biostudies-literatureRemaut HMedical Research CouncilBiotechnology and Biological Sciences Research Council831-842https://www.ebi.ac.uk/biostudies/studies/S-EPMC7617774biostudies-literatureUnknown22(6)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 Molecular cellDonor-strand exchange in chaperone-assisted pilus assembly proceeds through a concerted beta strand displacement mechanism.PMC7617774SF16972G0100442Radford SERose RJWaksman GHultgren SJRemaut HHannan TJAshcroft AEfalseDonor-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 2006-01-01T00:00:00Z2006 Jun2026-06-03T07:56:38.649Z2026-04-26T03:10:45.846ZS-EPMC76177741679355110.1016/j.molcel.2006.05.033