Donor-strand exchange in chaperone-assisted pilus assembly proceeds through a concerted beta strand displacement mechanism.
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.
Remaut
Han
H
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.
Rose
Rebecca J
RJ
Astbury Centre for Structural Molecular Biology, Garstang/Astbury Building, University of Leeds, Leeds, LS2 9JT, United Kingdom.
Hannan
Thomas J
TJ
Department of Molecular Microbiology, Washington University Medical School, 660 South Euclid Avenue, St. Louis, Missouri 63105.
Hultgren
Scott J
SJ
Department of Molecular Microbiology, Washington University Medical School, 660 South Euclid Avenue, St. Louis, Missouri 63105.
Radford
Sheena E
SE
Astbury Centre for Structural Molecular Biology, Garstang/Astbury Building, University of Leeds, Leeds, LS2 9JT, United Kingdom.
Ashcroft
Alison E
AE
Astbury Centre for Structural Molecular Biology, Garstang/Astbury Building, University of Leeds, Leeds, LS2 9JT, United Kingdom.
Waksman
Gabriel
G
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.
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
D001426:Bacterial Proteins; D018832:Molecular Chaperones; D046912:Multiprotein Complexes