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. 2016 Jan 14;164(1-2):269-278.
doi: 10.1016/j.cell.2015.11.049. Epub 2015 Dec 24.

Structure of a Chaperone-Usher Pilus Reveals the Molecular Basis of Rod Uncoiling

Manuela K Hospenthal  1 , Adam Redzej  1 , Karen Dodson  2 , Marta Ukleja  1 , Brandon Frenz  3 , Catarina Rodrigues  1 , Scott J Hultgren  2 , Frank DiMaio  3 , Edward H Egelman  4 , Gabriel Waksman  5
Affiliations

Structure of a Chaperone-Usher Pilus Reveals the Molecular Basis of Rod Uncoiling

Manuela K Hospenthal et al. Cell. .

Abstract

Types 1 and P pili are prototypical bacterial cell-surface appendages playing essential roles in mediating adhesion of bacteria to the urinary tract. These pili, assembled by the chaperone-usher pathway, are polymers of pilus subunits assembling into two parts: a thin, short tip fibrillum at the top, mounted on a long pilus rod. The rod adopts a helical quaternary structure and is thought to play essential roles: its formation may drive pilus extrusion by preventing backsliding of the nascent growing pilus within the secretion pore; the rod also has striking spring-like properties, being able to uncoil and recoil depending on the intensity of shear forces generated by urine flow. Here, we present an atomic model of the P pilus generated from a 3.8 Å resolution cryo-electron microscopy reconstruction. This structure provides the molecular basis for the rod's remarkable mechanical properties and illuminates its role in pilus secretion.

