---
pmid: '22437828'
title: Structural characterization of closely related O-antigen lipopolysaccharide
  (LPS) chain length regulators.
authors:
- Kalynych S
- Yao D
- Magee J
- Cygler M
journal: J Biol Chem
year: '2012'
full_text_available: true
full_text_extraction_method: html
pmcid: PMC3346132
doi: 10.1074/jbc.M112.354837
---

# Structural characterization of closely related O-antigen lipopolysaccharide (LPS) chain length regulators.
**Authors:** Kalynych S, Yao D, Magee J, Cygler M
**Journal:** J Biol Chem (2012)
**DOI:** [10.1074/jbc.M112.354837](https://doi.org/10.1074/jbc.M112.354837)
**PMC:** [PMC3346132](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3346132/)

## Abstract

1. J Biol Chem. 2012 May 4;287(19):15696-705. doi: 10.1074/jbc.M112.354837. Epub 
2012 Mar 21.

Structural characterization of closely related O-antigen lipopolysaccharide 
(LPS) chain length regulators.

Kalynych S(1), Yao D, Magee J, Cygler M.

Author information:
(1)Department of Biochemistry, McGill University, Montreal, Quebec H3G 0B1, 
Canada.

The surface O-antigen polymers of gram-negative bacteria exhibit a modal length 
distribution that depends on dedicated chain length regulator periplasmic 
proteins (polysaccharide co-polymerases, PCPs) anchored in the inner membrane by 
two transmembrane helices. In an attempt to determine whether structural changes 
underlie the O-antigen modal length specification, we have determined the 
crystal structures of several closely related PCPs, namely two chimeric PCP-1 
family members solved at 1.6 and 2.8 Å and a wild-type PCP-1 from Shigella 
flexneri solved at 2.8 Å. The chimeric proteins form circular octamers, whereas 
the wild-type WzzB from S. flexneri was found to be an open trimer. We also 
present the structure of a Wzz(FepE) mutant, which exhibits severe attenuation 
in its ability to produce very long O-antigen polymers. Our findings suggest 
that the differences in the modal length distribution depend primarily on the 
surface-exposed amino acids in specific regions rather than on the differences 
in the oligomeric state of the PCP protomers.

DOI: 10.1074/jbc.M112.354837
PMCID: PMC3346132
PMID: 22437828 [Indexed for MEDLINE]

## Full Text

Abstract

The surface O-antigen polymers of Gram-negative bacteria exhibit a modal length distribution that depends on dedicated chain length regulator periplasmic proteins (polysaccharide co-polymerases, PCPs) anchored in the inner membrane by two transmembrane helices. In an attempt to determine whether structural changes underlie the O-antigen modal length specification, we have determined the crystal structures of several closely related PCPs, namely two chimeric PCP-1 family members solved at 1.6 and 2.8 Å and a wild-type PCP-1 from Shigella flexneri solved at 2.8 Å. The chimeric proteins form circular octamers, whereas the wild-type WzzB from S. flexneri was found to be an open trimer. We also present the structure of a Wzz FepE mutant, which exhibits severe attenuation in its ability to produce very long O-antigen polymers. Our findings suggest that the differences in the modal length distribution depend primarily on the surface-exposed amino acids in specific regions rather than on the differences in the oligomeric state of the PCP protomers.

Introduction

Bacterial surface is lined with complex carbohydrate polymers that form a protective barrier against the external environment. For pathogenic bacteria, these polymers are an essential component of the survival toolkit within the host and frequently are critical determinants of pathogen-host interactions. In particular, the LPS O-antigen (OAg) 2 is emerging as an important pathogenesis factor contributing to virulence of a number of human pathogens ( 1 – 7 ). The O-antigen is the most chemically diverse component of the outer membrane lipopolysaccharide and varies among even the same species of bacteria. The mature O-antigen chain is a polymer made of the repeating units, with each O-unit containing 4–6 sugar residues ( 8 ). The proteins involved in the biosynthesis of the nucleotide sugars, glycosyl transfers, and OAg processing machinery are encoded on one or more gene clusters ( 9 , 10 ). The OAg processing machinery is responsible for the assembly of the polymer in the periplasmic space and for the transport to the cell surface ( 11 ). In a system referred to as Wzy-dependent synthesis, the individual O-units are synthesized at the cytoplasmic side of the inner membrane, then flipped to the periplasm by a flippase Wzx, and assemble into a high molecular mass polysaccharide by Wzy polymerase ( 9 ). The assembly is completed by the ligase WaaL, which transfers the mature O-antigen to the sugar moiety of the outer core of the lipid A molecule ( 9 ).

