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1.
Nat Commun ; 12(1): 4349, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34272394

RESUMO

Bacterial extracellular polysaccharides (EPSs) play critical roles in virulence. Many bacteria assemble EPSs via a multi-protein "Wzx-Wzy" system, involving glycan polymerization at the outer face of the cytoplasmic/inner membrane. Gram-negative species couple polymerization with translocation across the periplasm and outer membrane and the master regulator of the system is the tyrosine autokinase, Wzc. This near atomic cryo-EM structure of dephosphorylated Wzc from E. coli shows an octameric assembly with a large central cavity formed by transmembrane helices. The tyrosine autokinase domain forms the cytoplasm region, while the periplasmic region contains small folded motifs and helical bundles. The helical bundles are essential for function, most likely through interaction with the outer membrane translocon, Wza. Autophosphorylation of the tyrosine-rich C-terminus of Wzc results in disassembly of the octamer into multiply phosphorylated monomers. We propose that the cycling between phosphorylated monomer and dephosphorylated octamer regulates glycan polymerization and translocation.


Assuntos
Cápsulas Bacterianas/química , Cápsulas Bacterianas/metabolismo , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Membrana/química , Periplasma/metabolismo , Polissacarídeos Bacterianos/metabolismo , Proteínas Tirosina Quinases/química , Motivos de Aminoácidos , Domínio Catalítico , Microscopia Crioeletrônica , Citoplasma/metabolismo , Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Espectrometria de Massas , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Periplasma/química , Fosforilação , Conformação Proteica em alfa-Hélice , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Tirosina/química , Tirosina/metabolismo
2.
Nat Chem Biol ; 16(4): 450-457, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32152541

RESUMO

Lipopolysaccharide O-antigen is an attractive candidate for immunotherapeutic strategies targeting antibiotic-resistant Klebsiella pneumoniae. Several K. pneumoniae O-serotypes are based on a shared O2a-antigen backbone repeating unit: (→ 3)-α-Galp-(1 → 3)-ß-Galf-(1 →). O2a antigen is synthesized on undecaprenol diphosphate in a pathway involving the O2a polymerase, WbbM, before its export by an ATP-binding cassette transporter. This dual domain polymerase possesses a C-terminal galactopyranosyltransferase resembling known GT8 family enzymes, and an N-terminal DUF4422 domain identified here as a galactofuranosyltransferase defining a previously unrecognized family (GT111). Functional assignment of DUF4422 explains how galactofuranose is incorporated into various polysaccharides of importance in vaccine production and the food industry. In the 2.1-Å resolution structure, three WbbM protomers associate to form a flattened triangular prism connected to a central stalk that orients the active sites toward the membrane. The biochemical, structural and topological properties of WbbM offer broader insight into the mechanisms of assembly of bacterial cell-surface glycans.


Assuntos
Glicosiltransferases/metabolismo , Antígenos O/metabolismo , Antígenos O/ultraestrutura , Transportadores de Cassetes de Ligação de ATP/metabolismo , Sequência de Aminoácidos , Membrana Celular/metabolismo , Glicosiltransferases/fisiologia , Hexosiltransferases , Klebsiella pneumoniae/metabolismo , Lipopolissacarídeos/química , Polissacarídeos Bacterianos/química
3.
J Bacteriol ; 202(5)2020 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-31792013

