Bradley R. Clarke

Wza the translocon for E. coli capsular polysaccharides defines a new class of membrane protein

Many types of bacteria produce extracellular polysaccharides (EPSs). Some are secreted polymers and show only limited association with the cell surface, whereas others are firmly attached to the cell surface and form a discrete structural layer, the capsule, which envelopes the cell and allows the bacteria to evade or counteract the host immune system. EPSs have critical roles in bacterial colonization of surfaces, such as epithelia and medical implants; in addition some EPSs have important industrial and biomedical applications in their own right. Here we describe the 2.26 Å resolution structure of the 340 kDa octamer of Wza, an integral outer membrane lipoprotein, which is essential for group 1 capsule export in Escherichia coli. The transmembrane region is a novel α-helical barrel. The bulk of the Wza structure is located in the periplasm and comprises three novel domains forming a large central cavity. Wza is open to the extracellular environment but closed to the periplasm. We propose a route and mechanism for translocation of the capsular polysaccharide. This work may provide insight into the export of other large polar molecules such as DNA and proteins.

The 3D structure of a periplasm-spanning platform required for assembly of group 1 capsular polysaccharides in Escherichia coli

Capsular polysaccharides (CPSs) are essential virulence determinants of many pathogenic bacteria. Escherichia coli group 1 CPSs provide paradigms for widespread surface polysaccharide assembly systems in Gram-negative bacteria. In these systems, complex carbohydrate polymers must be exported across the periplasm and outer membrane to the cell surface. Group 1 CPS export requires oligomers of the outer membrane protein, Wza, for translocation across the outer membrane. Assembly also depends on Wzc, an inner membrane tyrosine autokinase known to regulate export and synthesis of group 1 CPS. Here, we provide a structural view of a complex comprising Wzc and Wza that spans the periplasm, connecting the inner and outer membranes. Examination of transmembrane sections of the complex suggests that the periplasm is compressed at the site of complex formation. An important feature of CPS production is the coupling of steps involved in biosynthesis and export. We propose that the Wza–Wzc complex provides the structural and regulatory core of a larger macromolecular machine. We suggest a mechanism by which CPS may move from the periplasm through the outer membrane.

Identification of an ATP-binding cassette transport system required for translocation of lipopolysaccharide O-antigen side-chains across the cytoplasmic membrane of Klebsiella pneumoniae serotype O1

The rfbkpO1 gene cluster of Klebsiella pneumoniae O1 directs synthesis of the D‐galactan I component of the lipopolysaccharide O‐antigen. The first two genes in the rfbkpO1cluster encode RrfbkpO1and RfbBKpO1, with predicted sizes of 29.5 or 30.0 kDa and 27.4 kDa, respectively. RfbBKpO1 contains a consensus ATP‐binding domain and shares homology with several proteins which function as ATP‐binding components of cell surface polysaccharide transporters. RfbAKpO1 is predicted to be an integral membrane protein with five putative membrane‐spanning domains and its transmembrane topology was confirmed by TnphoA mutagenesis. The hydropathy plot of RfbAKpO1 resembles KpsM, the transcytoplasmic membrane component of the capsular polysaccharide transporter from Escherichia coli K‐1 and K‐5. These relationships suggest that RfbAKpO1 and RfbBKpO1 belong to a family of two‐component ABC (ATP‐binding cassette) transporters. E. coli K‐12 containing a plasmid carrying an rfbKpO1 gene cluster deleted in rfbAKpO1 and rfbBKpO1 expresses rough lipopolysaccharide molecules on its surface and accumulates cytoplasmic O‐antigen. When RfbAKpO1 and RfbBKpO1 are supplied in trans by a compatible plasmid, O‐polysaccharide transport is restored and smooth D‐galactan l‐substituted lipopolysaccharide is produced. RfbAKpO1 and RfbBKpO1 are, therefore, proposed to constitute a system required for transport of D‐galactan I across the cytoplasmic membrane, where RfbAKpO1 represents the membrane‐spanning translocator and RfbBKpO1 couples the energy of ATP hydrolysis to the transport process.

