Domain Activities of Papc Usher Reveal the Mechanism of Action of an Escherichia Coli Molecular Machine

Domain activities of PapC usher reveal the mechanism of action of an Escherichia coli molecular machine

Ender Volkana,b, Bradley A. Fordb,c, Jerome S. Pinknera,b, Karen W. Dodsona,b, Nadine S. Hendersond, David G. Thanassid, Gabriel Waksmane, and Scott J. Hultgrena,b,1

aDepartment of Molecular Microbiology, bCenter for Women’s Infectious Diseases Research, cDepartment of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110; dCenter for Infectious Diseases, Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY 11794; and eInstitute of Structural and Molecular Biology, Birkbeck College and University College London, London WC1E 7HX, United Kingdom

Contributed by Scott J. Hultgren, April 26, 2012 (sent for review March 13, 2012) P pili are prototypical chaperone–usher pathway-assembled pili (PapD) and the integral outer membrane usher (PapC) in a used by Gram-negative bacteria to adhere to host tissues. The PapC specific order into bipartite P pilus fibers composing an open usher contains five functional domains: a transmembrane β-barrel, helical tip fibrillum made up of (from the tip) PapG (11), PapF, a β-sandwich Plug, an N-terminal (periplasmic) domain (NTD), and multiple PapEs, and PapK (12, 13), connected to a right-handed two C-terminal (periplasmic) domains, CTD1 and CTD2. Here, we helical rod made up of thousands of copies of PapA (14–17) and delineated usher domain interactions between themselves and terminated by a single copy of PapH (18, 19). Each pilin Ig fold is with chaperone–subunit complexes and showed that overexpres- missing the seventh C-terminal β-strand of the canonical Ig fold. sion of individual usher domains inhibits pilus assembly. Prior work Due to the lack of the C-terminal strand, pilin subunits are un- revealed that the Plug domain occludes the pore of the transmem- able to fold independently (12). Thus, folding of pilins in the brane domain of a solitary usher, but the chaperone–adhesin- periplasm is facilitated by the PapD chaperone (13, 20). PapD is bound usher has its Plug displaced from the pore, adjacent to the a two-domain periplasmic protein, with each domain having a NTD. We demonstrate an interaction between the NTD and Plug complete Ig fold (21). PapD binds and transiently donates a β- domains that suggests a biophysical basis for usher gating. Further- strand from its N-terminal domain to transiently complete the Ig fi more, we found that the NTD exhibits high-af nity binding to the fold of subunits, resulting in a noncanonical parallel interaction MICROBIOLOGY chaperone–adhesin (PapDG) complex and low-affinity binding to between the sixth strand of the subunit and the seventh strand the major tip subunit PapE (PapDE). We also demonstrate that provided by the chaperone (13). This process has been termed CTD2 binds with lower affinity to all tested chaperone–subunit donor strand complementation (DSC). The subunits in the complexes except for the chaperone–terminator subunit (PapDH) resulting complexes are stable. However, subunits in complex and has a catalytic role in dissociating the NTD–PapDG complex, with PapD are not completely condensed but instead are in an suggesting an interplay between recruitment to the NTD and trans- open conformation that is “primed” for assembly (20). DSC has fer to CTD2 during pilus initiation. The Plug domain and the NTD– been demonstrated both for P and type 1 pilus systems, as well as Plug complex bound all of the chaperone–subunit complexes tested for Sfa, Saf, Caf, Fml, and others (22), demonstrating the pro- including PapDH, suggesting that the Plug actively recruits chaper- totypical nature of P pili. one–subunit complexes to the usher and is the sole recruiter of Once formed, P pilus chaperone–subunit complexes are tar- PapDH. Overall, our studies reveal the cooperative, active roles geted to the outer membrane usher PapC (23, 24). PapC is an played by periplasmic domains of the usher to initiate, grow, and 809-residue outer membrane protein that forms an assembly terminate a prototypical chaperone–usher pathway pilus. platform for pilus biogenesis (23, 25). PapC has five functional domains, all of which are required for pilus biogenesis (23, 25– biolayer interferometry | macromolecular assembly | urinary tract 27). It has a 24-stranded β-barrel transmembrane domain that infections | virulence factor | bacterial pathogenesis allows translocation of the polymerized pilus fiber across the outer