RESUMO

Bacterial lipopolysaccharides are major components and contributors to the integrity of Gram-negative outer membranes. The more conserved lipid A-core part of this complex glycolipid is synthesized separately from the hypervariable O-antigenic polysaccharide (OPS) part, and they are joined in the periplasm prior to translocation to the outer membrane. Three different biosynthesis strategies are recognized for OPS biosynthesis, and one, the synthase-dependent pathway, is currently confined to a single example: the O:54 antigen from Salmonella enterica serovar Borreze. Synthases are complex enzymes that have the capacity to both polymerize and export bacterial polysaccharides. Although synthases like cellulose synthase are widespread, they typically polymerize a glycan without employing a lipid-linked intermediate, unlike the O:54 synthase (WbbF), which produces an undecaprenol diphosphate-linked product. This raises questions about the overall similarity between WbbF and conventional synthases. In this study, we examine the topology of WbbF, revealing four membrane-spanning helices, compared to the eight in cellulose synthase. Molecular modeling of the glycosyltransferase domain of WbbF indicates a similar architecture, and site-directed mutagenesis confirmed that residues important for catalysis and processivity in cellulose synthase are conserved in WbbF and required for its activity. These findings indicate that the glycosyltransferase mechanism of WbbF and classic synthases are likely conserved despite the use of a lipid acceptor for chain extension by WbbF.IMPORTANCE Glycosyltransferases play a critical role in the synthesis of a wide variety of bacterial polysaccharides. These include O-antigenic polysaccharides, which form the distal component of lipopolysaccharides and provide a protective barrier important for survival and host-pathogen interactions. Synthases are a subset of glycosyltransferases capable of coupled synthesis and export of glycans. Currently, the O:54 antigen of Salmonella enterica serovar Borreze involves the only example of an O-polysaccharide synthase, and its generation of a lipid-linked product differentiates it from classical synthases. Here, we explore features conserved in the O:54 enzyme and classical synthases to shed light on the structure and function of the unusual O:54 enzyme.


Assuntos
Domínio Catalítico , Glicosiltransferases/química , Modelos Moleculares , Salmonella enterica/enzimologia , Sequência de Aminoácidos , Catálise , Antígenos O/biossíntese , Proteínas Recombinantes de Fusão , Salmonella enterica/imunologia
4.
J Biol Chem ; 294(41): 14978-14990, 2019 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-31416837

RESUMO

Escherichia coli serotype O9a provides a model for export of lipopolysaccharide (LPS) O-antigen polysaccharide (O-PS) via ABC transporters. In O9a biosynthesis, a chain-terminator enzyme, WbdD, caps the nonreducing end of the glycan with a methylphosphate moiety and thereby establishes chain-length distribution. A carbohydrate-binding module (CBM) in the ABC transporter recognizes terminated glycans, ensuring that only mature O-PS is exported and incorporated into LPS. Here, we addressed two questions arising from this model. Are both residues in the binary terminator necessary for termination and export? And is a terminal methylphosphate moiety sufficient for export of heterologous glycans? To answer the first question, we uncoupled WbdD kinase and methyltransferase activities. WbdD mutants revealed that although the kinase activity is solely responsible for chain-length regulation, both activities are essential for CBM recognition and export. Consistent with this observation, a saturation transfer difference NMR experiment revealed a direct interaction between the CBM and the terminal methyl group. To determine whether methylphosphate is the sole determinant of substrate recognition by the CBM, we exploited Klebsiella pneumoniae O7, whose O-PS repeat-unit structure differs from O9a, but, as shown here, offers the second confirmed example of a terminal methylphosphate serving in substrate recognition. In vitro and in vivo experiments indicated that each CBM can bind the O-PS only with the native repeat unit, revealing that methylphosphate is essential but not sufficient for substrate recognition and export. Our findings provide important new insight into the structural determinants in a prototypical quality control system for glycan assembly and export.


Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Metabolismo dos Carboidratos , Escherichia coli/metabolismo , Antígenos O/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Sequência de Carboidratos , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Antígenos O/química , Organofosfatos/metabolismo , Polimerização , Ligação Proteica , Conformação Proteica
5.
J Biol Chem ; 294(28): 10863-10876, 2019 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-31138653