Nonreducing Terminal Modifications Determine the Chain Length of Polymannose O Antigens of Escherichia coli and Couple Chain Termination to Polymer Export via an ATP-binding Cassette Transporter

The chain length of bacterial lipopolysaccharide O antigens is regulated to give a modal distribution that is critical for pathogenesis. This paper describes the process of chain length determination in the ATP-binding cassette (ABC) transporter-dependent pathway, a pathway that is widespread among Gram-negative bacteria. Escherichia coli O8 and O9/O9a polymannans are synthesized in the cytoplasm, and an ABC transporter exports the nascent polymer across the inner membrane prior to completion of the LPS molecule. The polymannan O antigens have nonreducing terminal methyl groups. The 3-O-methyl group in serotype O8 is transferred from S-adenosylmethionine by the WbdDO8 enzyme, and this modification terminates polymerization. Methyl groups are added to the O9a polymannan in a reaction dependent on preceding phosphorylation. The bifunctional WbdDO9a catalyzes both reactions, but only the kinase activity controls chain length. Chain termination occurs in a mutant lacking the ABC transporter, indicating that it precedes export. An E. coli wbdDO9a mutant accumulated O9a polymannan in the cytoplasm, indicating that WbdD activity coordinates polymannan chain termination with export across the inner membrane.

Periplasmic Protein-Protein Contacts in the Inner Membrane Protein Wzc Form a Tetrameric Complex Required for the Assembly of Escherichia coli Group 1 Capsules

The K antigenic capsular polysaccharide forms a structural layer, the capsule, on the surfaces of Escherichia coli cells. The capsule provides an important protective covering that helps protect encapsulated bacteria from host immune defenses. The assembly and translocation of the capsule requires proteins in the inner and outer membranes. The inner membrane protein Wzc is a tyrosine autokinase that plays an essential role in what is believed to be a coordinated biosynthesis and secretion process. Mutants lacking Wzc can form K antigen oligosaccharides but are unable to polymerize high molecular weight capsular polymers. Wzc homologs have been identified in exopolymer biosynthesis systems in many different Gram-negative and -positive bacteria. Using single particle averaging on cryo-negatively stained samples, we have produced the first three-dimensional structure of this type of membrane protein in its phosphorylated state at ∼14 Å resolution. Perfluoro-octanoate-PAGE analysis of detergent-solubilized oligomeric Wzc and symmetry analysis of the transmission electron microscopy data clearly demonstrated that Wzc forms a tetrameric complex with C4 rotational symmetry. Viewed from the top of the complex, the oligomer is square with a diameter of ∼100 Å and can be divided into four separate densities. From the side, Wzc is ∼110 Å high and has a distinctive appearance similar to an extracted molar tooth. The upper “crown” region is ∼55 Å high and forms a continuous ring of density. Four unconnected “roots” (∼65 Å high) emerge from the underside of the crown. We propose that the crown is formed by protein-protein contacts from the four Wzc periplasmic domains, while each root represents an individual cytoplasmic tyrosine autokinase domain.

Coordination of Polymerization, Chain Termination, and Export in Assembly of the Escherichia coli Lipopolysaccharide O9a Antigen in an ATP-binding Cassette Transporter-dependent Pathway

The Escherichia coli O9a O-polysaccharide (O-PS) is a prototype for O-PS synthesis and export by the ATP-binding cassette transporter-dependent pathway. Comparable systems are widespread in Gram-negative bacteria. The polymannose O9a O-PS is assembled on a polyisoprenoid lipid intermediate by mannosyltransferases located at the cytoplasmic membrane, and the final polysaccharide chain length is determined by the chain terminating dual kinase/methyltransferase, WbdD. The WbdD protein is tethered to the membrane via a C-terminal region containing amphipathic helices located between residues 601 and 669. Here, we establish that the C-terminal domain of WbdD plays an additional pivotal role in assembly of the O-PS by forming a complex with the chain-extending mannosyltransferase, WbdA. Membrane preparations from a ΔwbdD mutant had severely diminished mannosyltransferase activity in vitro, and no significant amounts of the WbdA protein are targeted to the membrane fraction. Expression of a polypeptide comprising the WbdD C-terminal region was sufficient to restore both proper localization of WbdA and mannosyltransferase activity. In contrast to WbdA, the other required mannosyltransferases (WbdBC) are targeted to the membrane independent of WbdD. A bacterial two-hybrid system confirmed the interaction of WbdD and WbdA and identified two regions in the C terminus of WbdD that contributed to the interaction. Therefore, in the O9a assembly export system, the WbdD protein orchestrates the critical localization and coordination of activities involved in O-PS chain extension and termination at the cytoplasmic membrane.