membrane and four globular domains: a periplasmic N- he chaperone–usher pathway (CUP) is used by Gram-nega- terminal domain (NTD), two periplasmic C-terminal domains Ttive pathogens to assemble hundreds of different adhesive (CTD1 and CTD2), and a plug domain (Plug) (23, 28). Muta- proteinaceous surface fibers called pili or fimbriae (1). Pili are tions of the NTD or either CTD abolish pilus biogenesis (25, 27). important virulence factors, which in part determine tropism for The Plug domain is also required for pilus biogenesis (29, 30). A host tissues of many pathogens. In uropathogenic Escherichia coli Plug-deleted PapC folds but does not assemble pili, suggesting (UPEC), type 1 pili facilitate bladder colonization and invasion of that the Plug domain plays a direct role in catalyzing pilus bio- the bladder tissue in cystitis (2–5), and P pili mediate pyelone- genesis (29, 30). Binding studies have suggested that the chap- phritis by binding to the globoseries of glycolipids in the human erone–adhesin complex is initially targeted to the NTD of PapC kidney (6–9). Type 1 and P pili are prototypes for understanding (26), and this is thought to activate the usher protein such that β assembly by the CUP. Each of these pilus systems are encoded in the -sandwich Plug domain shifts from the channel, where it is apo a separate gene cluster (fim for type 1 and pap for P pili), with located in the usher, to the periplasmic space, resulting in an open translocation pore (28). This affinity of the NTD for the each operon encoding regulatory proteins, a tip adhesin, multiple – pilin subunits, and a dedicated chaperone and usher. The corre- chaperone adhesin complex has also been shown for the type 1 sponding proteins in each system share significant homology, and pilus system (31). However, the crystal structure of the type 1 studies have shown that the chaperone and usher proteins function in homologous ways. pap Author contributions: E.V., B.A.F., and S.J.H. designed research; E.V. performed research; The chromosomally encoded (pyelonephritis-associated E.V., J.S.P., K.D., N.S.H., D.G.T., and G.W. contributed new reagents/analytic tools; E.V. and pilus) gene cluster encodes five Ig-like pilin subunits (PapF, B.A.F. analyzed data; and E.V., B.A.F., and S.J.H. wrote the paper. PapE, PapK, PapA, PapH) and a two-domain adhesin, PapG, The authors declare no conflict of interest. with one Ig-like pilin domain and a ligand-binding domain. Upon 1To whom correspondence should be addressed. E-mail: [email protected]. their transfer to the periplasm (10), these structural subunits are This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. assembled through the actions of the periplasmic chaperone 1073/pnas.1207085109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1207085109 PNAS Early Edition | 1of6 Downloaded by guest on September 23, 2021 pilus usher (FimD) bound to its cognate chaperone–adhesin Table 1. Binding affinities of PapC periplasmic domains for complex (FimCH) revealed interactions between the chaperone– chaperone–subunit complexes adhesin complex and the CTDs, as suggested in an earlier study K (M) k [1/(M*s)] k (1/s) R2* by copurification after trypsin protection assays (25, 28). D on off In the absence of energy from either ATP hydrolysis or the NTD binding − − proton-motive force, PapC catalyzes the ordered polymerization PapDG 3.20 × 10 9 2.35 × 10+6 6.68 × 10 3 0.83 and translocation of subunits across the outer membrane to the PapG lectin No binding × −6 × +2 × −4 cell surface (1, 32, 33) via a process called donor strand exchange PapDEntd 1.19 10 4.64 10 6.11 10 0.99 (DSE). In addition to the incomplete Ig fold, every nonadhesin PapDK No binding pilin subunit has a short N-terminal extension (NTE) that is PapDAKnte No binding exchanged with the donated chaperone strand bound to the PapDH No binding previously incorporated subunit. The A and F strands of the PapD No binding 2 subunit condense around the newly donated NTE, driving fold- Plug binding KD (M) kon [1/(M*s)] koff (1/s) R * ing of the subunit into a canonical Ig domain with the seventh PapDG 5.11 × 10−8 2.45 × 10+3 1.25 × 10−4 0.97 strand provided by the next subunit (20). This polymerization PapG lectin No binding × −8 × +4 × −3 and translocation of the growing pilus fiber through the β-barrel PapDEntd 3.38 10 2.70 10 1.31 10 0.94 − − transmembrane pore to the cell surface appears to rely on the PapDK 1.79 × 10 8 2.23 × 10+4 3.19 × 10 4 0.99 × −8 × +4 × −4 energy stored in the incompletely condensed Ig folds