RESUMO

A limited range of different structures is observed in O-antigenic polysaccharides (OPSs) from Klebsiella pneumoniae lipopolysaccharides. Among these, several are based on modifications of a conserved core element of serotype O2a OPS, which has a disaccharide repeat structure [→3)-α-d-Galp-(1→3)-ß-d-Galf-(1→]. Here, we describe the enzymatic pathways for a highly unusual modification strategy involving the attachment of a second glycan repeat-unit structure to the nonreducing terminus of O2a. This occurs by the addition of the O1 [→3)-α-d-Galp-(1→3)-ß-d-Galp-(1→] or O2c [→3)-ß-d-GlcpNAc-(1→5)-ß-d-Galf-(1→] antigens. The organization of the enzyme activities performing these modifications differs, with the enzyme WbbY possessing two glycosyltransferase catalytic sites solely responsible for O1 antigen polymerization and forming a complex with the O2a glycosyltransferase WbbM. In contrast, O2c polymerization requires glycosyltransferases WbmV and WbmW, which interact with one another but apparently not with WbbM. Using defined synthetic acceptors and site-directed mutants to assign the activities of the WbbY catalytic sites, we found that the C-terminal WbbY domain is a UDP-Galp-dependent GT-A galactosyltransferase adding ß-(1→3)-linked d-Galp, whereas the WbbY N terminus includes a GT-B enzyme adding α-(1→3)-linked d-Galp These activities build the O1 antigen on a terminal Galp in the O2a domain. Using similar approaches, we identified WbmV as the UDP-GlcNAc transferase and noted that WbmW represents a UDP-Galf-dependent enzyme and that both are GT-A members. WbmVW polymerizes the O2c antigen on a terminal Galf. Our results provide mechanistic and conceptual insights into an important strategy for polysaccharide antigen diversification in bacteria.


Assuntos
Diversidade de Anticorpos/imunologia , Klebsiella pneumoniae/enzimologia , Antígenos O/imunologia , Diversidade de Anticorpos/fisiologia , Proteínas de Bactérias/metabolismo , Galactanos/metabolismo , Galactosiltransferases/metabolismo , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/metabolismo , Lipopolissacarídeos/química , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/metabolismo , Família Multigênica/genética , Antígenos O/química , Antígenos O/metabolismo , Sorotipagem/métodos
6.
J Biol Chem ; 293(13): 4666-4679, 2018 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-29602878

RESUMO

Klebsiella pneumoniae is a major health threat. Vaccination and passive immunization are considered as alternative therapeutic strategies for managing Klebsiella infections. Lipopolysaccharide O antigens are attractive candidates because of the relatively small range of known O-antigen polysaccharide structures, but immunotherapeutic applications require a complete understanding of the structures found in clinical settings. Currently, the precise number of Klebsiella O antigens is unknown because available serological tests have limited resolution, and their association with defined chemical structures is sometimes uncertain. Molecular serotyping methods can evaluate clinical prevalence of O serotypes but require a full understanding of the genetic determinants for each O-antigen structure. This is problematic with Klebsiella pneumoniae because genes outside the main rfb (O-antigen biosynthesis) locus can have profound effects on the final structure. Here, we report two new loci encoding enzymes that modify a conserved polysaccharide backbone comprising disaccharide repeat units [→3)-α-d-Galp-(1→3)-ß-d-Galf-(1→] (O2a antigen). We identified in serotype O2aeh a three-component system that modifies completed O2a glycan in the periplasm by adding 1,2-linked α-Galp side-group residues. In serotype O2ac, a polysaccharide comprising disaccharide repeat units [→5)-ß-d-Galf-(1→3)-ß-d-GlcpNAc-(1→] (O2c antigen) is attached to the non-reducing termini of O2a-antigen chains. O2c-polysaccharide synthesis is dependent on a locus encoding three glycosyltransferase enzymes. The authentic O2aeh and O2c antigens were recapitulated in recombinant Escherichia coli hosts to establish the essential gene set for their synthesis. These findings now provide a complete understanding of the molecular genetic basis for the known variations in Klebsiella O-antigen carbohydrate structures based on the O2a backbone.


