Crystal structure of the FimD usher bound to its cognate FimC–FimH substrate

Abstract

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.

Quality control of disulfide bond formation in pilus subunits by the chaperone FimC

“…Type 1 pili are assembled in vivo via the 'chaperone-usher pathway' 8 , involving the soluble periplasmic chaperone FimC 9 and the assembly platform ('usher') FimD in the outer membrane [10][11][12] . The pilus subunits enter the periplasm in an unfolded conformation and are recognized by the chaperone FimC, which catalyzes their folding 13 .…”

Molecular mechanism of bacterial type 1 and P pili assembly

“…In its active form, the translocation pore transits from an elongated kidney shape to a more round conformation (figure 3) and the plug domain swings out of the pore lumen into the periplasm close to the NTD to allow translocation of the pilus subunits. This transition from an closed/inactive to an open/active state of the usher is triggered by the recruitment of the first chaperone-subunit complex to the usher (the chaperone-adhesin complexes FimC-FimH in type 1 pili and PapD-PapG in P pili) [43]. Exactly how this recruitment induces pore opening or other conformational changes affecting the various usher domains is still unclear.…”

“…Thus, the two subunits are ready to undergo DSE. This and other results led us to propose a general subunit-incorporation cycle mechanism by a monomeric usher that is shown in figure 4 [43,57].…”

“…One major step in usher activation is the swinging out of the plug from its position within the usher pore lumen to its position in the periplasm where it interacts with the usher NTD. Electrophysiological studies suggested that both the NTD and the CTDs are involved in gating the usher, even in the absence of a translocation substrate [61], with only the FimH adhesin being able to stabilize the plug-NTD interaction in the open state while reaching out for the pore [43,47] ( figure 4, view b). Recently, it was proposed that the lectin domain of FimH actively displaces the plug domain, because the plug domain was calculated to have weaker interactions with the usher lumen than the lectin domain of FimH [27].…”

“…Thus, this visualizes the three main stages of subunit localization and interaction with its assembly catalyst: in the periplasm, after DSE traversing the OM through the usher, and in the extracellular milieu. [43]. In the FimD-FimC-FimF-FimG-FimH complex, where both FimH domains are outside on the extracellular milieu side of the pore, the angle between the two domains decreases by 37.5 • and the FimH L domain changes from a compressed and translocationcompetent state to a decompressed and receptor-competent state.…”