of PapDAKnte 3.02 10 1.86 10 4.54 10 0.99 chaperone-bound subunits. PapDH 1.65 × 10−8 1.62 × 10+4 2.70 × 10−4 0.96 − − Here, we investigated relevant protein–protein interactions PapD 2.26 × 10 8 2.19 × 10+4 5.38 × 10 4 0.98 2 that occur at the usher. We characterized the interactions of the CTD2 binding KD (M) kon [1/(M*s)] koff (1/s) R * periplasmic PapC domains, NTD, CTD2, and Plug to obtain insight PapDG 5.47 × 10−6 4.43 × 10+3 9.81 × 10−3 0.79 into the molecular basis of how the usher catalyzes pilus biogenesis PapG lectin No binding × −6 × +2 × −4 and how these domains function as a molecular machine. We PapDEntd 1.05 10 6.90 10 6.92 10 0.98 demonstrate that the NTD is the initial site of recruitment of PapDK 1.87 × 10−6 3.38 × 10+3 7.33 × 10−3 0.88 × −6 × +3 × −3 chaperone–adhesin complexes and that CTD2 allosterically PapDAKnte 1.10 10 5.95 10 6.24 10 0.91 destabilizes this complex, resulting in transfer to the CTD domains. PapDH No binding − − Subsequent chaperone–pilin complexes are recruited to the NTD– PapD 1.70 × 10 6 3.49 × 10+3 5.91 × 10 3 0.71 Plug complex or the Plug domain alone, rationalizing an active role *R2 is the coefficient of determination estimating the goodness of a curve fit for this domain in pilus assembly. Finally, CTD2 binds non- reported by ForteBio Data Analysis software version 6.4. selectively to all chaperone–subunit complexes except the terminator complex, reflecting its second role as a transient docking site – for chaperone subunit complexes (28) and suggesting that pilus including apo-PapD and PapDH (Fig. S1, Table 1). PapGtrunc termination and/or anchoring may in part occur by failure of the did not bind to CTD2, Plug, or NTD (Table 1). These data argue terminator to transfer from the Plug domain. that PapDG and PapDEntd are selectively recruited to the usher – Results by the NTD and that recruitment of all other chaperone subunit PapC Periplasmic Domains Interact Selectively with Chaperone– complexes involves the Plug domain. The CTD domains are not Subunit Complexes. It has been previously shown that ushers bind to chaperone–subunit complexes with differing affinities (34–36). Thus, we used biolayer interferometry to assess the binding affinities of chaperone–subunit complexes with the various usher domains of PapC, hypothesizing that one or more of the periplasmic domains of the usher would recapitulate the discriminatory binding demonstrated for the full-length usher. Chaperone alone (PapD) and chaperone complexes with the adhesin (PapDG), major tip subunit (PapDE), the rod subunit (PapDA), the tip adaptor to the rod (PapDK), and the terminator (PapDH) were purified as previously described (11, 13) (SI Materials and Methods). To prevent homopolymerization of PapA and PapE subunits, we used PapA and PapE constructs in which the PapA NTE was replaced with the PapK NTE (PapAKnte) and the PapE NTE was deleted to create PapEntd.A truncate of PapG containing the galabiose-binding N-terminal domain of PapG (PapGtrunc) was also purified. A total of 50 μg/ mL of biotinylated NTD, Plug, or CTD2 was incubated with Super Streptavidin pins (ForteBio Inc.). Super Streptavidin pins coated with biotinylated NTD, CTD2, or Plug were then incubated with each of the purified chaperone–subunit complexes. – The NTD bound only to the PapDG and PapDEntd complexes Fig. 1. PapC periplasmic domain CTD2 mediates binding with chaperone with nanomolar and micromolar affinity, respectively, consistent subunit complexes. PapC CTD2 mediates concentration-dependent, micro- with their order of assembly at the usher (Table 1). The NTD did molar affinity interactions with chaperone–subunit complexes PapDG, Pap- fi not bind to apo-PapD, PapG , any of the other chaperone– DEntd, PapDAKnte, and apo-PapD, but has no af nity for PapDH. BSA was trunc used as a binding specificity control. The tips of Super Streptavidin pins were subunit complexes, or bovine serum albumin (BSA). In contrast, coated with 50 μg/mL of biotinylated PapC CTD2 and washed to remove CTD2 bound to apo-PapD and all of the chaperone–subunit fi unbound protein. These pins were dipped in increasing concentrations of complexes with micromolar af nities, with the exception of chaperone–subunit complexes (shown at 2 μM each) to measure binding of PapDH (Fig. 1). The Plug domain bound to each of the chap- chaperone–subunit complexes to CTD2 (left side of graph) and then moved erone–subunit complexes with high affinity (∼20–50 nM), to wells containing HBS to measure dissociation rates (right side of graph).