Assuntos
Proteínas de Bactérias , Klebsiella pneumoniae , Antígenos O , Animais , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Configuração de Carboidratos , Klebsiella pneumoniae/química , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/metabolismo , Antígenos O/biossíntese , Antígenos O/química , Antígenos O/genética , Coelhos
7.
J Biol Chem ; 291(18): 9748-61, 2016 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-26934919

RESUMO

Export of the Escherichia coli serotype O9a O-antigenic polysaccharides (O-PS) involves an ATP-binding cassette (ABC) transporter. The process requires a non-reducing terminal residue, which is recognized by a carbohydrate-binding module (CBM) appended to the C terminus of the nucleotide-binding domain of the transporter. Here, we investigate the process in Klebsiella pneumoniae serotype O12 (and Raoultella terrigena ATCC 33257). The O12 polysaccharide is terminated at the non-reducing end by a ß-linked 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) residue. The O12 ABC transporter also binds its cognate O-PS via a CBM, and export is dependent on the presence of the terminal ß-Kdo residue. The overall structural architecture of the O12 CBM resembles the O9a prototype, but they share only weak sequence similarity, and the putative binding pocket for the O12 glycan is different. Removal of the CBM abrogated O-PS transport, but export was restored when the CBM was expressed in trans with the mutant CBM-deficient ABC transporter. These results demonstrate that the CBM-mediated substrate-recognition mechanism is evolutionarily conserved and can operate with glycans of widely differing structures.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/metabolismo , Klebsiella pneumoniae/metabolismo , Mutação , Antígenos O/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Proteínas de Bactérias/genética , Klebsiella pneumoniae/genética , Antígenos O/genética
8.
Nat Struct Mol Biol ; 22(1): 50-56, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25504321

RESUMO

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.


Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/química , Escherichia coli/enzimologia , Antígenos O/química , Antígenos O/metabolismo , Cristalografia por Raios X , Lipopolissacarídeos/química , Lipopolissacarídeos/metabolismo , Manosiltransferases/química , Manosiltransferases/metabolismo , Conformação Proteica , Espalhamento a Baixo Ângulo
9.
J Biol Chem ; 290(2): 1075-85, 2015 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-25422321

RESUMO

The Escherichia coli O9a O-polysaccharide (O-PS) is a prototype for bacterial glycan synthesis and export by an ATP-binding cassette transporter-dependent pathway. The O9a O-PS possesses a tetrasaccharide repeat unit comprising two α-(1→2)- and two α-(1→3)-linked mannose residues and is extended on a polyisoprenoid lipid carrier by the action of a polymerase (WbdA) containing two glycosyltransferase active sites. The N-terminal domain of WbdA possesses α-(1→2)-mannosyltransferase activity, and we demonstrate in this study that the C-terminal domain is an α-(1→3)-mannosyltransferase. Previous studies established that the size of the O9a polysaccharide is determined by the chain-terminating dual kinase/methyltransferase (WbdD) that is tethered to the membrane and recruits WbdA into an active enzyme complex by protein-protein interactions. Here, we used bacterial two-hybrid analysis to identify a surface-exposed α-helix in the C-terminal mannosyltransferase domain of WbdA as the site of interaction with WbdD. However, the C-terminal domain was unable to interact with WbdD in the absence of its N-terminal partner. Through deletion analysis, we demonstrated that the α-(1→2)-mannosyltransferase activity of the N-terminal domain is regulated by the activity of the C-terminal α-(1→3)-mannosyltransferase. In mutants where the C-terminal catalytic site was deleted but the WbdD-interaction site remained, the N-terminal mannosyltransferase became an unrestricted polymerase, creating a novel polymer comprising only α-(1→2)-linked mannose residues. The WbdD protein therefore orchestrates critical localization and coordination of activities involved in chain extension and termination. Complex domain interactions are needed to position the polymerase components appropriately for assembly into a functional complex located at the cytoplasmic membrane.


Assuntos
Glicosiltransferases/química , Metiltransferases/química , Antígenos O/biossíntese , Polissacarídeos Bacterianos/biossíntese , Escherichia coli/química , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Glicosiltransferases/metabolismo , Manose/química , Manose/metabolismo , Metiltransferases/metabolismo , Complexos Multiproteicos/biossíntese , Complexos Multiproteicos/química , Antígenos O/química , Antígenos O/genética , Polissacarídeos Bacterianos/química , Polissacarídeos Bacterianos/genética , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
10.
Proc Natl Acad Sci U S A ; 111(17): 6407-12, 2014 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-24733938

RESUMO

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.


