Structural biology of the chaperone-usher pathway of pilus biogenesis

Abstract

The chaperone-usher (CU) pathway of pilus biogenesis is the most widespread of the five pathways that assemble adhesive pili at the surface of Gram-negative bacteria. Recent progress in the study of the structural biology of the CU pathway has unravelled the molecular basis of chaperone function and elucidated the mechanisms of fibre assembly at the outer membrane, leading to a comprehensive description of each step in the biogenesis pathway. Other studies have provided the molecular basis of host recognition by CU pili. The knowledge that has been gathered about both the assembly of and host recognition by CU pili has been harnessed to design promising antibiotic compounds.

Figure 1. P and type I pili

A schematic of P (part a) and type 1 (part b) pili, represented by the Pap and Fim systems, respectively. Numbers indicate the number of copies of each subunit in the pilus. The chaperones attached to the last subunit to be incorporated into each pilus are shown in yellow. P pili are terminated at the outer membrane (OM) by the termination subunit, PapH. No such subunit is known in the Fim system. The usher dimers are indicated in purple and blue. E, extracellular space; P, periplasm. Figure is modified, with permission, from REF. © (2008) Elsevier.

Figure 2. Donor strand complementation and donor strand exchange

a | A ribbon diagram of the PapD–PapK complex structure. PapD and PapK are in green and cyan, respectively. The carboxyl terminus of PapK is shown as a ball-and-stick representation. The conserved chaperone residues that contact the subunit C terminus (Arg8 and Lys112) and the P2, P3 and P4 residues in the chaperone G1 β -strand are shown in the same representation. b | A topology diagram of PapK. Arrows and cylinders represent β-strands and α-helices, respectively. The C and amino termini are indicated. Note that in the chaperone–subunit complex structure, the N-terminal extension (Nte) is disordered and thus not represented in the topology diagram of the subunit prior to donor strand exchange (DSE). The red arrow represents the G1β-strand donated by the chaperone. c | The structure of the pilus subunit after DSE. The subunit is shown in a ribbon diagram (cyan). The donor strand (or Nte) from the incoming subunit is shown in red. The P2, P3, P4 and P5 residues in the donor strand are shown in a ball-and-stick representation (red). d | A topological diagram of the subunits in the pilus. The representation is as in part b. The Nte (red) of the following subunit complements in trans the fold of the previously assembled subunit. e | A close-up of the P5 pocket (circled in green) and P4 pocket of PapA. f | A sequence alignment of the Ntes of all Pap subunits (except for PapG, which does not have an Nte). The alternating hydrophobic residue motif (DSE region) is highlighted in yellow, with the conserved Gly in magenta. Parts b and d are modified, with permission, from REF. © (2008) Elsevier. Part e is reproduced, with permission, from REF © (2008) Elsevier.

Figure 3. The concerted ‘zip-in, zip-out’ mechanism for donor strand exchange

a | A schematic diagram for donor strand exchange (DSE). Progressive insertion of the P5, P4, P3 and P2 residues of the attacking amino-terminal extension (Nte) is shown in pink. b | The presence of a P5 pocket in PapK (left panel) and the absence of a corresponding pocket in PapH (right panel). All Pap subunits except PapH have a P5 pocket, which serves as an initiation site for the progressive zipping-in of the Nte of the subunit that comes next in assembly. As PapH does not have a P5 pocket, an Nte cannot challenge the PapD–PapH complex. Part b is reproduced, with permission, from EMBO Reports REF. © (2006) Macmillan Publishers Ltd. All rights reserved.

Figure 4. The structural biology of the usher

a | The domain structure of PapC. The translocation pore and plug domains are shown in blue and magenta, respectively. The amino-terminal domain (NTD) and carboxy-terminal domain (CTD) are also indicated. b | The structure of the FimD N-terminal domain bound to the FimC–FimH complex. c | The structure of the translocation domain of the PapC usher, shown as a ribbon representation, with the barrel in cyan, the single helix in yellow, the trigger hairpin in orange and the plug domain in magenta. d | The cryo-electron microscopy structure of the FimD₂–FimH–FimG–FimF–FimC complex. The left panel shows a ribbon diagram of the structure. The two usher protomers are labelled usher 1 and usher 2.Their N-terminal domains are labelled N1 and N2, respectively. In the grey area, a model of FimC bound to FimA and N2 is shown. This model is oriented in such a way that the N-terminal extension (Nte) of FimA is within range for donor strand exchange with the FimF groove (that is, the P5 residue of FimA (not shown) is within interaction distance of the P5 pocket of FimF). The structure of the FimD₂–FimH–FimG–FimF–FimC complex is shown in the right panel in a schematic representation. The plug of usher 1 (the secretion pore) is shown in two different conformations: one in which it is still within the lumen of the pore but pushed to the side, and another in which it is pushed out into the periplasm (P). e | The subunit incorporation cycle, mediated by the twinned ushers. An incoming chaperone–subunit (FimC–FimA) complex is recruited by binding to N2 (step 1). Donor strand exchange with FimF causes the FimF-bound chaperone (C1) to be released, and N1 dissociates (steps 2 and 3) to recruit another FimC–FimA complex (step 4), adding it to the N2-bound FimC–FimA complex (step 5). Donor strand exchange then releases N2, which can recruit the next chaperone–subunit complex (step 6). Alternating binding to the released usher N-terminal domains and donor strand exchange with the next chaperone–subunit complex leads to stepwise growth of the pilus (steps 1–6). The plug domains are indicated as P in usher 2 and P′ and P″ in the alternative orientations shown for usher 1. C, FimC; E, extracellular space; F, FimF; G, FimG; H, FimH; OM, outer membrane. Parts a, d and e are modified, with permission, from REF. © (2008) Elsevier.

Figure 5. Receptor binding and pilus biogenesis inhibition

a | The structure of FimH bound to α-d-mannose. FimH is shown as a ribbon representation, and α-d-mannose is shown as a ball-and-stick representation (magenta). b | The structure of the PapG receptor domain, in a ribbon representation, bound to galabiose, in a ball-and-stick representation.