Donor-strand exchange in chaperone-assisted pilus assembly proceeds through a concerted beta strand displacement mechanism

Gram-negative pathogens commonly use the chaperone-usher pathway to assemble adhesive multisubunit fibers on their surface. In the periplasm, subunits are stabilized by a chaperone that donates a beta strand to complement the subunits' truncated immunoglobulin-like fold. Pilus assembly proceeds through a "donor-strand exchange" (DSE) mechanism whereby this complementary beta strand is replaced by the N-terminal extension (Nte) of an incoming pilus subunit. Using X-ray crystallography and real-time electrospray ionization mass spectrometry (ESI-MS), we demonstrate that DSE requires the formation of a transient ternary complex between the chaperone-subunit complex and the Nte of the next subunit to be assembled. The process is crucially dependent on an initiation site (the P5 pocket) needed to recruit the incoming Nte. The data also suggest a capping reaction displacing DSE toward product formation. These results support a zip-in-zip-out mechanism for DSE and a catalytic role for the usher, the molecular platform at which pili are assembled.     

The Outer Membrane Usher Guarantees the Formation of Functional Pili by Selectively Catalyzing Donor-Strand Exchange between Subunits That Are Adjacent in the Mature Pilus

Type 1 pili from uropathogenic Escherichia coli are a prototype of adhesive surface organelles assembled and secreted by the conserved chaperone/usher pathway. They are composed of four different homologous protein subunits that need to be assembled in a defined order. In the periplasm, the pilus chaperone FimC donates a β-strand segment to the subunits to complete their imperfect immunoglobulin-like fold. During subunit assembly, this segment of the chaperone is displaced by an amino-terminal extension of an incoming subunit in a reaction termed donor-strand exchange. To date, the molecular mechanisms underlying the coordinated subunit assembly, in particular the role of the outer membrane usher FimD, are still poorly understood. Here we show that the binding of complexes between FimC and the different pilus subunits to the amino-terminal substrate recognition domain of FimD is an extremely fast process, with association rate constants in the range of 10⁷-10⁸ M^− 1 s^− 1 at 20 °C. Furthermore, we demonstrate that the ordered assembly of pilus subunits is a consequence of the usher's ability to selectively catalyze the assembly of defined subunit-subunit pairs that are adjacent in the mature pilus. The usher therefore coordinates the assembly of pilus subunits at the stage of donor-strand exchange between pairs of subunits and not at the level of the initial binding of chaperone-subunit complexes.     

Crystal structure of the P pilus rod subunit PapA

P pili are important adhesive fibres involved in kidney infection by uropathogenic Escherichia coli strains. P pili are assembled by the conserved chaperone-usher pathway, which involves the PapD chaperone and the PapC usher. During pilus assembly, subunits are incorporated into the growing fiber via the donor-strand exchange (DSE) mechanism, whereby the chaperone's G1 beta-strand that complements the incomplete immunoglobulin-fold of each subunit is displaced by the N-terminal extension (Nte) of an incoming subunit. P pili comprise a helical rod, a tip fibrillum, and an adhesin at the distal end. PapA is the rod subunit and is assembled into a superhelical right-handed structure. Here, we have solved the structure of a ternary complex of PapD bound to PapA through donor-strand complementation, itself bound to another PapA subunit through DSE. This structure provides insight into the structural basis of the DSE reaction involving this important pilus subunit. Using gel filtration chromatography and electron microscopy on a number of PapA Nte mutants, we establish that PapA differs in its mode of assembly compared with other Pap subunits, involving a much larger Nte that encompasses not only the DSE region of the Nte but also the region N-terminal to it.     

