
  9. Javadpour M. M., Eilers M., Groesbeek M., Smith S. O.. 1999; Helix packing in polytopic membrane proteins: role of glycine in transmembrane helix association. Biophys J77:1609–1618 [CrossRef][PubMed]
    [Google Scholar]
  10. Jenei Z. A., Borthwick K., Zammit V. A., Dixon A. M.. 2009; Self-association of transmembrane domain 2 (TM2), but not TM1, in carnitine palmitoyltransferase 1A: role of GXXXG(A) motifs. J Biol Chem284:6988–6997 [CrossRef][PubMed]
    [Google Scholar]
  11. Jenkins F. A., Parrington F. R.. 1976; The postcranial skeletons of the triassic mammals eozostrodon, megazostrodon and erythrotherium. Philos Trans R Soc Lond B Biol Sci273: 12276– 12283[PubMed][CrossRef]
    [Google Scholar]
  12. Kalynych S., Cherney M., Bostina M., Rouiller I., Cygler M.. 2015; Quaternary structure of WzzB and WzzE polysaccharide copolymerases. Protein Sci24:58–69 [CrossRef][PubMed]
    [Google Scholar]
  13. Kalynych S., Yao D., Magee J., Cygler M.. 2012; Structural characterization of closely related O-antigen lipopolysaccharide (LPS) chain length regulators. J Biol Chem287:15696–15705 [CrossRef][PubMed]
    [Google Scholar]
  14. Larue K., Kimber M. S., Ford R., Whitfield C.. 2009; Biochemical and structural analysis of bacterial O-antigen chain length regulator proteins reveals a conserved quaternary structure. J Biol Chem284:7395–7403 [CrossRef][PubMed]
    [Google Scholar]
  15. Lugtenberg B., Meijers J., Peters R., van der Hoek P., van Alphen L.. 1975; Electrophoretic resolution of the "major outer membrane protein" of Escherichia coli K12 into four bands. FEBS Lett58:254–258 [CrossRef][PubMed]
    [Google Scholar]
  16. Mackenzie K. R., Engelman D. M.. 1998; Structure-based prediction of the stability of transmembrane helix-helix interactions: the sequence dependence of glycophorin A dimerization. Proc Natl Acad Sci U S A95:3583–90 [CrossRef][PubMed]
    [Google Scholar]
  17. Morona R., Mavris M., Fallarino A., Manning P. A.. 1994; Characterization of the rfc region of Shigella flexneri . J Bacteriol176:733–747[PubMed]
    [Google Scholar]
  18. Morona R., Purins L., Tocilj A., Matte A., Cygler M.. 2009; Sequence-structure relationships in polysaccharide co-polymerase (PCP) proteins. Trends Biochem Sci34:78–84 [CrossRef][PubMed]
    [Google Scholar]
  19. Morona R., Van Den Bosch L., Daniels C.. 2000; Evaluation of Wzz/MPA1/MPA2 proteins based on the presence of coiled-coil regions. Microbiology146:1–4 [CrossRef][PubMed]
    [Google Scholar]
  20. Morona R., van den Bosch L., Manning P. A.. 1995; Molecular, genetic, and topological characterization of O-antigen chain length regulation in Shigella flexneri . J Bacteriol177:1059–1068[PubMed]
    [Google Scholar]
  21. Murray G. L., Attridge S. R., Morona R.. 2003; Regulation of Salmonella typhimurium lipopolysaccharide O antigen chain length is required for virulence; identification of FepE as a second Wzz. Mol Microbiol47:1395–1406 [CrossRef][PubMed]
    [Google Scholar]
  22. Overton M. C., Chinault S. L., Blumer K. J.. 2003; Oligomerization, biogenesis, and signaling is promoted by a glycophorin A-like dimerization motif in transmembrane domain 1 of a yeast G protein-coupled receptor. J Biol Chem278:49369–49377 [CrossRef][PubMed]
    [Google Scholar]
  23. Papadopoulos M., Morona R.. 2010; Mutagenesis and chemical cross-linking suggest that Wzz dimer stability and oligomerization affect lipopolysaccharide O-antigen modal chain length control. J Bacteriol192:3385–3393 [CrossRef][PubMed]
    [Google Scholar]
  24. Purins L., Van Den Bosch L., Richardson V., Morona R.. 2008; Coiled-coil regions play a role in the function of the Shigella flexneri O-antigen chain length regulator WzzpHS2. Microbiology154:1104–1116 [CrossRef][PubMed]
    [Google Scholar]
  25. Raetz C. R., Whitfield C.. 2002; Lipopolysaccharide endotoxins. Annu Rev Biochem71:635–700 [CrossRef][PubMed]
    [Google Scholar]
  26. Russ W. P., Engelman D. M.. 2000; The GxxxG motif: a framework for transmembrane helix-helix association. J Mol Biol296:911–919 [CrossRef][PubMed]
    [Google Scholar]
  27. Samuel G., Reeves P.. 2003; Biosynthesis of O-antigens: genes and pathways involved in nucleotide sugar precursor synthesis and O-antigen assembly. Carbohydr Res338:2503–2519 [CrossRef][PubMed]
    [Google Scholar]
  28. Sperandeo P., Dehò G., Polissi A.. 2009; The lipopolysaccharide transport system of gram-negative bacteria. Biochim Biophys Acta1791:594–602 [CrossRef][PubMed]
    [Google Scholar]
  29. Stevenson G., Kessler A., Reeves P. R.. 1995; A plasmid-borne O-antigen chain length determinant and its relationship to other chain length determinants. FEMS Microbiol Lett125:23–30 [CrossRef][PubMed]
    [Google Scholar]
  30. Tocilj, Munger C., Proteau A., Morona R., Purins L., Ajamian E., Wagner J., Papadopoulos M., Van Den Bosch L., other authors. 2008; Bacterial polysaccharide co-polymerases share a common framework for control of polymer length. Nat Struct Mol Biol15:130–138 [CrossRef][PubMed]
    [Google Scholar]
  31. Tran E. N., Morona R.. 2013; Residues located inside the Escherichia coli FepE protein oligomer are essential for lipopolysaccharide O-antigen modal chain length regulation. Microbiology159:701–714 [CrossRef][PubMed]
    [Google Scholar]
  32. Tran E. N., Papadopoulos M., Morona R.. 2014; Relationship between O-antigen chain length and resistance to colicin E2 in Shigella flexneri . Microbiology160:589–601 [CrossRef][PubMed]
    [Google Scholar]
  33. Van den Bosch L., Morona R.. 2003; The actin-based motility defect of a Shigella flexneri rmID rough LPS mutant is not due to loss of IcsA polarity. Microb Pathog35:11–18 [CrossRef][PubMed]
    [Google Scholar]
  34. Van den Bosch L., Manning P. A., Morona R.. 1997; Regulation of O-antigen chain length is required for Shigella flexneri virulence. Mol Microbiol23:765–775 [CrossRef][PubMed]
    [Google Scholar]
  35. Weinglass A. B., Smirnova I. N., Kaback H. R.. 2001; Engineering conformational flexibility in the lactose permease of Escherichia coli: Use of glycine-scanning mutagenesis to rescue mutant Glu325-->Asp. Biochemistry40:769–776 [CrossRef][PubMed]
    [Google Scholar]
  36. Whitfield C., Naismith J. H.. 2008; Periplasmic export machines for outer membrane assembly. Curr Opin Struct Biol18:466–474 [CrossRef][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.000282
Loading
/content/journal/micro/10.1099/mic.0.000282
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error