Assuntos
Escherichia coli/metabolismo , Antígenos O/química , Proteínas de Escherichia coli/metabolismo , Manosiltransferases/metabolismo , Modelos Biológicos , Peso Molecular
11.
Mol Microbiol ; 86(3): 730-42, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22970759

RESUMO

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.


Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Metiltransferases/química , Metiltransferases/metabolismo , Antígenos O/química , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Sequência de Aminoácidos , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Metiltransferases/genética , Modelos Moleculares , Dados de Sequência Molecular , Antígenos O/metabolismo , Polissacarídeos Bacterianos/química , Polissacarídeos Bacterianos/metabolismo , Proteínas Quinases/genética , Estrutura Terciária de Proteína , Alinhamento de Sequência , Especificidade por Substrato
12.
Acta Crystallogr D Biol Crystallogr ; 68(Pt 10): 1371-9, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22993091

RESUMO

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.


Assuntos
Desidratação , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Metiltransferases/química , Complexos Multienzimáticos/química , Fosfotransferases (Aceptor do Grupo Álcool)/química , Cristalização , Proteínas de Escherichia coli/metabolismo , Metiltransferases/metabolismo , Complexos Multienzimáticos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Difração de Raios X
13.
J Biol Chem ; 286(48): 41391-41401, 2011 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-21990359

RESUMO

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-(33)P]ATP. Methylation of the acceptor was dependent on phosphorylation and was demonstrated by radiolabeling using S-[methyl-(3)H]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.


Assuntos
Metabolismo dos Carboidratos/fisiologia , Escherichia coli/metabolismo , Polissacarídeos Bacterianos/biossíntese , Configuração de Carboidratos , Sequência de Carboidratos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Espectroscopia de Ressonância Magnética , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , Organofosfatos/metabolismo , Fosforilação/fisiologia , Polissacarídeos Bacterianos/genética
14.
J Biol Chem ; 284(44): 30662-72, 2009 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-19734145

RESUMO

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 DeltawbdD 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.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Endotoxinas/biossíntese , Escherichia coli/metabolismo , Lipopolissacarídeos/biossíntese , Redes e Vias Metabólicas , Transporte Biológico , Endotoxinas/metabolismo , Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Lipopolissacarídeos/metabolismo , Manosiltransferases , Metiltransferases , Ligação Proteica
15.
J Struct Biol ; 166(2): 172-82, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19236919

RESUMO

The outer membrane protein Wza, from Escherichia 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 alpha-helices, rather than being a beta-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 destabilized 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.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Microscopia Crioeletrônica/métodos , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/ultraestrutura , Western Blotting , Dicroísmo Circular , Eletroforese em Gel de Poliacrilamida , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/ultraestrutura , Microscopia de Fluorescência , Dados de Sequência Molecular , Mutagênese , Polissacarídeos Bacterianos/química , Estabilidade Proteica , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade
16.
Proc Natl Acad Sci U S A ; 104(7): 2390-5, 2007 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-17283336

RESUMO

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.


Assuntos
Cápsulas Bacterianas/biossíntese , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Periplasma/química , Polissacarídeos Bacterianos/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas da Membrana Bacteriana Externa/fisiologia , Transporte Biológico , Dimerização , Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/fisiologia , Proteínas de Membrana/fisiologia , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/fisiologia , Periplasma/metabolismo , Proteínas Tirosina Quinases/fisiologia
17.
Nature ; 444(7116): 226-9, 2006 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-17086202

RESUMO

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 A 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 alpha-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.