The Structure of the PapD-PapGII Pilin Complex Reveals an Open and Flexible P5 Pocket

P pili are hairlike polymeric structures that mediate binding of uropathogenic Escherichia coli to the surface of the kidney via the PapG adhesin at their tips. PapG is composed of two domains: a lectin domain at the tip of the pilus followed by a pilin domain that comprises the initial polymerizing subunit of the 1,000-plus-subunit heteropolymeric pilus fiber. Prior to assembly, periplasmic pilin domains bind to a chaperone, PapD. PapD mediates donor strand complementation, in which a beta strand of PapD temporarily completes the pilin domain''s fold, preventing premature, nonproductive interactions with other pilin subunits and facilitating subunit folding. Chaperone-subunit complexes are delivered to the outer membrane usher where donor strand exchange (DSE) replaces PapD''s donated beta strand with an amino-terminal extension on the next incoming pilin subunit. This occurs via a zip-in–zip-out mechanism that initiates at a relatively accessible hydrophobic space termed the P5 pocket on the terminally incorporated pilus subunit. Here, we solve the structure of PapD in complex with the pilin domain of isoform II of PapG (PapGIIp). Our data revealed that PapGIIp adopts an immunoglobulin fold with a missing seventh strand, complemented in parallel by the G1 PapD strand, typical of pilin subunits. Comparisons with other chaperone-pilin complexes indicated that the interactive surfaces are highly conserved. Interestingly, the PapGIIp P5 pocket was in an open conformation, which, as molecular dynamics simulations revealed, switches between an open and a closed conformation due to the flexibility of the surrounding loops. Our study reveals the structural details of the DSE mechanism.     

Mechanism of fibre assembly through the chaperone-usher pathway

The chaperone-usher pathway directs the formation of adhesive surface fibres in numerous pathogenic Gram-negative bacteria. The fibres or pili consist exclusively of protein subunits that, before assembly, form transient complexes with a chaperone in the periplasm. In these chaperone:subunit complexes, the chaperone donates one beta-strand to complete the imperfect immunoglobulin-like fold of the subunit. During pilus assembly, the chaperone is replaced by a polypeptide extension of another subunit in a process termed 'donor strand exchange' (DSE). Here we show that DSE occurs in a concerted reaction in which a chaperone-bound acceptor subunit is attacked by another chaperone-bound donor subunit. We provide evidence that efficient DSE requires interactions between the reacting subunits in addition to those involving the attacking donor strand. Our results indicate that the pilus assembly platforms in the outer membrane, referred to as ushers, catalyse fibre formation by increasing the effective concentrations of donor and acceptor subunits.     

Chaperone-subunit-usher interactions required for donor strand exchange during bacterial pilus assembly

The assembly of type 1 pili on the surface of uropathogenic Escherichia coli proceeds via the chaperone-usher pathway. Chaperone-subunit complexes interact with one another via a process termed donor strand complementation whereby the G1beta strand of the chaperone completes the immunoglobulin (Ig) fold of the pilus subunit. Chaperone-subunit complexes are targeted to the usher, which forms a channel across the outer membrane through which pilus subunits are translocated and assembled into pili via a mechanism known as donor strand exchange. This is a mechanism whereby chaperone uncapping from a subunit is coupled with the simultaneous assembly of the subunit into the pilus fiber. Thus, in the pilus fiber, the N-terminal extension of every subunit completes the Ig fold of its neighboring subunit by occupying the same site previously occupied by the chaperone. Here, we investigated details of the donor strand exchange assembly mechanism. We discovered that the information necessary for targeting the FimC-FimH complex to the usher resides mainly in the FimH protein. This interaction is an initiating event in pilus biogenesis. We discovered that the ability of an incoming subunit (in a chaperone-subunit complex) to participate in donor strand exchange with the growing pilus depended on a previously unrecognized function of the chaperone. Furthermore, the donor strand exchange assembly mechanism between subunits was found to be necessary for subunit translocation across the outer membrane usher.     

