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 system1. EPSs have critical roles in bacterial colonization of surfaces2, such as epithelia and medical implants; in addition some EPSs have important industrial and biomedical applications in their own right3. Here we describe the 2.26 Å 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 α-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.
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J.H.N. is a Biotechnology and Biology Sciences Research Council (BBSRC) Career Development Fellow, and acknowledges funding from a Wellcome Trust programme grant for biological aspects of the research. C.W. holds a Canada Research Chair and acknowledges funding from the Canadian Institutes of Health Research. The experimental structural biology was performed by the Scottish Structural Proteomics Facility, which is funded by the Scottish Higher Education Funding Council and the BBSRC. We acknowledge the use of ESRF beamlines and are grateful for assistance from D. Bourgeois and G. Leonard with data collection. We thank L. Cuthbertson for assistance with bioinformatic analyses, R. Clarke for fluorescence-activated cell sorting, and G. Taylor, M. White and B. Hunter for a critical review of the manuscript.
Author Contributions C.D. optimized crystals, collected data, and solved, refined and analysed the Wza structure. K.B. and J.N. grew the first crystals. K.B. devised and refined the SeMet protocol. A.L.B.-L. and B.R.C. made and analysed the site-directed mutants and Wza–Flag fusion, respectively. C.W. oversaw the project, analysed the data and wrote the paper. J.H.N. oversaw the project, collected X-ray data, solved and refined the structure, analysed the data and wrote the paper.
This file contains Supplementary Methods, Supplementary Figures 1–7 and additional references. The Supplementary Methods detail the molecular biology and crystallography. The Supplementary Figures give different views of the structure including experimental density in wall eye stereo. Extra experimental data is also shown in the Supplementary Figures. (PDF 4670 kb)
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