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1207085109 Volkan et al. Downloaded by guest on September 23, 2021 thought to be involved in subunit recruitment, and the lower no affinity. Despite structural homology between PapC’s Plug affinity of CTD2 for chaperone–subunit complexes likely reflects and CTD2 domains (37), CTD2 showed no affinity for NTD its role as a docking site for the assembling pilus fiber (28). (Table 2) and no interaction was observed between the Plug and CTD2 domains. Intrausher Interactions of PapC Plug Domain, CTD2, and NTD. Crys- The interaction between the Plug and NTD may stabilize the tallographic studies of the type 1 pilus usher, FimD, in complex open, ungated conformation of the usher and help position the with its chaperone–adhesin complex FimC–FimH showed that NTD (and possibly the Plug) in a proper orientation to recruit the Plug domain had relocated to a site in close proximity to the chaperone–subunit complexes and facilitate pilus biogenesis. NTD domain of the usher, thus representing the usher’s active To investigate the hypothesis that the stable PapC NTD–Plug state (28). Because the individual domains of FimD do not ex- complex is active in recruiting chaperone–subunit complexes, we press well or are unstable, we used the Pap system to directly carried out an additional biolayer interferometry assay by in- investigate the interaction that the NTD makes with the Plug cubating Super Streptavidin pins in 50 μg/mL of biotinylated domain and/or CTD2 of the PapC usher using biolayer in- Plug, which were then washed and incubated in 24.6 μΜ NTD to terferometry assays. We were unable to test the CTD1 domain in establish a stable NTD–Plug complex. This complex was briefly this assay because this domain is unstable in isolation, likely due washed and incubated in chaperone–subunit complexes (PapDG, to its close association with the transmembrane barrel domain as PapDEntd, PapDK, PapDAKnte, PapDH) and apo-PapD to test seen in the FimDCH structure (28). its ability to recruit chaperone–subunit complexes. NTD–Plug Purified PapC Plug domain was biotinylated and immobilized complex was able to bind to all tested chaperone–subunit com- on Super Streptavidin pins (ForteBio Inc.) at 50 μg/mL, washed, plexes, even to those that have no affinity for NTD alone such as and incubated in wells containing pure PapC NTD in a dilution PapDK, PapDAKnte, PapDH, and apo-PapD (Fig. 2B). CTD2 series ranging from 0.2 to 13.2 μM (Fig. 2A). We found that the was used as a binding control as it has no affinity for apo-NTD Plug domain of PapC binds the NTD with high affinity (KD: 4.12 × or Plug domains (Fig. 2B). Binding affinities of apo-PapD and − 10 10) (Fig. 2A, Table 2), whereas a control protein (BSA) showed chaperone–subunit complexes for the NTD–Plug complex were MICROBIOLOGY

Fig. 2. PapC Plug domain mediates a high-afﬁnity, stable interaction with PapC NTD, and this complex is capable of recruiting chaperone–subunit complexes. (A)In a biolayer interferometry assay, Super Streptavidin pins incubated in 50 μg/mL of biotinylated PapC Plug domain were incubated with increasing concentrations of puriﬁed PapC NTD for 2 min (0.2, 0.4, 3.2, 6.6, and 13.2 μM) to detect NTD–Plug association. The pins with NTD–Plug complex were then moved to wells containing HBS to measure dissociation for 30 min. A concentration-dependent, stable interaction between NTD and Plug domains of PapC was observed. (B) The stable complex that forms between PapC NTD and Plug domains is active in recruiting all tested chaperone–subunit complexes and apo-PapD. Using biolayer interferometry as in A, a stable complex between NTD and Plug domain was obtained by incubating Super Streptavidin pins coated with 50 μg/mL of Plug domain in 24.6 μM NTD and then washing them. The pins coated with NTD–Plug complex were incubated in chaperone–subunit complexes for 5 min in increasing concentrations of chaperone–subunit complexes or chaperone alone (shown at 1 μM each) and then moved to wells containing HBS for measurement of dissociation rates.