The number of the O-units in a given O-antigen polymer exposed on bacterial surface falls within a narrow range distribution, resulting in polymers of a well defined modal length. Much like the chemical composition of the individual sugar moieties making up the polymer, the length of the polysaccharide is species- and serotype-specific ( 10 , 12 ). Early genetic studies identified a single gene wzzB (previously cld or rol ) to be responsible for defining the specific number of the repeat units in a given O-antigen polymer ( 13 ). Subsequent biochemical investigations have found that the product of wzzB gene is a homo-oligomeric protein embedded in the inner membrane through two transmembrane helices and containing a large periplasmic domain ( 14 ). Shortly after, the presence of similar structural features was discovered in seemingly unrelated proteins involved in trafficking a range of complex polysaccharides in both Gram-negative and Gram-positive bacteria ( 14 – 16 ). These functionally related proteins were named p olysaccharide c o- p olymerases (PCPs) ( 17 ) and are further subdivided into various subfamilies depending on the type of the polysaccharide biosynthetic pathway they are part of and the presence of a cytosolic tyrosine kinase domain ( 11 , 17 ).

Currently, atomic-level structural data are available only for periplasmic domains of a few family members. Crystallographic studies of the WzzB from Salmonella typhimurium (Wzz ST ), FepE (Wzz FepE ) from Escherichia coli O157:H7, and WzzE from E. coli revealed that these proteins adopt a very similar three-dimensional fold despite a very limited sequence similarity ( 18 ). The oligomeric chain length regulators resemble elongated bell-shaped structures, with the protomers composed of an α-β base domain and a long α-helix extending about 100 Å away from the inner membrane into the periplasm ( 18 ). In the crystal structures, soluble domains of WzzB, Wzz FepE , and WzzE were found in different oligomeric organizations composed of five, nine, and eight protomers, respectively. The size of the oligomers was addressed independently also by electron microscopy and small angle scattering. The study by electron microscopy of the full-length WzzB and Wzz FepE reconstituted in proteoliposomes led Larue et al. ( 19 ) to suggest a hexameric structure for these oligomers. A small angle x-ray scattering study of the full-length E. coli (O86:H2) WzzB solubilized in n -dodecyl-β-maltoside suggested on the other hand the presence of tetramers in solution ( 20 ), although higher order oligomers were observed when the salt concentration was increased. The contradictory results of these studies performed away from the natural environment within the bacterial membrane leaves uncertain the native oligomeric state of the PCPs within the inner membrane.

Despite the current functional and structural data, little is understood about the precise molecular mechanism underlying the chain length control. The specific modal length appears to be associated with each chain length regulator because when expressed in a closely related bacterial species, the PCP imposes its own characteristic modal length distribution despite the drastically different chemical structure of the carbohydrate polymer ( 14 , 21 , 22 ). Previously, we have used a structure-guided chimeric protein approach to establish which regions of the chain length regulator are responsible for the control of modal length. By swapping various structural elements between two closely WzzB molecules yet imposing distinctly different modal length distributions, Shigella flexneri WzzB SF and S. typhimurium WzzB ST , we were able to produce a number of functional chimeric proteins exhibiting a range of intermediate modal lengths ( 22 ). These findings posed a series of further questions, one of which was whether the observed differences in phenotypes could be the result of significant local conformational differences between the wild-type protein and the corresponding chimeric chain length regulators.

To address this question, we determined the structures of the periplasmic domain of the S. flexneri WzzB SF and three chimeric chain length regulator proteins generated from parental WzzB SF and WzzB ST and compared them with the parental structures. The structural alignments demonstrated little differences in the main-chain conformations, suggesting that a given modal length is determined by the side chains of residues in selected regions. Most of these residues were found to be surface-exposed on the external face of the oligomer. The chimeras form oligomers with a variable number of protomers, bell-shaped octamers similar to the oligomers of WzzE, pentamers similar to the previously observed E. coli WzzB, and open-face trimers. The size of the octamers corresponds very well to the electron microscopy data, and we surmise that this is the most likely arrangement of WzzB in the bacterial cell. Lastly, we determined the structure of a Wzz FepE loop deletion mutant, which confers strikingly different modal length from the wild-type protein, and discovered that it forms a nonameric structure very similar to its wild-type counterpart. All of these observations suggest that the modality is dictated predominantly by the nature of the external surface amino acids rather then by structural alterations of the chain length regulator itself.