A coiled-coil domain acts as a molecular ruler to regulate O-antigen chain length in lipopolysaccharide.

Long-chain bacterial polysaccharides play important roles in pathogenicity. In Escherichia coli O9a, a model for ABC transporter dependent polysaccharide assembly, a large extracellular carbohydrate with a narrow distribution of size is polymerized from monosaccharides by a complex of two proteins, WbdA (polymerase) and WbdD (terminating protein). Such careful control of polymerization is recurring theme in biology. Combining crystallography and small angle X-ray scattering, we show that the C-terminal domain of WbdD contains an extended coiled-coil that physically separates WbdA from the catalytic domain of WbdD. The effects of insertions and deletions within the coiled-coil region were analyzed in vivo, revealing that polymer size is controlled by varying the length of the coiled-coil domain. Thus, the coiled-coil domain of WbdD functions as a molecular ruler that, along with WbdA:WbdD stoichiometry, controls the chain length of a model bacterial polysaccharide.Long-chain bacterial polysaccharides have important roles in pathogenicity. In Escherichia coli O9a, a model for ABC transporter–dependent polysaccharide assembly, a large extracellular carbohydrate with a narrow size distribution is polymerized from monosaccharides by a complex of two proteins, WbdA (polymerase) and WbdD (terminating protein). Combining crystallography and small-angle X-ray scattering, we found that the C-terminal domain of WbdD contains an extended coiled-coil that physically separates WbdA from the catalytic domain of WbdD. The effects of insertions and deletions in the coiled-coil region were analyzed in vivo, revealing that polymer size is controlled by varying the length of the coiled-coil domain. Thus, the coiled-coil domain of WbdD functions as a molecular ruler that, along with WbdA:WbdD stoichiometry, controls the chain length of a model bacterial polysaccharide.

In Vitro Reconstruction of the Chain Termination Reaction in Biosynthesis of the Escherichia coli O9a O-Polysaccharide THE CHAIN-LENGTH REGULATOR, WbdD, CATALYZES THE ADDITION OF METHYL PHOSPHATE TO THE NON-REDUCING TERMINUS OF THE GROWING GLYCAN

Background: WbdD is a chain-length regulator that modifies the non-reducing terminus of the Escherichia coli O9a glycan. Results: WbdD phosphorylates and methylates a synthetic O9a repeating unit acceptor. Conclusion: The acceptor is modified with a terminal methyl phosphate. Significance: Determining the terminal structure is crucial to understanding the quality control processes of chain-length regulation and export of this prototypical glycan. The Escherichia coli O9a O-polysaccharide (O-PS) represents a model system for glycan biosynthesis and export by the ATP-binding cassette (ABC) transporter-dependent pathway. The polymannose O9a O-PS is synthesized using an undecaprenol-diphosphate-linked acceptor by mannosyltransferases located at the cytoplasmic membrane. An ABC-transporter subsequently exports the polymer to the periplasm where it is assembled onto lipopolysaccharide prior to translocation to the cell surface. The chain length of the O9a O-PS is regulated by the dual kinase/methyltransferase activity of the WbdD enzyme and modification of the polymer is crucial for binding and export by the ABC-transporter. Previous biochemical data provided evidence for phosphorylation/methylation at the non-reducing end of the O9a O-PS but the structure of the terminus has not been determined. Here, we describe the exploitation of a synthetic O9a O-PS repeating unit carrying a fluorescent tag as an acceptor for in vitro phosphorylation and methylation by a purified soluble form of WbdD. Phosphorylation of the acceptor was evident by both a mobility shift in thin layer chromatography and radiolabeling of the acceptor using [γ-33P]ATP. Methylation of the acceptor was dependent on phosphorylation and was demonstrated by radiolabeling using S-[methyl-3H]adenosyl-methionine as a substrate, in the presence of ATP. NMR spectroscopic and mass spectrometric methods were used to determine the precise structure of the terminal modification, leading to the conclusion that WbdD catalyzes the addition of a novel methyl phosphate group to the 3-position of the non-reducing terminal mannose of the O9a O-PS repeating unit.