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Figures

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Graphical abstract
Figure 1
Figure 1
Purification of PapD:PapA and Electron Microscopy of the P Pilus Rod (A) SDS-PAGE of the purified PapD:PapA complex. M, molecular weight markers. (B) Electron micrograph of P pilus rods. Red rectangles indicate pilus rods. Scale bar, 100 nm. (C) Side-view of the experimentally derived electron density of the P pilus rod. The density was contoured at a 1.5 σ level and is shown as a semi-transparent surface colored in gray. A ribbon diagram of the refined atomic model is shown in cyan. (D) Top-view of the experimentally derived electron density. Density and model are as in (C). (E) Details of a representative region of the experimentally derived electron density. Electron density contoured at a 1.5 σ level is shown in chicken wire representation colored in blue. Only two PapA subunits of the final model are shown in stick representation with carbon atoms colored either in cyan or orange, while all oxygen and nitrogen atoms are colored in red and blue, respectively. Secondary structural elements are indicated as well as some side chains.
Figure 2
Figure 2
Structure of the P Pilus Rod (A) Surface diagram of the rod. Each subunit is shown in surface representation, color coded differently. The rod is oriented in such a way that the N termini of each subunit (the staples) are directed toward the top. In that orientation, the OM and tip fibrillum are toward the bottom and top, respectively. The subunit in cyan is the reference subunit and is numbered “0.” Subunits above or below this subunit are assembled before or after subunit 0, respectively, and are therefore numbered negatively (−1 to −6) or positively (+1 to +6), respectively. (B) Surface diagram focusing on the Ntes. The orientation of the pilus rod structure and the colorcoding of subunits are the same as in (A), but only the Ntes are shown, clearly illustrating the ascending path that the Ntes form within the structure. The rise from one subunit to another is indicated. (C) Top view of the pilus rod. The rod is represented as in (A). The Nte of the last subunit (−6) has been removed for clarity. (D) Ribbon diagram of the structure of PapA in the rod (subunit 0) in donor-strand exchange with the subunit next in assembly (subunit +1). The subunit is shown in cyan (labeled “PapA subunit 0”) with the Nte of subunit +1 colored in orange (labeled “PapA Nte subunit +1”). Secondary structure elements are labeled. The orientation of the subunit is the same as in (A). In that orientation, the staple extends approximately parallel to the pilus axis (indicated by an arrow).
Figure 3
Figure 3
Details of Subunit-Subunit Interaction Interfaces (A) Surface diagram of the pilus rod and localization of the regions depicted in (B– D). Color coding and representation of subunits are as in Figure 2A. Black boxes labeled B, C, and D locate the region depicted in detail in panels (B–D). (B) Details of secondary structures involved in interactions between the staple of subunit 0 and subunits −1, −2, −4, and −5. Details of residues involved in these interactions are reported in Figures S3A and S3D. Subunits are in ribbon representation color coded as in (A). (C) Details of the secondary structures involved in interactions between subunits 0 and −1 in the region around the C-terminal part of the Nte and the Nte-αA1 loop of subunit 0. Representation and labeling are as in (B). Details of interacting residues are shown in corresponding Figures S3B and S3D. (D) Details of the secondary structures involved in interactions between subunits 0 and +3. Representation and labeling are as in (B). Details of interacting residues are shown in corresponding Figures S3C and S3D.
Figure 4
Figure 4
Probing the Structure by Site-Directed Mutagenesis and Site-Specific Labeling (A) Location of the residues targeted for site-directed incorporation of AzF. Surfaces in blue locate residues involved in subunit-subunit interactions as defined in Figures 3 and S3. (B) NS-EM of wild-type and mutant PapA rods. The full set of NS-EM micrographs is reported in Figure S4A. Here, only representative micrographs of three mutants are shown: one for a mutant not affected in pilus rod formation (Lys27), one for a mutant only partially affected in rod formation (Lys50), and one severely affected in rod formation (Asn96). Scale, 100 nm. (C) Summary of pilus rod formation and solvent accessibility of various residues within the rod structure. Each PapA variant is categorized and color coded according to its pilus rod formation and labeling efficiency. The quantification of these parameters is described in Experimental Procedures and the data are represented in graphical form in Figure S4B. Dash (-), no data available. (D) Size exclusion chromatography of mutants unable to form rods. The identity of each peak was evaluated by SEC-MALS (Figure S4C) and is indicated above the peak. (E) Summary of haemagglutination results (full results in Figure S4D). pPAP5 wild-type, untransformed HB101 cells (HB101 alone), and PBS served as controls for this experiment. All PapA mutants tested, with the exception of Val18Tyr, show a positive haemagglutination reaction with rabbit red blood cells.
Figure S1
Figure S1
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 Figure 1 (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.
Figure S2
Figure S2
Interactions between PapA and Its Nte after Rod Formation and Superposition of PapA before and after Rod Insertion, Related to Figure 2 (A) Overview of the subunit-Nte interaction. The subunit and Nte are in cyan and orange respectively. Boxed areas labeled B and C locate the areas shown in panels B and C. Surfaces in dark teal locate residues mentioned in the main text. In panels B and C, residues in the subunit and in the Nte are labeled black and orange, respectively. The P1-P5 residues of the Nte are labeled both P1 to P5 and also by their residue numbering in the PapA sequence. The Nte of the subunit in cyan has been removed for clarity. (B) Area B of panel A. (C) Area C of panel A. (D) Superposition of the structure of PapA in the rod and that of PapA before rod formation. Both structures are shown in ribbon representation. PapA in the rod is labeled “PapA subunit 0” and is shown in cyan. PapA before rod formation is labeled “PapA DSE (PDB2UY6)” and is in magenta. The Nte depicted here is that inserted into PapA DSE (PDB2UY6) as determined in the structure by Verger et al. (2007).
