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1.  Structure of WbdD: a bifunctional kinase and methyltransferase that regulates the chain length of the O antigen in Escherichia coli O9a 
Molecular Microbiology  2012;86(3):730-742.
Summary
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.
doi:10.1111/mmi.12014
PMCID: PMC3482155  PMID: 22970759
2.  Structure-function relationships of the outer membrane translocon Wza investigated by cryo-electron microscopy and mutagenesis 
Journal of structural biology  2009;166(2):172-182.
Summary
The outer membrane protein, Wza from E. coli K30, forms an octameric complex that is essential for capsular polysaccharide export. Homologs of Wza are widespread in gram-negative bacterial pathogens where capsules are critical virulence determinants. Wza is unusual in that it spans the outer membrane using a barrel composed of amphipathic α-helices, rather than being a β-barrel like almost all other outer membrane channels. The transmembrane helical barrel of Wza also forms the external opening to a hydrophilic translocation pathway that spans the periplasm. Here, we have probed the structure and function of the Wza complex using both cryo-electron microscopy and mutagenesis. The helical barrel structure is stable in detergent micelles under mildly acidic conditions but is destabilised at basic pH, although the overall quaternary structure is retained. Truncation of the C-terminal region that forms the helical barrel by 4 residues has no effect on the ability of Wza to oligomerize and support capsule export, but larger truncations of 18, 24 or 35 amino acids abolish its function. The bulk of the C-terminal domain is essential for the stability and assembly of the E. coli Wza complex.
doi:10.1016/j.jsb.2009.02.005
PMCID: PMC3498625  PMID: 19236919
Outer membrane protein; capsular polysaccharide; transport; Wza; cryo-electron microscopy
3.  Structure of WbdD: a bifunctional kinase and methyltransferase that regulates the chain length of the O antigen in Escherichia coli O9a 
Molecular microbiology  2012;86(3):730-742.
Summary
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.
doi:10.1111/mmi.12014
PMCID: PMC3482155  PMID: 22970759
4.  Crystallization, dehydration and experimental phasing of WbdD, a bifunctional kinase and methyltransferase from Escherichia coli O9a 
The optimization of WbdD crystals using a novel dehydration protocol and experimental phasing at 3.5 Å resolution by cross-crystal averaging followed by molecular replacement of electron density into a non-isomorphous 3.0 Å resolution native data set are reported.
WbdD is a bifunctional kinase/methyltransferase that is responsible for regulation of lipopolysaccharide O antigen polysaccharide chain length in Escherichia coli serotype O9a. Solving the crystal structure of this protein proved to be a challenge because the available crystals belonging to space group I23 only diffracted to low resolution (>95% of the crystals diffracted to resolution lower than 4 Å and most only to 8 Å) and were non-isomorphous, with changes in unit-cell dimensions of greater than 10%. Data from a serendipitously found single native crystal that diffracted to 3.0 Å resolution were non-isomorphous with a lower (3.5 Å) resolution selenomethionine data set. Here, a strategy for improving poor (3.5 Å resolution) initial phases by density modification and cross-crystal averaging with an additional 4.2 Å resolution data set to build a crude model of WbdD is desribed. Using this crude model as a mask to cut out the 3.5 Å resolution electron density yielded a successful molecular-replacement solution of the 3.0 Å resolution data set. The resulting map was used to build a complete model of WbdD. The hydration status of individual crystals appears to underpin the variable diffraction quality of WbdD crystals. After the initial structure had been solved, methods to control the hydration status of WbdD were developed and it was thus possible to routinely obtain high-resolution diffraction (to better than 2.5 Å resolution). This novel and facile crystal-dehydration protocol may be useful for similar challenging situations.
doi:10.1107/S0907444912029599
PMCID: PMC3447403  PMID: 22993091
WbdD; crystal dehydration
5.  The structure of Wza, the translocon for group 1 capsular polysaccharides in Escherichia coli, identifies a new class of outer membrane protein 
Nature  2006;444(7116):226-229.