Volkan et al. PNAS Early Edition | 3of6 Downloaded by guest on September 23, 2021 Table 2. Binding kinetics of intrausher interactions 2 Interaction KD kon [1/(M*s)] koff (1/s) R *

Plug–NTD 4.12 × 10−10 4.10 × 10+4 1.34 × 10−5 0.985 NTD–CTD2 No binding Plug–CTD2 No binding

*R2 is the coefﬁcient of determination estimating the goodness of a curve ﬁt reported by ForteBio Data Analysis software version 6.4.

similar to that of the Plug domain alone (Table 1). The apparent afﬁnity of the NTD–Plug complex for PapDG was two orders of magnitude lower than that for NTD alone and one order of magnitude lower than that for the Plug domain alone, suggesting that NTD in the context of a closed pore may be the initial targeting site for PapDG, thus initiating pilus formation, and that subsequent recruitment of chaperone–subunit complexes requires the NTD–Plug complex or the Plug domain alone.

CTD2 Mediates Dissociation of the NTD/Chaperone–Adhesin Complex. Fig. 3. CTD2 mediates dissociation of the PapC NTD/chaperone–adhesin The PapC NTD has been implicated as the initial targeting site for complex. Super Streptavidin pins were incubated in 50 μg/mL of biotinylated – NTD, washed, and incubated in 0.5 μM PapDG to measure the NTD–PapDG chaperone subunit complexes (26), and in the present study we – show that NTD selectively binds to the PapDG chaperone– interaction (left side of graph). The pins containing the NTD PapDG complex were moved to wells containing HBS only, HBS with 0.5 μM CTD, or 0.5 μM adhesin complex. However, the FimDCH crystal structure – – Plug domain (right side of graph). Incubation of the bound NTD PapDG showed the chaperone adhesin complex (FimCH) binding to the complex with CTD2 (blue, CTD2) causes faster dissociation of chaperone– usher’s CTDs (28). Knowing that CTD2 directly interacts with the adhesin from the NTD compared with buffer alone (red, HBS). Incubating chaperone–adhesin complex (Fig. 1), we hypothesized that CTD2 NTD–PapDG complex with the Plug domain causes the Plug domain’s asso- is capable of causing the dissociation of the initial PapDG com- ciation with the bound complex (green, Plug). plex from NTD. We set up a biolayer interferometry experiment, conjugating Super Streptavidin pins with 50 μg/mL of biotinylated PapC NTD and incubating these pins in 0.5 μM PapDG complex. – also revealed that overexpression of the CTD2, Plug, or NTD Pins containing the NTD PapDG complex were moved to either A μ resulted in decreased piliation (Fig. 4 ). Overexpression of peri- buffer only to measure an off rate or to buffer containing 0.5 M fi of CTD2 to determine its effect on dissociation of the NTD– plasmic PapC domains did not cause a signi cant change in type 1 PapDG complex. PapC Plug domain (0.5 μM) was used as pilus assembly (Fig. S2), suggesting that overexpression of these a control. We observed that when CTD2 was present the dissociation rate of the NTD–PapDG complex (fit in the ForteBio software version 6.4) increased by over an order of magnitude − − from 2.75 × 10 3 ± 1.25(1/s) to 6.77 × 10 2 ± 5.2 (1/s) compared with buffer alone, suggesting that CTD2 is involved in catalyzing the dissociation of NTD–PapDG complex (Fig. 3). Further analysis of the data by fitting (Origin 8; OriginLab) the curves to the sum of two exponentials revealed that the Hepes buffered saline (HBS) data were best fit as a double exponential with fast (0.057/s ± 0.003) and slow (0.00035/s ± 0.00014) rates, with the slow phase accounting for five times the amplitude of the fast phase, whereas the CTD2-catalyzed reaction was best fit as a single exponential with a fast rate (0.054/s ± 0.004), confirming that CTD2 accelerates the rate of dissociation by over an order of magnitude. Interestingly, however, the Plug domain of PapC associated only with the NTD–PapDG complex. Thus, the catalysis of the dissociation of the NTD–PapDG complex was specificto CTD2 (Fig. 3).