Figure S3
Figure S3
Details of the Interactions between Subunits within the Pilus Rod, Related to Figure 3 (A) Side chains involved in the interaction of adjacent subunits with the staple of subunit 0. Orientation and secondary structures are as in Figure 3B. At the very N terminus of the staple region, residues Ala1 and Pro2 (subunit 0) make interactions with Ala45 and Phe42 (subunit −5), respectively. Next, residue Thr3 (subunit 0) interacts with residue Asp62 (subunit −4), while Ile4 (subunit 0) interacts with Glu149 (subunit −2) and Val18 (subunit −1). Residue Pro5 (subunit 0) is in close proximity to Lys125 and Asp126 (subunit −4). Gln6 (subunit 0) is positioned at the interface between the donor-strand and the staple region and interacts with Asp19, which is positioned at the end of the complemented subunit’s Nte (subunit −1). Lastly Gln8 (subunit 0) interacts with Gln37 (subunit −2), as well as Glu128 and Lys125 (subunit −4). (B) Side chains involved in interactions between the C-terminal region of subunit 0’s Nte with subunit −1. Orientation and secondary structures are as in Figure 3C. Starting at the top of the panel, residue Asp19 (subunit 0) interacts with Glu149 and is also in close proximity to Lys40 (subunit −1). Next, Val18 (subunit 0) interacts with Leu38 and Lys40 (subunit −1). Residues Ala20, Pro21 and Ser23 (subunit 0) are in close proximity to Ser39 (subunit −1), while Ile24 and Ala29 (subunit 0) interact with Gln37 (subunit −1). In addition, both Ser25 and Gln26 (subunit 0) are positioned close to Gly36 (subunit −1), Residue Asn60 (subunit 0) makes contacts with both Phe42 and Ser49 (subunit −1). Lastly, Glu128 (subunit 0) makes a salt bridge interaction with Lys50 (subunit −1). (C) Side chains involved in interactions between subunit 0 and +3. Orientation and secondary structures are as in Figure 3D. Going from left to right, Gln106 (subunit 0) is positioned at the edge of the interface and is in close proximity to Asn96 and Gly98 (subunit +3). Next, Pro51 (subunit 0) interacts with Ala151 (subunit +3), while Thr134 (subunit 0) makes extensive contacts with Asn96 (subunit +3). Gly109 (subunit 0) interacts with Asp94 (subunit +3), while His132 (subunit 0) is crucial and makes contacts with Asn96, Ser153 and Val155 (subunit +3). Meanwhile, Ala108 (subunit 0) contacts Asp94 and Gly97 (subunit +3). Asn122 (subunit 0) interacts with Pro84 (subunit +3), while Val130 (subunit 0) interacts with Asn157 and is also in close proximity to Val155 (both of subunit +3). Lastly, Thr123 and Lys125 (subunit 0) interact with Thr82 and Asn159 (subunit +3), respectively. The main chains in panels A, B, and C are in ribbon representation while side chains are in stick representation with oxygen and nitrogen atoms color-coded in red and blue, respectively. Color-coding of ribbons and carbon atoms follows the color code established in Figure 2A for each subunit. (D) Table listing interactions shown in panels A, B, and C. (E) Surface mapping of subunit 0’s residues involved in interaction with other subunits. Color-coding indicates which subunits each residue is interacting with. The color code established in Figure 2A is used here. Residues colored in black represent regions which are interacting with multiple subunits.
Figure S4
Figure S4
Biochemical Characterization of P Pilus Rod Mutants and In Vivo Assessment of Mutations of Interface Residues, Related to Figure 4 (A) The full set of negative stain images of all rod mutants after assembly into pilus rods and purification by ultracentrifugation. The scale bar represents 100 nm. (B) Quantification of pilus rod formation (left) and labeling efficiency (right). For pilus rod formation (left), the Sypro Ruby signal was converted into a percentage and normalized to the wild-type (100%). For labeling efficiency (right), if PapA in pilus rod samples labeled with Alexa 647 to the same extent as in the corresponding non-piliated control samples, the final ratio would be 1.0 (indicated by a red line). These data were used to categorise and color-code each PapA variant according to its pilus rod formation and labeling efficiency as shown in Figure 4C. For pilus rod formation, category 1 (+), 0%–29%; category 2 (++), 30%–59%; category 3 (+++), 60%–89%; category 4 (++++), > 90%. Dash (-), no data available. For labeling efficiency, category 1 (+), 0-0.25; category 2 (++), 0.26-0.5; category 3 (+++), 0.51-0.75; category 4 (++++), > 0.76. A detailed description of the quantitation can be found in the Experimental Procedures. The results presented in Figure 4C are from two independent experiments. (C) SEC-MALS results for each of the peaks reported for the PapD:PapA Val18AzF in Figure 4D and comparison to expected molecular weights. (D) Haemagglutination result of HB101 cells expressing the entire Pap operon (pPAP5 plasmid) with wild-type PapA or mutant versions of PapA. All pPAP5 PapA mutants showed a positive haemagglutination result, with the exception of Val18Tyr which haemagglutinated to a much lesser extent. Untransformed HB101 cells (HB101 alone) and PBS serve as negative controls. (E) Location of residues mutated in panel F. Representation is as in Figure 4A. (F) Summary of analysis of pilus translocation to the bacterial surface and assessment of the strength of helical interaction in the pilus. The residues mutated in this experiment are shown in panel E. The first column (surface appendage) indicates whether the PapA mutant has incorporated into pili displayed on the bacterial surface and the second column (urea requirement for uncoiling) is an assessment of the helical strength of these pili produced in vivo. (+) indicates results comparable to wild-type; (+/−) indicates either lesser pilus production and/or rod formation; (-) indicates either no pilus production or no rod formation. A description of the how these experiments were performed can be found in the Experimental Procedures section.
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