Type 1 pili are the archetypal representative of a widespread class of adhesive multisubunit fibres in Gram-negative bacteria. During pilus assembly, subunits dock as chaperone-bound complexes to an usher, which catalyzes their polymerization and mediates pilus translocation across the outer membrane. We report the crystal structure of the full-length FimD usher bound to the FimC:FimH chaperone:adhesin complex and that of the unbound form of the FimD translocation domain. The FimD:FimC:FimH structure shows FimH inserted inside the FimD 24-stranded β-barrel translocation channel. FimC:FimH is held in place through interactions with the two C-terminal periplasmic domains of FimD, a binding mode confirmed in solution by electron paramagnetic resonance spectroscopy. To accommodate FimH, the usher plug domain is displaced from the barrel lumen to the periplasm, concomitant with a dramatic conformational change in the β-barrel. The N-terminal domain of FimD is observed in an ideal position to catalyse incorporation of a newly recruited chaperone:subunit complex. The FimD:FimC:FimH structure provides unique insights into the pilus subunit incorporation cycle, and captures the first view of a protein transporter in the act of secreting its cognate substrate.

The tripartite multidrug efflux system MexAB-OprM is a major actor in Pseudomonas aeruginosa antibiotic resistance by exporting a large variety of antimicrobial compounds. Crystal structures of MexB and of its Escherichia coli homolog AcrB had revealed asymmetric trimers depicting a directional drug pathway by a conformational interconversion (from Loose and Tight binding pockets to Open gate (LTO) for drug exit). It remains unclear how MexB acquires its LTO form. Here by performing functional and cryo-EM structural investigations of MexB at various stages of the assembly process, we unveil that MexB inserted in lipid membrane is not set for active transport because it displays an inactive LTC form with a Closed exit gate. In the tripartite complex, OprM and MexA form a corset-like platform that converts MexB into the active form. Our findings shed new light on the resistance nodulation cell division (RND) cognate partners which act as allosteric factors eliciting the functional drug extrusion.