Assuntos
Cápsulas Bacterianas/química , Proteínas da Membrana Bacteriana Externa/classificação , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/classificação , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Polissacarídeos Bacterianos/metabolismo , Cápsulas Bacterianas/metabolismo , Transporte Biológico , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Conformação Proteica , Propriedades de Superfície
18.
Proc Natl Acad Sci U S A ; 103(18): 7088-93, 2006 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-16641107

RESUMO

The PglB oligosaccharyltransferase (OTase) of Campylobacter jejuni can be functionally expressed in Escherichia coli, and its relaxed oligosaccharide substrate specificity allows the transfer of different glycans from the lipid carrier undecaprenyl pyrophosphate to an acceptor protein. To investigate the substrate specificity of PglB, we tested the transfer of a set of lipid-linked polysaccharides in E. coli and Salmonella enterica serovar Typhimurium. A hexose linked to the C-6 of the monosaccharide at the reducing end did not inhibit the transfer of the O antigen to the acceptor protein. However, PglB required an acetamido group at the C-2. A model for the mechanism of PglB involving this functional group was proposed. Previous experiments have shown that eukaryotic OTases have the same requirement, suggesting that eukaryotic and prokaryotic OTases catalyze the transfer of oligosaccharides by a conserved mechanism. Moreover, we demonstrated the functional transfer of the C. jejuni glycosylation system into S. enterica. The elucidation of the mechanism of action and the substrate specificity of PglB represents the foundation for engineering glycoproteins that will have an impact on biotechnology.


Assuntos
Proteínas de Bactérias/metabolismo , Campylobacter jejuni/enzimologia , Hexosiltransferases/metabolismo , Proteínas de Membrana/metabolismo , Polissacarídeos/metabolismo , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/genética , Transporte Biológico/fisiologia , Configuração de Carboidratos , Sequência de Carboidratos , Glicosilação , Hexosiltransferases/genética , Lipoproteínas/metabolismo , Proteínas de Membrana/genética , Dados de Sequência Molecular , Estrutura Molecular , Salmonella enterica/metabolismo , Especificidade por Substrato
19.
J Biol Chem ; 281(4): 2144-50, 2006 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-16172129

RESUMO

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 approximately 14 A 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 approximately 100 A and can be divided into four separate densities. From the side, Wzc is approximately 110 A high and has a distinctive appearance similar to an extracted molar tooth. The upper "crown" region is approximately 55 A high and forms a continuous ring of density. Four unconnected "roots" ( approximately 65 A 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.


Assuntos
Proteínas de Bactérias/química , Escherichia coli/metabolismo , Proteínas de Membrana/química , Proteínas Tirosina Quinases/química , Proteínas de Bactérias/metabolismo , Caprilatos/farmacologia , Membrana Celular/metabolismo , Citoplasma/enzimologia , Citoplasma/metabolismo , Detergentes/farmacologia , Eletroforese em Gel de Poliacrilamida , Fluorcarbonetos/farmacologia , Imuno-Histoquímica , Proteínas de Membrana/metabolismo , Microscopia Eletrônica de Transmissão , Modelos Biológicos , Peso Molecular , Mutação , Oligossacarídeos/química , Proteínas Periplásmicas/química , Ligação Proteica , Conformação Proteica , Mapeamento de Interação de Proteínas , Proteínas Tirosina Quinases/metabolismo , Especificidade da Espécie , Tirosina/química
20.
J Biol Chem ; 279(34): 35709-18, 2004 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-15184370

RESUMO

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 WbdD(O8) enzyme, and this modification terminates polymerization. Methyl groups are added to the O9a polymannan in a reaction dependent on preceding phosphorylation. The bifunctional WbdD(O9a) 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 wbdD(O9a) mutant accumulated O9a polymannan in the cytoplasm, indicating that WbdD activity coordinates polymannan chain termination with export across the inner membrane.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Escherichia coli/metabolismo , Mananas/química , Antígenos O/química , Sequência de Aminoácidos , Catálise , Membranas Intracelulares/metabolismo , Mananas/genética , Mananas/metabolismo , Dados de Sequência Molecular , Antígenos O/genética , Antígenos O/metabolismo , Fosforilação , Polímeros , Transporte Proteico , Alinhamento de Sequência , Relação Estrutura-Atividade
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