title: Substrate binding by a bacterial ABC transporter involved in polysaccharide export.
abstract: ATP-binding-cassette (ABC) transporters are responsible for the export of a wide variety of cell-surface glycoconjugates in both Gram-positive and Gram-negative bacteria. These include the O-antigenic polysaccharide (O-PS) portion of lipopolysaccharide, a crucial virulence determinant in Gram-negative pathogens. O-PSs are synthesized by one of two fundamentally different pathways. Escherichia coli O serotypes O8 and O9a provide the prototype systems for studying O-PS export via ABC transporters. The transporter is composed of the transmembrane component Wzm and the nucleotide-binding component Wzt. Although the N-terminal domain of Wzt is a conventional ABC protein, the C-terminal domain of Wzt (C-Wzt) is a unique structural element that determines the specificity of the transporter for either the O8 or O9a O-PS. We show here that the two domains of Wzt can function when expressed as separate polypeptides; both are essential for export. In vitro, C-Wzt binds its cognate O-PS by recognizing a residue located at the nonreducing end of the polymer. The crystal structure of C-Wzt(O9a) is reported here and reveals a beta sandwich with an immunoglobulin-like topology that contains the O-PS-binding pocket. Substrate interactions with nucleotide-binding domains have been demonstrated in an ABC exporter previously. However, to our knowledge substrate binding by a discrete, cytoplasmic accessory domain in an extended nucleotide-binding domain polypeptide has not previously been demonstrated. Elucidation of the substrate-recognition system involved in O-PS export provides insight into the mechanism that coordinates polymer biosynthesis, termination, and export.
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada.
authors.forename: Leslie
authors.initials: L
authors.lastname: Cuthbertson
authors.forename: Matthew S
authors.initials: MS
authors.lastname: Kimber
authors.forename: Chris
authors.initials: C
authors.lastname: Whitfield
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D018360:Crystallography, X-Ray; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D019081:O Antigens / Q000737:chemistry*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
chemical_list: D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D019081:O Antigens; C108433:Wzt protein, bacteria