Conserved Hydrophobic Clusters on the Surface of the Caf1A Usher C-Terminal Domain Are Important for F1 Antigen Assembly |
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Authors: | Anatoly P. Dubnovitsky,Zoe Duck,Torleif Hä rd,Stefan D. Knight |
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Affiliation: | 1 Department of Molecular Biology, Uppsala BioCenter, Swedish University of Agricultural Sciences, Box 590, SE-75124 Uppsala, Sweden2 School of Biological Sciences, University of Reading, Reading RG6 6AJ, UK |
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Abstract: | The outer membrane usher protein Caf1A of the plague pathogen Yersinia pestis is responsible for the assembly of a major surface antigen, the F1 capsule. The F1 capsule is mainly formed by thin linear polymers of Caf1 (capsular antigen fraction 1) protein subunits. The Caf1A usher promotes polymerization of subunits and secretion of growing polymers to the cell surface. The usher monomer (811 aa, 90.5 kDa) consists of a large transmembrane β-barrel that forms a secretion channel and three soluble domains. The periplasmic N-terminal domain binds chaperone-subunit complexes supplying new subunits for the growing fiber. The middle domain, which is structurally similar to Caf1 and other fimbrial subunits, serves as a plug that regulates the permeability of the usher. Here we describe the identification, characterization, and crystal structure of the Caf1A usher C-terminal domain (Caf1AC). Caf1AC is shown to be a periplasmic domain with a seven-stranded β-barrel fold. Analysis of C-terminal truncation mutants of Caf1A demonstrated that the presence of Caf1AC is crucial for the function of the usher in vivo, but that it is not required for the initial binding of chaperone-subunit complexes to the usher. Two clusters of conserved hydrophobic residues on the surface of Caf1AC were found to be essential for the efficient assembly of surface polymers. These clusters are conserved between the FGL family and the FGS family of chaperone-usher systems. |
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Keywords: | Caf1AC, Caf1A usher C-terminal domain CUP, chaperone-usher pathway OM, outer membrane SEC, size-exclusion chromatography TM, transmembrane ZW3-14, Zwittergent 3-14 wt, wild type rCaf1AC, refolded Caf1AC PapCC, PapC usher C-terminal domain IMAC, immobilized metal-ion affinity chromatography Bicine, N,N-bis(2-hydroxyethyl)glycine PBS, phosphate-buffered saline PEG, polyethylene glycol ESRF, European Synchrotron Radiation Facility EDTA, ethylenediaminetetraacetic acid |
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