Pathogenic bacteria frequently cloak themselves with a capsular polysaccharide layer. Escherichia coli group 1 capsules are formed from repeat-unit polysaccharides with molecular weights exceeding 100 kDa. The export of such a large polar molecule across the hydrophobic outer membrane in Gram-negative bacteria presents a formidable challenge, given that the permeability barrier of the membrane must be maintained. We describe the 2.26 Å structure of Wza, an integral outer membrane protein, that is essential for capsule export. Wza is an octamer, with a composite molecular weight of 340 kDa, and it forms an “amphora”-like structure. The protein has a large central cavity 100 Å long and 30 Å wide. The transmembrane region is a novel α-helical barrel, and is linked to three additional novel periplasmic domains, marking Wza as the representative of a new class of membrane protein. Although Wza is open to the extracellular environment, a flexible loop in the periplasmic region occludes the cavity and may regulate the opening of the channel. The structure defines the route taken by the capsular polymer as it exits the cell, using the structural data we propose a mechanism for the translocation of the large polar capsular polysaccharide.
doi:10.1038/nature05267
PMCID: PMC3315050  PMID: 17086202
6.  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 Journal of biological chemistry  2005;281(4):2144-2150.
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.
doi:10.1074/jbc.M508078200
PMCID: PMC3315051  PMID: 16172129
7.  Cloning, Expression, and Purification of the K5 Capsular Polysaccharide Lyase (KflA) from Coliphage K5A: Evidence for Two Distinct K5 Lyase Enzymes 
Journal of Bacteriology  2000;182(13):3761-3766.
The Escherichia coli K5 capsular polysaccharide [-4)-βGlcA-(1,4)-αGlcNAc-(1-] is a receptor for the capsule-specific bacteriophage K5A. Associated with the structure of bacteriophage K5A is a polysaccharide lyase which degrades the K5 capsule to expose the underlying bacterial cell surface. The bacteriophage K5A lyase gene (kflA) was cloned and sequenced. The kflA gene encodes a polypeptide with a predicted molecular mass of 66.9 kDa and which exhibits amino acid homology with ElmA, a K5 polysaccharide lyase encoded on the chromosome of E. coli SEBR 3282. There was only limited nucleotide homology between the kflA and elmA genes, suggesting that these two genes are distinct and either have been derived from separate progenitors or have diverged from a common progenitor for a considerable length of time. Southern blot analysis revealed that kflA was not present on the chromosome of the E. coli strains examined. In contrast, elmA was present in a subset of E. coli strains. Homology was observed between DNA flanking the kflA gene of bacteriophage K5A and DNA flanking a small open reading frame (ORFL) located 5′ of the endosialidase gene of the E. coli K1 capsule-specific bacteriophage K1E. The DNA homology between these noncoding sequences indicated that bacteriophages K5A and K1E were related. The deduced polypeptide sequence of ORFL in bacteriophage K1E exhibited homology to the N terminus of KflA from bacteriophage K5A, suggesting that ORFL is a truncated remnant of KflA. The presence of this truncated kflA gene implies that bacteriophage K1E has evolved from bacteriophage K5A by acquisition of the endosialidase gene and subsequent loss of functional kflA. A (His)6-KflA fusion protein was overexpressed in E. coli and purified to homogeneity with a yield of 4.8 mg per liter of bacterial culture. The recombinant enzyme was active over a broad pH range and NaCl concentration and was capable of degrading K5 polysaccharide into a low-molecular-weight product.
PMCID: PMC94548  PMID: 10850992
8.  Genetic Organization of the Escherichia coli K10 Capsule Gene Cluster: Identification and Characterization of Two Conserved Regions in Group III Capsule Gene Clusters Encoding Polysaccharide Transport Functions 
Journal of Bacteriology  1999;181(7):2279-2285.
Analysis of the Escherichia coli K10 capsule gene cluster identified two regions, regions 1 and 3, conserved between different group III capsule gene clusters. Region 1 encodes homologues of KpsD, KpsM, KpsT, and KpsE proteins, and region 3 encodes homologues of the KpsC and KpsS proteins. An rfaH mutation abolished K10 capsule production, suggesting that expression of the K10 capsule was regulated by RfaH in a manner analogous to group II capsule gene clusters. An IS3 element and a φR73-like prophage, both of which may have played a role in the acquisition of group III capsule gene clusters, were detected flanking the K10 capsule genes.
PMCID: PMC93645  PMID: 10094710

Results 1-8 (8)