RESUMO
Complex carbohydrates are essential for many biological processes, from protein quality control to cell recognition, energy storage, and cell wall formation. Many of these processes are performed in topologically extracellular compartments or on the cell surface; hence, diverse secretion systems evolved to transport the hydrophilic molecules to their sites of action. Polyprenyl lipids serve as ubiquitous anchors and facilitators of these transport processes. Here, we summarize and compare bacterial biosynthesis pathways relying on the recognition and transport of lipid-linked complex carbohydrates. In particular, we compare transporters implicated in O antigen and capsular polysaccharide biosyntheses with those facilitating teichoic acid and N-linked glycan transport. Further, we discuss recent insights into the generation, recognition, and recycling of polyprenyl lipids.
Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Glicolipídeos/biossíntese , Antígenos O/biossíntese , Poliprenois/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/química , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transporte Biológico , Carbono-Oxigênio Ligases/química , Carbono-Oxigênio Ligases/genética , Carbono-Oxigênio Ligases/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Glicosiltransferases/química , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Modelos Moleculares , Estrutura Secundária de Proteína , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Ácidos Teicoicos/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismoRESUMO
Capsular polysaccharides (CPSs) fortify the cell boundaries of many commensal and pathogenic bacteria1. Through the ABC-transporter-dependent biosynthesis pathway, CPSs are synthesized intracellularly on a lipid anchor and secreted across the cell envelope by the KpsMT ABC transporter associated with the KpsE and KpsD subunits1,2. Here we use structural and functional studies to uncover crucial steps of CPS secretion in Gram-negative bacteria. We show that KpsMT has broad substrate specificity and is sufficient for the translocation of CPSs across the inner bacterial membrane, and we determine the cell surface organization and localization of CPSs using super-resolution fluorescence microscopy. Cryo-electron microscopy analyses of the KpsMT-KpsE complex in six different states reveal a KpsE-encaged ABC transporter, rigid-body conformational rearrangements of KpsMT during ATP hydrolysis and recognition of a glycolipid inside a membrane-exposed electropositive canyon. In vivo CPS secretion assays underscore the functional importance of canyon-lining basic residues. Combined, our analyses suggest a molecular model of CPS secretion by ABC transporters.
Assuntos
Cápsulas Bacterianas , Proteínas de Escherichia coli , Escherichia coli , Polissacarídeos Bacterianos , Trifosfato de Adenosina/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/ultraestrutura , Cápsulas Bacterianas/metabolismo , Cápsulas Bacterianas/química , Cápsulas Bacterianas/ultraestrutura , Membrana Celular/química , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Microscopia Crioeletrônica , Escherichia coli/química , Escherichia coli/metabolismo , Escherichia coli/ultraestrutura , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/ultraestrutura , Glicolipídeos/química , Glicolipídeos/metabolismo , Hidrólise , Microscopia de Fluorescência , Modelos Moleculares , Polissacarídeos Bacterianos/metabolismo , Polissacarídeos Bacterianos/química , Especificidade por SubstratoRESUMO
Hyaluronan is an acidic heteropolysaccharide comprising alternating N-acetylglucosamine and glucuronic acid sugars that is ubiquitously expressed in the vertebrate extracellular matrix1. The high-molecular-mass polymer modulates essential physiological processes in health and disease, including cell differentiation, tissue homeostasis and angiogenesis2. Hyaluronan is synthesized by a membrane-embedded processive glycosyltransferase, hyaluronan synthase (HAS), which catalyses the synthesis and membrane translocation of hyaluronan from uridine diphosphate-activated precursors3,4. Here we describe five cryo-electron microscopy structures of a viral HAS homologue at different states during substrate binding and initiation of polymer synthesis. Combined with biochemical analyses and molecular dynamics simulations, our data reveal how HAS selects its substrates, hydrolyses the first substrate to prime the synthesis reaction, opens a hyaluronan-conducting transmembrane channel, ensures alternating substrate polymerization and coordinates hyaluronan inside its transmembrane pore. Our research suggests a detailed model for the formation of an acidic extracellular heteropolysaccharide and provides insights into the biosynthesis of one of the most abundant and essential glycosaminoglycans in the human body.