Abstract
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.
Highlights
Escherichia coli serotype O9a provides a model for export of lipopolysaccharide (LPS) O-antigen polysaccharide (O-PS) via ATP-binding cassette (ABC) transporters
To determine whether methylphosphate is the sole determinant of substrate recognition by the carbohydrate-binding module (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
Many O-PSs are assembled by a mechanism that requires an ATP-binding cassette (ABC) transporter to export lipid-linked O-PS intermediates from the site of synthesis in the cytosol to the periplasm [7, 8], where the LPS molecule is completed before being translocated to the cell surface by the Lpt (LPS transport) machinery [9, 10]
Summary
WbdD kinase activity is solely responsible for arresting O9a O-PS polymerization. The modal distribution of O9a-PS chain lengths is established by the coiled-coil domain, which physically separates the WbdD termination enzymes from membrane-anchored WbdA [20]. The most intense signal from the obtained STDD spectrum (aside from a differencing artifact arising from the Tris buffer) was a doublet at ␦H 3.63 ppm with coupling constant J ϭ 11 Hz (Fig. 5B), which precisely corresponds to the chemical shift of the methyl protons on the nonreducing terminal methylphosphate group from O9a O-PS [33] These findings offer clear and direct data for the interaction of the nonreducing terminal methyl group with WztO9a-C and are entirely consistent with its requirement for WztO9a-Cbinding in LPS pulldown experiments as well as its requirement for display of the O9a polysaccharide on the cell surface. To examine whether the nonreducing terminal modification is the sole contributor to WztO9a-C O-PS recognition, we performed a pulldown experiment using the E. coli O9a CBM with K. pneumoniae O7 LPS (Fig. 8, A and B).
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