Molecular mechanism of P pilus termination in uropathogenic Escherichia coli

P pili are important adhesive fibres that are assembled by the conserved chaperone-usher pathway. During pilus assembly, the subunits are incorporated into the growing fibre by the donor-strand exchange mechanism, whereby the beta-strand of the chaperone, which complements the incomplete immunoglobulin fold of each subunit, is displaced by the amino-terminal extension of an incoming subunit in a zip-in-zip-out exchange process that is initiated at the P5 pocket, an exposed hydrophobic pocket in the groove of the subunit. In vivo, termination of P pilus growth requires a specialized subunit, PapH. Here, we show that PapH is incorporated at the base of the growing pilus, where it is unable to undergo donor-strand exchange. This inability is not due to a stronger PapD-PapH interaction, but to a lack of a P5 initiator pocket in the PapH structure, suggesting that PapH terminates pilus growth because it is lacking the initiation point by which donor-strand exchange proceeds.     

Structure, folding and stability of FimA, the main structural subunit of type 1 pili from uropathogenic Escherichia coli strains

Filamentous type 1 pili are responsible for attachment of uropathogenic Escherichia coli strains to host cells. They consist of a linear tip fibrillum and a helical rod formed by up to 3000 copies of the main structural pilus subunit FimA. The subunits in the pilus interact via donor strand complementation, where the incomplete, immunoglobulin-like fold of each subunit is complemented by an N-terminal donor strand of the subsequent subunit. Here, we show that folding of FimA occurs at an extremely slow rate (half-life: 1.6 h) and is catalyzed more than 400-fold by the pilus chaperone FimC. Moreover, FimA is capable of intramolecular self-complementation via its own donor strand, as evidenced by the loss of folding competence upon donor strand deletion. Folded FimA is an assembly-incompetent monomer of low thermodynamic stability (− 10.1 kJ mol^− 1) that can be rescued for pilus assembly at 37 °C because FimC selectively pulls the fraction of unfolded FimA molecules from the FimA folding equilibrium and allows FimA refolding on its surface. Elongation of FimA at the C-terminus by its own donor strand generated a self-complemented variant (FimAa) with alternative folding possibilities that spontaneously adopts the more stable conformation (− 85.0 kJ mol^− 1) in which the C-terminal donor strand is inserted in the opposite orientation relative to that in FimA. The solved NMR structure of FimAa revealed extensive β-sheet hydrogen bonding between the FimA pilin domain and the C-terminal donor strand and provides the basis for reconstruction of an atomic model of the pilus rod.     

Chaperone priming of pilus subunits facilitates a topological transition that drives fiber formation

Periplasmic chaperones direct the assembly of adhesive, multi-subunit pilus fibers that play critical roles in bacterial pathogenesis. Pilus assembly occurs via a donor strand exchange mechanism in which the N-terminal extension of one subunit replaces the chaperone G(1) strand that transiently occupies a groove in the neighboring subunit. Here, we show that the chaperone primes the subunit for assembly by holding the groove in an open, activated conformation. During donor strand exchange, the subunit undergoes a topological transition that triggers the closure of the groove and seals the N-terminal extension in place. It is this topological transition, made possible only by the priming action of the chaperone that drives subunit assembly into the fiber.     

Structure of the full-length major pilin from Streptococcus pneumoniae: implications for isopeptide bond formation in gram-positive bacterial pili

The surface of the pneumococcal cell is adorned with virulence factors including pili. The major pilin RrgB, which forms the pilus shaft on pathogenic Streptococcus pneumoniae, comprises four immunoglobulin (Ig)-like domains, each with a common CnaB topology. The three C-terminal domains are each stabilized by internal Lys-Asn isopeptide bonds, formed autocatalytically with the aid of an essential Glu residue. The structure and orientation of the crucial N-terminal domain, which provides the covalent linkage to the next pilin subunit in the shaft, however, remain incompletely characterised. We report the crystal structure of full length RrgB, solved by X-ray crystallography at 2.8 Å resolution. The N-terminal (D1) domain makes few contacts with the rest of the RrgB structure, and has higher B-factors. This may explain why D1 is readily lost by proteolysis, as are the N-terminal domains of many major pilins. D1 is also found to have a triad of Lys, Asn and Glu residues in the same topological positions as in the other domains, yet mass spectrometry and the crystal structure show that no internal isopeptide bond is formed. We show that this is because β-strand G of D1, which carries the Asn residue, diverges from β-strand A, carrying the Lys residue, such that these residues are too far apart for bond formation. Strand G also carries the YPKN motif that provides the essential Lys residue for the sortase-mediated intermolecular linkages along the pilus shaft. Interaction with the sortase and formation of the intermolecular linkage could result in a change in the orientation of this strand, explaining why isopeptide bond formation in the N-terminal domains of some major pilins appears to take place only upon assembly of the pili.     