Overexpression of Periplasmic Usher Domains in Trans Inhibits Pilus Assembly. On the basis of interactions that we observed with the CTD2, Plug, and NTD within the usher and with chaperone– subunit complexes, we hypothesized that, if these interactions were physiological, overexpression of these speciﬁc usher Fig. 4. In vivo overexpression of usher periplasmic domains interferes with domains would interfere with pilus biogenesis. Therefore, the P pilus biogenesis. Electron microscopy (A) and hemagglutination assays (B) pilus operon was expressed from its own promoter in the pFJ3 reveal that overexpression of the PapC domains CTD2, NTD, and Plug in the plasmid (38) on Tryptic Soy Agar (TSA) plates, and the CTD2, periplasmic space decreases pilus biogenesis. E. coli C600 cells carrying the pFJ3 plasmid were transformed with pKDC3 (CTD), pKDC5 (Plug), and Plug, or NTD domains were expressed by isopropyl-β-D-thio-ga- pKDC16 (NTD). These strains were grown on TSA plates with IPTG for in- lactoside (IPTG) induction of plasmids pKDC3, pKDC5, or duction of P pili from the pFJ3 plasmid and expression of Plug, NTD, and pKDC16, respectively. Overexpression of any of these PapC CTD. A lawn of cells was grown and collected for electron microscopy (A)and domains interfered with pilus biogenesis as determined by hem- hemagglutination assays (HA) (B) to investigate the impact of overexpressed agglutination titers of human type A erythrocytes (Fig. 4B). Mor- PapC domains on pilus biogenesis. HA titer is the highest dilution of bacteria phologic investigation of these bacteria via electron microscopy that still provides agglutination of human type A erythrocytes.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1207085109 Volkan et al. Downloaded by guest on September 23, 2021 domains is not cytotoxic and that the effects observed are specific to the P pilus system. Overexpressed PapC domains may titrate the relevant chaperone–subunit complexes away from PapC or block aspects of usher gating and/or transfer of complexes to the CTD2 domain, thus interfering with functional pilus biogenesis and in- directly supporting our in vitro results. Discussion Chaperone–usher pilus assembly requires strict organization and reorganization of usher domains to coordinate the transfer of multiple pilin subunits from chaperone–subunit complexes to various usher domains and their subsequent movement, in a tightly regulated process, through the gated outer membrane usher into pili via a donor strand exchange mechanism. Using PapC as a prototype system, we evaluated interactions between the globular NTD, Plug, and CTD2 domains of the usher with each other and with chaperone–subunit complexes. Each of these usher domains is required for PapC function and, when expressed in excess in trans, inhibit pilus assembly. Using biolayer interferometry, we found that NTD selectively binds the PapDG and PapDEntd complexes with nanomolar and micromolar affinities, respectively, but does not bind to any of the other tested chaperone–subunit complexes (PapDK, PapDAKnte, or PapDH) or to the chaperone alone. Interestingly, CTD2 bound with an Fig. 5. Model of pilus biogenesis at the usher. The plug domain resides in affinity in the micromolar range to all of the tested chaperone– the translocation pore in the inactive usher (A). Upon chaperone–adhesin subunit complexes, except for the PapDH terminator complex, binding to the NTD, the plug domain extends to the periplasm where it – whereas the Plug domain bound to all of the tested chaperone– stably binds to the NTD (B). CTD2 mediates binding to the chaperone adhesin complex at the NTD (C) where it catalyzes dissociation of the NTD– subunit complexes, including PapDH as well as the chaperone PapDG complex and remains bound to PapDG (D). Subsequent interactions MICROBIOLOGY alone. This argues for direct involvement of the PapC Plug do- of incoming subunits with the CTDs and Plug result in pilus assembly (E–G). main