class="head no_bottom_margin" id="sec1title">IntroductionChaperone-usher (CU) pili are ubiquitous appendages displayed on the surface of bacterial pathogens (). They play crucial roles in infection, being responsible for recognition and adhesion to host tissues. Types 1 and P pili are archetypal CU pili produced by uropathogenic Escherichia coli (UPEC) that mediate host-pathogen interactions critical in disease and biofilm formation (). Types 1 and P pili are composed of a short tip fibrillum made of three to four different subunits (FimH, FimG, and FimF for type 1 pili and PapG, PapF, PapE, and PapK for P pili) mounted on a 1–2 μM long and helically wound rod, which is composed of ∼1,000 copies of the major pilus subunit FimA or PapA for type 1 or P pili, respectively (A) (, ).Architecture of Type 1 and P Pili, Their Assembly Mechanism via Donor-Strand Complementation and Donor-Strand Exchange, and Model Building and Refinement of the P Pilus Rod, Related to (A) Pilus subunits are transported to the periplasm through the SecYEG transporter in the inner membrane (IM), where a chaperone (FimC or PapD) assists in the folding and transport of subunits to the usher situated in the outer membrane (OM). Here, the subunits polymerize and are assembled into a pilus which can be divided into a thin ‘tip fibrillum’ and a ‘helically wound rod’.(B) Subunits are incorporated into the growing pilus through sequential steps of donor-strand exchange (DSE). The subunits are unstable on their own, as they consist of C-terminally truncated Ig-folds lacking strand G. As a result of the missing strand, a large hydrophobic groove is created where the strand G would have been if the fold had been complete. As they emerge from the SecYEG transporter, subunits are captured by the chaperone, which inserts its G1 β strand into the hydrophobic groove of the subunit thereby completing and stabilizing its fold. This is termed donor-strand complementation (DSC). The chaperone’s P1 to P4 residues are positioned in the subunits groove’s P1 to P4 pockets. The P5 pocket remains empty in DSC. In the pilus, the N-terminal extension (Nte; 10-20 residues) of each subunit provides the ‘complementing’ β strand and is thus inserted into the preceding subunit’s groove, thereby stabilizing it structurally. This is termed donor-strand exchange (DSE). The transition from DSC to DSE occurs via a zip-in-zip-out mechanism whereby the Nte of the incoming subunit occupies the previously empty P5 pocket, before inserting into the P4, P3, P2 and P1 pockets.(C) Topology diagrams of a pilus subunit during DSC and DSE. The key difference is the orientation of the inserted β strand, in DSC the chaperone’s β strand is inserted in a parallel fashion, whereas in DSE the subunit’s Nte is inserted in a more stable anti-parallel fashion. The P1-P5 pockets are indicated by filled circles, note that the P5 pocket is vacant during DSC. DS, donor strand.(D) Two independent reconstructions (map1 and map2) were used to guide refinement: models were refined against each, and evaluated against both the reconstruction used for refinement, as well as the independent reconstruction. These results indicate that the models are not overfit to the data: the difference in the agreement of models fit to map1 compared to map2 is explained by a relatively lower quality of map2. Finally, the agreement of the model fit to map1 and the full map confirms the claimed resolution of 3.8 Å.(E) Model validation statistics.
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