RESULTS

In our previous work, we reported construction of several chimeric chain length regulators based on two parental WzzBs, WzzB SF and WzzB ST , which share 70% amino acid identity and yet lead to distinctly different O-antigen modal length distributions ( 22 ). The chimeric WzzBs confer the O-antigen modal lengths that are in between those of the two parental WzzBs. We have previously reported the structure of the WzzB ST ( 18 ). In an effort to understand the structural basis for the observed phenotypic outcomes, we first determined the crystal structure of the periplasmic domain of WzzB SF (amino acids 52–293) and subsequently of two chimeras, which we refer to as SF4, WzzB SF (1–200)-WzzB ST (201:327), and SF5, WzzB SF (1–255)-WzzB ST (256:327). Both chimeras were composed of larger N-terminal portions of WzzB SF and shorter C-terminal regions of WzzB ST ( Fig. 1 A ). The two chimeric proteins share 96% sequence identity ( Fig. 1 B ) and are over 80% identical to the WzzB SF and WzzB ST parents ( Fig. 1 B ). Remarkably, these minor differences in the amino acid sequence are sufficient to cause pronounced differences in modal lengths of the resulting O-antigen chains; WzzB SF leads to a modal length of ∼13 repeating units, WzzB ST leads to ∼20 repeating units, SF4 leads to ∼17 repeating units, and SF5 leads to ∼15 repeating units ( Fig. 1 C ).

DISCUSSION

What is the oligomeric state of WzzBs in the periplasm? In the previous crystallographic studies ( 18 ), the periplasmic domains of the three different PCPs exhibited different oligomerization states, with Wzz FepE being a nonamer, WzzE being an octamer, and WzzB ST being a pentamer. Although the protomer-protomer packing in Wzz FepE and WzzE was similar and the contacts extended along the entire length of the protomers, those of WzzB ST interacted only through their base domains. The observations by crystallography of a variety of oligomeric sizes as well as circular and open oligomers, by electron microscopy of circular oligomers interpreted as hexamers ( 19 ), and by cross-linking studies of higher molecular weight oligomers for the Wzz from S. flexneri ( 14 , 35 ) raise the question of what the in vivo oligomerization state of the polysaccharide co-polymerases really is. In solution, the purified soluble periplasmic domains of PCPs form smaller oligomers, likely dimers as judged by size exclusion chromatography (Ref. 18 and present work, data not shown). Moreover, while pursuing these studies, we have obtained other crystal forms of these and other chimeric WzzB proteins. Although the poor quality of these crystals did not allow for the high resolution structure determination, the molecular replacement solutions indicated a different oligomeric assembly from that seen for either WzzB chimera SF4 or WzzB chimera SF5. The periplasmic domain of ST1 chimeric protein (WzzB ST (1–95)-WzzB SF (96–325) was found to be a trimer, whereas that of ST5 chimeric protein (WzzB ST (1–255)-WzzB SF (256–325) was found to be a pentamer ( Table 2 ). Although these soluble domains exhibit a propensity to assemble into the oligomers of various compositions, comparing crystallization conditions of the crystals ( Table 2 ), we conclude that the number of subunits and the assembly into the open or the closed forms is not strictly dependent on the given crystallization condition. Both high salt and high polyethylene glycol conditions appear to be conducive to the formation of closed and open oligomers ( Table 2 ).

Because we have observed the circular octameric oligomers of WzzB chimeras in the crystals, we have reanalyzed the electron micrographs of Larue et al. ( 19 ) of the two-dimensional crystals of various detergent-solubilized O-antigen chain length regulators. Their estimate of the largest diameter of WzzB from the electron microscopy reconstructions was ∼100 Å. This was smaller than the dimensions of the Wzz FepE nonamer, slightly smaller than the WzzE octamer, and larger than the WzzB ST pentamer. The authors derived a model of a hexamer based on the crystal structure of WzzE that measured 100 Å at the base, 95 Å in length, and 55 Å at the top and concluded that this model had the size compatible with the EM reconstruction. Here, we show that the WzzB chimeras SF4 and SF5 form octamers in the crystal with dimensions of 95 Å across the base, 100 Å in length, and 50 Å across the top of the octameric ring. These octamers are therefore in a very good agreement with the EM data. Moreover, removing two protomers from this octamer would result in a smaller oligomer size than constructed by Larue et al. ( 19 ), too small to fit the EM data. To further investigate this, we have analyzed the curvature of the open trimers of WzzB SF . Superposition of this trimer on the SF4 octamer shows that it displays higher curvature (smaller radius) ( Fig. 3 C ). We have computationally extended the trimer following its curvature and found that this accommodates a hexamer. However, the dimensions of this artificially constructed hexamer would be ∼70 Å, which is inconsistent with the EM studies of the full-length proteins ( 19 ) and with the number of protomers in a circular oligomers observed in the crystal structures. Clearly, a crystal structure of the full-length WzzB is needed to clarify the controversy surrounding the real oligomerization state of these molecules and to determine whether the size of the oligomer is indeed playing a role in the determination of the length of the carbohydrate polymer. However, the fact that the size of the octamers is in agreement with that derived for the full-length proteins resuspended in proteoliposomes supports the view that octamers represent the physiologically relevant assembly of WzzB. Interestingly, the cytosolic kinase domain from a PCP-IIa family member Wzc was also found to assemble as octamers ( 36 ). Wza, an outer membrane lipoprotein, proposed to form a trans-envelope complex with Wzc, also adopts the octameric organization in the crystals ( 37 ).