in pilus biogenesis beyond being merely a pore gate. Rod and tip components interact with Plug and CTD2, but the terminator Crystallographic studies of the type 1 pilus usher FimD, in complex PapDH is directly targeted to the Plug domain without transfer to complex with its chaperone–adhesin complex FimC-FimH, the NTD or CTD2 (G). showed that the Plug domain had relocated to a site adjacent to the NTD domain of the usher in the usher’s active state (28). Using the Pap system, we elucidated an intrausher interaction shows no binding with any of the usher domains tested. We between the NTD and Plug domains. The interaction of Plug discovered that the CTD2 was capable of catalyzing the disso- with NTD may be part of a mechanism to open the PapC pore ciation of PapDG from the NTD in a competitive binding assay. upon PapDG binding. Despite the structural homology between This function of CTD2 in the dissociation of the NTD–PapDG the Plug and the CTD2 (37), NTD showed no affinity for CTD2. complex is a catalytic activity for this highly conserved domain. Similarly, no interaction between CTD2 and the Plug was ob- This function is likely the result of an allosteric mechanism be- served. Because we found that the Plug–NTD complex was able cause CTD2 binds to chaperone–subunit complexes but not to to bind apo-PapD and PapD subunit complexes, this reorga- the NTD, ruling out direct competition. Thus, PapDG is trans- nization of usher domains may serve to recruit subsequent ferred to the CTDs via allosteric destabilization by CTD2 (Fig. 5 chaperone–subunit complexes. This finding emphasizes that the C and D). This frees the NTD/Plug for the recruitment of sub- NTD–Plug complex, in addition to its likely role in retaining an sequent chaperone–subunit complexes. Allosteric handover of open PapC pore, is also of physiological importance in the es- the chaperone–adhesin complex to the CTDs is the last step of sential usher function of chaperone–subunit recruitment and, pilus initiation at the usher because, upon transfer to the CTDs, hence, in the catalysis of pilus biogenesis. Finally, we showed that the adhesin domain likely starts emerging through the usher −9 the affinity of NTD alone for PapDG (KD =10 M) is two transmembrane domain as seen in the FimDCH structure (28). orders of magnitude higher than that of the NTD–Plug complex During the assembly of the pilus rod, the NTD in its apo state is −7 (KD =10 M), suggesting that PapDG is more likely to target bypassed, and subunits are instead recruited to the Plug, or the NTD before opening of the pore and formation of an NTD– NTD–Plug complex, and CTD2 (Fig. 5 E and F). Considering its Plug complex. equal affinity for apo-PapD, the Plug domain may also be in- CTD1 is another periplasmic domain of PapC that is likely volved in catalysis by removing PapD after DSE. Interestingly, involved in docking of chaperone–subunit complexes along with Plug is the only domain capable of binding the PapDH termi- CTD2 after the complexes are competitively transferred to the nator complex. Incorporation of PapH is known to terminate CTDs from the NTD by the action of CTD2. This Ig-like domain pilus assembly due in part to the occluded P5 pocket of PapH of PapC is likely involved in extensive interactions with the lectin (19). The unique interaction of PapDH with Plug implicates the domain of PapG and the chaperone as this was shown to be the usher’s Plug domain in pilus termination and anchoring, where case for the homologous type 1 system (28). CTD1, as a sub- PapDH is directly targeted to the Plug domain or NTD–Plug domain of the transmembrane β-barrel itself, is likely also im- complex bypassing CTD2 (Fig. 5G) and resulting in a unique and portant for the overall conformation of PapC. irreversible (by DSE) interaction that terminates pilus assembly. Our results suggest a mechanism whereby PapDG is targeted Overall, our studies provide insight into how ordered assembly of to the NTD, triggering