Large is fast, small is tight: determinants of speed and affinity in subunit capture by a periplasmic chaperone

The chaperone/usher pathway assembles surface virulence organelles of Gram-negative bacteria, consisting of fibers of linearly polymerized protein subunits. Fiber subunits are connected through 'donor strand complementation': each subunit completes the immunoglobulin (Ig)-like fold of the neighboring subunit by donating the seventh β-strand in trans. Whereas the folding of Ig domains is a fast first-order process, folding of Ig modules into the fiber conformation is a slow second-order process. Periplasmic chaperones separate this process in two parts by forming transient complexes with subunits. Interactions between chaperones and subunits are also based on the principle of donor strand complementation. In this study, we have performed mutagenesis of the binding motifs of the Caf1M chaperone and Caf1 capsular subunit from Yersinia pestis and analyzed the effect of the mutations on the structure, stability, and kinetics of Caf1M-Caf1 and Caf1-Caf1 interactions. The results suggest that a large hydrophobic effect combined with extensive main-chain hydrogen bonding enables Caf1M to rapidly bind an early folding intermediate of Caf1 and direct its partial folding. The switch from the Caf1M-Caf1 contact to the less hydrophobic, but considerably tighter and less dynamic Caf1-Caf1 contact occurs via the zip-out-zip-in donor strand exchange pathway with pocket 5 acting as the initiation site. Based on these findings, Caf1M was engineered to bind Caf1 faster, tighter, or both faster and tighter. To our knowledge, this is the first successful attempt to rationally design an assembly chaperone with improved chaperone function.     

The Structure of Aβ42 C-Terminal Fragments Probed by a Combined Experimental and Theoretical Study

The C-terminus of amyloid β-protein (Aβ) 42 plays an important role in this protein's oligomerization and may therefore be a good therapeutic target for the treatment of Alzheimer's disease. Certain C-terminal fragments (CTFs) of Aβ42 have been shown to disrupt oligomerization and to strongly inhibit Aβ42-induced neurotoxicity. Here we study the structures of selected CTFs [Aβ(x-42); x = 29-31, 39] using replica exchange molecular dynamics simulations and ion mobility mass spectrometry. Our simulations in explicit solvent reveal that the CTFs adopt a metastable β-structure: β-hairpin for Aβ(x-42) (x = 29-31) and extended β-strand for Aβ(39-42). The β-hairpin of Aβ(30-42) is converted into a turn-coil conformation when the last two hydrophobic residues are removed, suggesting that I41 and A42 are critical in stabilizing the β-hairpin in Aβ42-derived CTFs. The importance of solvent in determining the structure of the CTFs is further highlighted in ion mobility mass spectrometry experiments and solvent-free replica exchange molecular dynamics simulations. A comparison between structures with solvent and structures without solvent reveals that hydrophobic interactions are critical for the formation of β-hairpin. The possible role played by the CTFs in disrupting oligomerization is discussed.     