The EM images of full-length Wzz FepE reconstituted in lipid bilayers indicated particles of ∼100 Å diameter at the base ( 19 ), as compared with 115 Å of the nonamer in the crystal, which the authors suggested would be more consistent with a hexamer. Here, we report the crystal structure of a Wzz FepE mutant crystallized under significantly different crystallization conditions than the wild type and in a different space group (C2 versus P6 3 22 or P3 2 21) and yet forming a nonamer with the same overall dimensions as that of the wild-type Wzz FepE oligomer. Although we cannot exclude the possibility that the presence of the transmembrane regions might affect the number of protomers within the oligomer through their interaction with the surrounding lipid bilayers, we surmise that the same oligomeric arrangement with nine protomers is also likely for the full-length protein. We have remeasured the distances between the particles in the negatively stained EM micrographs ( Fig. 4 F in Ref. 19 ) using the specified calibrated pixel size of 3.84 Å and obtained the distance between the centers of the neighboring subunits of 115 Å for a full-length Wzz FepE , which is very similar to the size of the nonamer observed in the crystals.

Another interesting finding that emerged from the structural analysis of Wzz FepE (Δ258:266::GSG) mutant is that it exhibits the same main-chain conformation and oligomeric state, yet leads to a drastically different modal length distribution. We previously considered that the oligomer size might be a significant factor in the OAg modal length determination mechanism, with the larger oligomers producing polymers containing more repeating O-units ( 12 , 18 ). Kintz and Goldberg ( 41 ) recently reported that a residue involved in maintaining the oligomer stability of Wzz FepE homologue from Pseudomonas aeruginosa (Wzz2) is partially responsible for the specification of a given O-antigen chain length. The structure of our Wzz FepE mutant, which leads to much shorter O-antigen chains and yet forms the same oligomer as the wild-type Wzz FepE , strongly suggests that the oligomeric size alone is not a sole factor of the modal length determination. Instead, our finding reiterates the importance of the flexible region at the top of the molecule in the mechanism of the OAg chain length control. This is interesting in light of the reported observation that OAg modality can be reproduced in vitro with only Wzy polymerase and WzzB chain regulator present ( 38 ). Loop L4 region is positioned too far from the inner membrane to form physical protein-protein interactions with the Wzy polymerase, and therefore, L4 is likely to serve as one of the sugar-binding faces interacting with the polar O-antigen subunits. Indeed, most of the differences between SF4 and SF5 chimeric proteins, which produce the O-antigens with distinctly different modal lengths, also fall in this region ( Fig. 4 A ). As subtle as the differences are, they would be sufficient to confer differential affinities to the O-antigen-PCP interaction. Recently, it was shown that the Wzy polymerase-O-antigen interactions in Pseudomonas are very sensitive to even conservative amino acid changes in specific positions as even Arg-to-Lys mutation led to a complete loss of the polymerase function, which was attributed to the loss of sugar binding ( 39 ). Clearly, the top of the molecule is not the only site of O-antigen-PCP interaction. In our previous study, we established the importance of the 269–274 segment. This region is the same in SF4 and SF5 and contains only one negatively charged residue (Asp-273); however, in WzzB SF , it contains three negatively charged residues (Asp-270, Asp-271, and Asp-274). This patch would thus be expected to form a different network of interactions with the incoming repeat units, perhaps leading to a weaker affinity between the O-antigen and the chain length regulator.

In summary, our work provides another structural insight into the PCP-1 machinery involved in the secretion of high molecular weight sugar polysaccharides at the atomic resolution. It demonstrates previously unseen oligomerization states for the WzzB periplasmic domains, of which the octameric assembly appears to be more physiologically relevant than the previously reported pentameric arrangement, and is in agreement with the EM data reported for the full-length proteins. The structures of the chimeric domains show for the first time the structural details of the top region of the oligomer, which is involved in PCP function. Our structural analysis of the Wzz FepE mutant also brings forth two important points. First, it shows that the mutation of the L4 region does not exert its phenotypic effect through the structural effects on the individual protomers. Second, the finding that the periplasmic domains of both wild-type and mutant FepE were found to be nonamers despite the wide differences of the crystallization conditions provides the evidence that this arrangement may indeed represent the preferred physiological state of the Wzz FepE O-antigen chain length regulator.