the ungating of the usher, likely via al- a prototypical chaperone–usher pilus takes place at the usher via losteric interactions that occur upon PapDG binding. PapC is coordinated participation of each of its periplasmic domains. We subsequently stabilized in an open conformation by the NTD– have defined the differential affinities of the usher domains for Plug interaction (Fig. 5 A and B). The full-length PapDG is chaperone–subunit complexes, facilitating subunit ordering. This needed for this to occur as the lectin domain of PapG alone allows efficient transfer of chaperone–subunit complexes around

Volkan et al. PNAS Early Edition | 5of6 Downloaded by guest on September 23, 2021 the usher molecular machine from NTD/Plug to CTD2 (Fig. 5). bound pins were incubated in wells containing chaperone–subunit com- These studies provide evidence on the collective, direct roles of plexes (PapDG, PapDEntd, PapDK, PapDAKnte, PapDH) or PapC NTD at con- NTD, CTD2, and Plug domains of PapC in pilus biogenesis. The centrations of 0.18, 0.5, 1.66, 2, 5, and 12.5 μMfor5–10 min or 0.2, 0.4, 3.2, results afford insight into how the usher catalyzes pilus assembly 6.6, or 13.2 μM, respectively. They were then incubated in wells containing in the absence of ATP, an important virulence event in many HBS for measurement of dissociation rates. Inverse experiments where Gram-negative pathogens, which rely on adhesive pili to pro- chaperone–subunits were biotinylated and then incubated in PapC domains mote adhesion to host tissues and subsequent infection. were also carried out. For NTD–Plug complex-binding experiments, the tips of Super Streptavidin pins were coated with 50 μg/mL of biotinylated Plug Materials and Methods domain and incubated in 24.6 μM of NTD. Once a stable interaction is – Strains and Constructs. All PapC constructs were cloned into the pTrc99a established, the pins were washed and incubated in chaperone subunit vector with PapD’s signal sequence and a 6His-tag using standard PCR and complexes, chaperone alone, or CTD2 (negative control) for 5 min followed recombinant techniques and expressed in E. coli strain BL21. Plug domain by incubation in HBS for 5 min to measure dissociation rates. was expressed from pKDC5, CTD2 was expressed from pKDC3, and NTD was expressed from pKDC16 (Table S1). E. coli strain C600, carrying appropriate PapC Plug, NTD, and CTD2 in Vivo Overexpression Studies. E. coli C600 cells plasmids, was used for HA titer and EM assays. The pFJ3 plasmid was used to were transformed with pFJ3 and pKDC5 (Plug), pKDC3 (CTD2), pKDC16 express WT pap operon under its own promoter on TSA plates (38). (NTD), or the empty vector pTrc99a. Cells were grown for 8 h in LB broth at 37 °C with shaking and plated on TSA plates containing 1 μM IPTG and the Biolayer Interferometry Assays. All interaction experiments were conducted appropriate antibiotics. A lawn of cells was grown at 37 °C overnight, col- at 30 °C in HBS (20 mM Hepes, 150 mM NaCl, pH 7.5) using an Octet Red lected, and used for HA titers (39) and negative-stain electron microscopy. instrument (ForteBio Inc.). Purified PapC Plug and NTD and CTD2 domains were dialyzed overnight in HBS and biotinylated using NHS-PEO4-biotin at fi μ ACKNOWLEDGMENTS. We gratefully acknowledge the expertise of W. Beatty a ratio of 1:1 (Thermo Scienti c). A total of 50 g/mL of CTD2, NTD, or Plug (Imaging Facility, Washington University School of Medicine). This study was fi were bound to Super Streptavidin pins (ForteBio Inc.). The puri ed chaper- funded by National Institutes of Health RO1 Grant AI029549 and AI049450 (to – one subunit complexes PapDG, PapDEntd, PapDK, PapDAKnte, PapDH, or S.J.H.). D.G.T. is supported by National Institutes of Health Grant GM62987. PapD alone were dialyzed overnight in HBS. The biotinylated PapC domain- B.A.F. is supported by National Institutes of Health K08 Grant 1K08DK093707-01.

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