Lipopolysaccharide O antigen size distribution is determined by a chain extension complex of variable stoichiometry in Escherichia coli O9a

Significance Lipopolysaccharides (LPSs) are unique glycolipids that are characteristic components of outer membranes in Gram negative bacteria and play important roles in pathogenesis. Many LPSs contain a long-chain polysaccharide (known as the O antigen) whose length can be an important factor in bacterial resistance to complement-mediated killing. While components involved in chain-length determination are known in many systems, the underlying regulatory mechanism is not. Here we apply a mathematical modeling approach that integrates the existing structural and biochemical data to develop a new model, variable geometry, for chain-length regulation using the prototype for O antigens whose synthesis involves the widespread ATP-binding cassette transporter-dependent pathway. The lengths of bacterial polysaccharides can be critical for their biological function. Unlike DNA or protein synthesis, where polymer length is implicit in the nucleic acid template, the molecular mechanisms for regulating polysaccharide length are poorly understood. Two models are commonly cited: a “molecular clock” regulates length by controlling the duration of the polymer extension process, whereas a “molecular ruler” determines length by measurement against a physical structure in the biosynthetic complex. Escherichia coli O9a is a prototype for the biosynthesis of O polysaccharides by ATP-binding cassette transporter-dependent processes. The length of the O9a polysaccharide is determined by two proteins: an extension enzyme, WbdA, and a termination enzyme, WbdD. WbdD is known to self-oligomerize and also to interact with WbdA. Changing either enzyme’s concentration can alter the polysaccharide length. We quantified the O9a polysaccharide length distribution and the enzyme concentration dependence in vivo, then made mathematical models to predict the polymer length distributions resulting from hypothetical length-regulation mechanisms. Our data show qualitative features that cannot be explained by either a molecular clock or a molecular ruler model. Therefore, we propose a “variable geometry” model, in which a postulated biosynthetic WbdA–WbdD complex assembles with variable stoichiometry dependent on relative enzyme concentration. Each stoichiometry produces polymers with a distinct, geometrically determined, modal length. This model reproduces the enzyme concentration dependence and modality of the observed polysaccharide length distributions. Our work highlights limitations of previous models and provides new insight into the mechanisms of length control in polysaccharide biosynthesis.

Structure of WbdD : a bifunctional kinase and methyltransferase that regulates the chain length of the O antigen in Escherichia coli O9a

The Escherichia coli serotype O9a O‐antigen polysaccharide (O‐PS) is a model for glycan biosynthesis and export by the ATP‐binding cassette transporter‐dependent pathway. The polymannose O9a O‐PS is synthesized as a polyprenol‐linked glycan by mannosyltransferase enzymes located at the cytoplasmic membrane. The chain length of the O9a O‐PS is tightly regulated by the WbdD enzyme. WbdD first phosphorylates the terminal non‐reducing mannose of the O‐PS and then methylates the phosphate, stopping polymerization. The 2.2 Å resolution structure of WbdD reveals a bacterial methyltransferase domain joined to a eukaryotic kinase domain. The kinase domain is again fused to an extended C‐terminal coiled‐coil domain reminiscent of eukaryotic DMPK (Myotonic Dystrophy Protein Kinase) family kinases such as Rho‐associated protein kinase (ROCK). WbdD phosphorylates 2‐α‐d‐mannosyl‐d‐mannose (2α‐MB), a short mimic of the O9a polymer. Mutagenesis identifies those residues important in catalysis and substrate recognition and the in vivo phenotypes of these mutants are used to dissect the termination reaction. We have determined the structures of co‐complexes of WbdD with two known eukaryotic protein kinase inhibitors. Although these are potent inhibitors in vitro, they do not show any in vivo activity. The structures reveal new insight into O‐PS chain‐length regulation in this important model system.