title: Membrane topology and identification of critical amino acid residues in the Wzx O-antigen translocase from Escherichia coli O157:H4.
abstract: Wzx belongs to a family of membrane proteins involved in the translocation of isoprenoid lipid-linked glycans, which is loosely related to members of the major facilitator superfamily. Despite Wzx homologs performing a conserved function, it has been difficult to pinpoint specific motifs of functional significance in their amino acid sequences. Here, we elucidate the topology of the Escherichia coli O157 Wzx (Wzx(EcO157)) by a combination of bioinformatics and substituted cysteine scanning mutagenesis, as well as targeted deletion-fusions to green fluorescent protein and alkaline phosphatase. We conclude that Wzx(EcO157) consists of 12 transmembrane (TM) helices and six periplasmic and five cytosolic loops, with N and C termini facing the cytoplasm. Four TM helices (II, IV, X, and XI) contain polar residues (aspartic acid or lysine), and they may form part of a relatively hydrophilic core. Thirty-five amino acid replacements to alanine or serine were targeted to five native cysteines and most of the aspartic acid, arginine, and lysine residues. From these, only replacements of aspartic acid-85, aspartic acid-326, arginine-298, and lysine-419 resulted in a protein unable to support O-antigen production. Aspartic acid-85 and lysine-419 are located in TM helices II and XI, while arginine-298 and aspartic acid-326 are located in periplasmic and cytosolic loops 4, respectively. Further analysis revealed that the charge at these positions is required for Wzx function since conservative substitutions maintaining the same charge polarity resulted in a functional protein, whereas those reversing or eliminating polarity abolished function. We propose that the functional requirement of charged residues at both sides of the membrane and in two TM helices could be important to allow the passage of the Und-PP-linked saccharide substrate across the membrane.
authors.affiliation: Center for Human Immunology, Siebens-Drake Research Institute, Departments of Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada.
authors.forename: Cristina L
authors.initials: CL
authors.lastname: Marolda
authors.forename: Bo
authors.initials: B
authors.lastname: Li
authors.forename: Michael
authors.initials: M
authors.lastname: Lung
authors.forename: Mei
authors.initials: M
authors.lastname: Yang
authors.forename: Anna
authors.initials: A
authors.lastname: Hanuszkiewicz
authors.forename: Amanda Roa
authors.initials: AR
authors.lastname: Rosales
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Valvano
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution / Q000235:genetics; D000596:Amino Acids / Q000235:genetics*; D002462:Cell Membrane / Q000201:enzymology*; D019295:Computational Biology; D019453:Escherichia coli O157 / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D019081:O Antigens / Q000096:biosynthesis; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000596:Amino Acids; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D019081:O Antigens; D011993:Recombinant Fusion Proteins; C543926:Wzx protein, E coli; D049452:Green Fluorescent Proteins; D000469:Alkaline Phosphatase