Intramolecular Donor Strand Complementation in the E. coli Type 1 Pilus Subunit FimA Explains the Existence of FimA Monomers As Off-Pathway Products of Pilus Assembly That Inhibit Host Cell Apoptosis

Type 1 pili are filamentous organelles mediating the attachment of uropathogenic Escherichia coli to epithelial cells of host organisms. The helical pilus rod consists of up to 3000 copies of the main structural subunit FimA that interact via donor strand complementation, where the incomplete Ig-like fold of FimA is completed by insertion of the N-terminal extension (donor strand) of the following FimA subunit. Recently, it was shown that FimA also exists in a monomeric, assembly-incompetent form and that FimA monomers act as inhibitors of apoptosis in infected host cells. Here we present the NMR structure of monomeric wild-type FimA with its natural N-terminal donor strand complementing the Ig fold. Compared to FimA subunits in the assembled pilus, intramolecular self-complementation in the monomer stabilizes the FimA fold with significantly less interactions, and the natural FimA donor strand is inserted in the opposite orientation. In addition, we show that a motif of two glycine residues in the FimA donor strand, separated by five residues, is the prerequisite of the alternative, parallel donor strand insertion mechanism in the FimA monomer and that this motif is preserved in FimA homologs of many enteroinvasive pathogens. We conclude that FimA is a unique case of a protein with alternative, functionally relevant folding possibilities, with the FimA polymer forming the highly stable pilus rod and the FimA monomer promoting pathogen propagation by apoptosis suppression of infected epithelial target cells.     

A hydrophilic microenvironment in the substrate-translocating groove of the YidC membrane insertase is essential for enzyme function

The YidC family of proteins are membrane insertases that catalyze the translocation of the periplasmic domain of membrane proteins via a hydrophilic groove located within the inner leaflet of the membrane. All homologs have a strictly conserved, positively charged residue in the center of this groove. In Bacillus subtilis, the positively charged residue has been proposed to be essential for interacting with negatively charged residues of the substrate, supporting a hypothesis that YidC catalyzes insertion via an early-step electrostatic attraction mechanism. Here, we provide data suggesting that the positively charged residue is important not for its charge but for increasing the hydrophilicity of the groove. We found that the positively charged residue is dispensable for Escherichia coli YidC function when an adjacent residue at position 517 was hydrophilic or aromatic, but was essential when the adjacent residue was apolar. Additionally, solvent accessibility studies support the idea that the conserved positively charged residue functions to keep the top and middle of the groove sufficiently hydrated. Moreover, we demonstrate that both the E. coli and Streptococcus mutans YidC homologs are functional when the strictly conserved arginine is replaced with a negatively charged residue, provided proper stabilization from neighboring residues. These combined results show that the positively charged residue functions to maintain a hydrophilic microenvironment in the groove necessary for the insertase activity, rather than to form electrostatic interactions with the substrates.     

Interactive surface in the PapD chaperone cleft is conserved in pilus chaperone superfamily and essential in subunit recognition and assembly.

The assembly of adhesive pili in Gram-negative bacteria is modulated by specialized periplasmic chaperone systems. PapD is the prototype member of the superfamily of periplasmic pilus chaperones. Previously, the alignment of chaperone sequences superimposed on the three dimensional structure of PapD revealed the presence of invariant, conserved and variable amino acids. Representative residues that protruded into the PapD cleft were targeted for site directed mutagenesis to investigate the pilus protein binding site of the chaperone. The ability of PapD to bind to fiber-forming pilus subunit proteins to prevent their participation in misassembly interactions depended on the invariant, solvent-exposed arginine-8 (R8) cleft residue. This residue was also essential for the interaction between PapD and a minor pilus adaptor protein. A mutation in the conserved methionine-172 (M172) cleft residue abolished PapD function when this mutant protein was expressed below a critical threshold level. In contrast, radical changes in the variable residue glutamic acid-167 (E167) had little or no effect on PapD function. These studies provide the first molecular details of how a periplasmic pilus chaperone binds to nascently translocated pilus subunits to guide their assembly into adhesive pili.     

