title: Donor-strand exchange in chaperone-assisted pilus assembly proceeds through a concerted beta strand displacement mechanism.
abstract: Gram-negative pathogens commonly use the chaperone-usher pathway to assemble adhesive multisubunit fibers on their surface. In the periplasm, subunits are stabilized by a chaperone that donates a beta strand to complement the subunits' truncated immunoglobulin-like fold. Pilus assembly proceeds through a "donor-strand exchange" (DSE) mechanism whereby this complementary beta strand is replaced by the N-terminal extension (Nte) of an incoming pilus subunit. Using X-ray crystallography and real-time electrospray ionization mass spectrometry (ESI-MS), we demonstrate that DSE requires the formation of a transient ternary complex between the chaperone-subunit complex and the Nte of the next subunit to be assembled. The process is crucially dependent on an initiation site (the P5 pocket) needed to recruit the incoming Nte. The data also suggest a capping reaction displacing DSE toward product formation. These results support a zip-in-zip-out mechanism for DSE and a catalytic role for the usher, the molecular platform at which pili are assembled.
authors.affiliation: Institute of Structural Molecular Biology at UCL/Birkbeck, Malet Street, London WC1E 7HX, United Kingdom; School of Crystallography, Malet Street, London WC1E 7HX, United Kingdom.
authors.forename: Han
authors.initials: H
authors.lastname: Remaut
authors.affiliation: Astbury Centre for Structural Molecular Biology, Garstang/Astbury Building, University of Leeds, Leeds, LS2 9JT, United Kingdom.
authors.forename: Rebecca J
authors.initials: RJ
authors.lastname: Rose
authors.affiliation: Department of Molecular Microbiology, Washington University Medical School, 660 South Euclid Avenue, St. Louis, Missouri 63105.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Hannan
authors.affiliation: Department of Molecular Microbiology, Washington University Medical School, 660 South Euclid Avenue, St. Louis, Missouri 63105.
authors.forename: Scott J
authors.initials: SJ
authors.lastname: Hultgren
authors.affiliation: Astbury Centre for Structural Molecular Biology, Garstang/Astbury Building, University of Leeds, Leeds, LS2 9JT, United Kingdom.
authors.forename: Sheena E
authors.initials: SE
authors.lastname: Radford
authors.affiliation: Astbury Centre for Structural Molecular Biology, Garstang/Astbury Building, University of Leeds, Leeds, LS2 9JT, United Kingdom.
authors.forename: Alison E
authors.initials: AE
authors.lastname: Ashcroft
authors.affiliation: Institute of Structural Molecular Biology at UCL/Birkbeck, Malet Street, London WC1E 7HX, United Kingdom; School of Crystallography, Malet Street, London WC1E 7HX, United Kingdom; Department of Biochemistry and Molecular Biology, University College London, Gower Street, London WC1E 6BT, United Kingdom. Electronic address: g.waksman@bbk.ac.uk.
authors.forename: Gabriel
authors.initials: G
authors.lastname: Waksman
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray; D010861:Fimbriae, Bacterial / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000737:chemistry* / Q000378:metabolism; D046912:Multiprotein Complexes / Q000737:chemistry; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D019779:Salmonella enterica / Q000737:chemistry* / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D001426:Bacterial Proteins; D018832:Molecular Chaperones; D046912:Multiprotein Complexes