A molecular dynamics study of pilus subunits: insights into pilus biogenesis

Biogenesis of pili in the uropathogenic Echerichia coli, essential to the bacterial pathogenicity, is a complex molecular process, which involves several protein components of the Pap gene cluster. A crucial role in the process is played by the chaperone PapD and by the PapE pilus subunit. Interestingly, PapE exhibits an Ig-like fold with a missing strand. The missing G strand is donated by the chaperone during pilin folding and by adjacent pilus subunits in the final fibre. In order to obtain a detailed picture at atomic level of the molecular events related to this process, we undertook molecular dynamics studies of the non-canonical immuno-globulin-like PapE in its unliganded state. These analyses were extended to the complexes of PapE with the complementary G(1) strand of PapD and with the N-terminal extension of PapK. All three systems investigated were stable in the time interval considered (20 ns). However, significant differences in their local and overall flexibilities were detected. Notably, the equilibrated structure of unliganded PapE, which is difficult to characterise experimentally, displays unexpected features. Indeed, a significant rearrangement of the local structure of the groove, which hosts the complementary strands, is observed. This reorganisation, characterised by the formation of several new hydrogen bonds, leads to a closure of the groove that likely makes pilin polymerisation more difficult. These data suggest that chaperone release and pilin-pilin association must be concerted processes and that chaperone plays an important role in preventing pilin transitions towards states that are not prone to polymerise.     

PapD-like chaperones and pilus biogenesis

The assembly of adhesive pili from individual subunits by periplasmic PapD-like chaperones in Gram-negative bacteria offers insight into the complex process of organelle biogenesis. PapD-like chaperones bind, stabilize, and cap interactive surfaces of subunits until they are assembled into the pilus. Subunits lack the seventh *gb-strand necessary to complete their immunoglobulin-like folds; the chaperone supplies this missing strand. Indeed, the chaperone may act as a template, providing steric information to facilitate subunit folding. In the mature pilus, each subunit is thought to supply the missing strand to complete the fold of its neighbor. Thus, one general function of chaperones in organelle biogenesis may be to cap highly interactive surfaces of subunits until they reach the proper assembly site.     

Structural analysis of the Saf pilus by electron microscopy and image processing

Bacterial pili are important virulence factors involved in host cell attachment and/or biofilm formation, key steps in establishing and maintaining successful infection. Here we studied Salmonella atypical fimbriae (or Saf pili), formed by the conserved chaperone/usher pathway. In contrast to the well-established quaternary structure of typical/FGS-chaperone assembled, rod-shaped, chaperone/usher pili, little is known about the supramolecular organisation in atypical/FGL-chaperone assembled fimbriae. In our study, we have used negative stain electron microscopy and single-particle image analysis to determine the three-dimensional structure of the Salmonella typhimurium Saf pilus. Our results show atypical/FGL-chaperone assembled fimbriae are composed of highly flexible linear multi-subunit fibres that are formed by globular subunits connected to each other by short links giving a “beads on a string”-like appearance. Quantitative fitting of the atomic structure of the SafA pilus subunit into the electron density maps, in combination with linker modelling and energy minimisation, has enabled analysis of subunit arrangement and intersubunit interactions in the Saf pilus. Short intersubunit linker regions provide the molecular basis for flexibility of the Saf pilus by acting as molecular hinges allowing a large range of movement between consecutive subunits in the fibre.     

Probing conserved surfaces on PapD

PapD is the periplasmic chaperone required for the assembly of P pili in pyelonephritic strains of Escherichia coli. It consists of two immunoglobulin-like domains bisected by a subunit binding cleft. PapD is the prototype member of a super family of immunoglobulin-like chaperones that work in concert with their respective ushers to assemble a plethora of adhesive organelles including pilus- and non-pilus-associated adhesins. Three highly conserved residue clusters have been shown to play critical roles in the structure and function of PapD, as determined by site-directed mutagenesis. The in vivo stability of the chaperone depended on the formation of a buried salt bridge within the cleft. Residues along the G1 beta strand were required for efficient binding of subunits consistent with the crystal structure of PapD-peptide complexes. Finally, Thr-53, a residue that is part of a conserved band of residues located on the amino-terminal domain surface opposite the subunit binding cleft, was also found to be critical for pilus assembly, but mutations at Thr-53 did not interfere with chaperone-subunit complex formation.