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.     

Adaptor function of PapF depends on donor strand exchange in P-pilus biogenesis of Escherichia coli

P-pilus biogenesis occurs via the highly conserved chaperone-usher pathway and involves the strict coordination of multiple subunit proteins. All nonadhesin structural P-pilus subunits possess the same topology, consisting of two domains: an incomplete immunoglobulin-like fold (pilin body) and an N-terminal extension. Pilus subunits form interactions with one another through donor strand exchange, occurring at the usher, in which the N-terminal extension of an incoming subunit completes the pilin body of the preceding subunit, allowing the incorporation of the subunit into the pilus fiber. In this study, pilus subunits in which the N-terminal extension was either deleted or swapped with that of another subunit were used to examine the role of each domain of PapF in functions involving donor strand exchange and hierarchical assembly. We found that the N-terminal extension of PapF is required to adapt the PapG adhesin to the tip of the fiber. The pilin body of PapF is required to efficiently initiate assembly of the remainder of the pilus, with the assistance of the N-terminal extension. Thus, distinct functions were assigned to each region of the PapF subunit. In conclusion, all pilin subunits possess the same overall architectural topology; however, each N-terminal extension and pilin body has specific functions in pilus biogenesis.     

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.     

A receptor-binding site as revealed by the crystal structure of CfaE, the colonization factor antigen I fimbrial adhesin of enterotoxigenic Escherichia coli

CfaE is the minor, tip-localized adhesive subunit of colonization factor antigen I fimbriae (CFA/I) of enterotoxigenic Escherichia coli and is thought to be essential for the attachment of enterotoxigenic E. coli to the human small intestine early in diarrhea pathogenesis. The crystal structure of an in cis donor strand complemented CfaE was determined, providing the first atomic view of a fimbrial subunit assembled by the alternate chaperone pathway. The in cis donor strand complemented variant of CfaE structure consists of an N-terminal adhesin domain and a C-terminal pilin domain of similar size, each featuring a variable immunoglobulin-like fold. Extensive interactions exist between the two domains and appear to rigidify the molecule. The upper surface of the adhesin domain distal to the pilin domain reveals a depression consisting of conserved residues including Arg(181), previously shown to be necessary for erythrocyte adhesion. Mutational analysis revealed a cluster of conserved, positively charged residues that are required for CFA/I-mediated hemagglutination, implicating this as the receptor-binding pocket. Mutations in a few subclass-specific residues that surround the cluster displayed differential effects on the two red cell species used in hemagglutination, suggesting that these residues play a role in host or cell specificity. The C-terminal donor strand derived from the major subunit CfaB is folded as a beta-strand and fits into a hydrophobic groove in the pilin domain to complete the immunoglobulin fold. The location of this well ordered donor strand suggests the positioning and orientation of the subjacent major fimbrial subunit CfaB in the native assembly of CFA/I fimbriae.     

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.     

Donor-strand exchange in chaperone-assisted pilus assembly revealed in atomic detail by molecular dynamics

Adhesive multi-subunit fibres are assembled on the surface of many pathogenic bacteria via the chaperone-usher pathway. In the periplasm, a chaperone donates a β-strand to a pilus subunit to complement its incomplete immunoglobulin-like fold. At the outer membrane, this is replaced with a β-strand formed from the N-terminal extension (Nte) of an incoming pilus subunit by a donor-strand exchange (DSE) mechanism. This reaction has previously been shown to proceed via a concerted mechanism, in which the Nte interacts with the chaperone:subunit complex before the chaperone has been displaced, forming a ternary intermediate. Thereafter, the pilus and chaperone β-strands have been postulated to undergo a strand swap by a ‘zip-in-zip-out’ mechanism, whereby the chaperone strand zips out, residue by residue, as the Nte simultaneously zips in, although direct experimental evidence for a zippering mechanism is still lacking. Here, molecular dynamics simulations have been used to probe the DSE mechanism during formation of the Saf pilus from Salmonella enterica at the atomic level, allowing the direct investigation of the zip-in-zip-out hypothesis. The simulations provide an explanation of how the incoming Nte is able to dock and initiate DSE due to inherent dynamic fluctuations within the chaperone:subunit complex. In the simulations, the chaperone donor strand was seen to unbind from the pilus subunit, residue by residue, in direct support of the zip-in-zip-out hypothesis. In addition, an interaction of a residue towards the N-terminus of the Nte with a specific binding pocket (P*) on the adjacent pilus subunit was seen to stabilise the DSE product against unbinding, which also proceeded in the simulations by a zippering mechanism. Together, the study provides an in-depth picture of DSE, including the first atomistic insights into the molecular events occurring during the zip-in-zip-out mechanism.     

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.     

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.     

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.     

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.     

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.     

Donor strand complementation governs intersubunit interaction of fimbriae of the alternate chaperone pathway

Fimbrial filaments assembled by distinct chaperone pathways share a common mechanism of intersubunit interaction, as elucidated for colonization factor antigen I (CFA/I), archetype of enterotoxigenic Escherichia coli (ETEC) Class 5 fimbriae. We postulated that a highly conserved beta-strand at the major subunit N-terminus represents the donor strand, analogous to interactions within Class I pili. We show here that CFA/I fimbriae utilize donor strand complementation to promote proper folding of and interactions between CFA/I subunits. We constructed a series of genetic variants of CfaE, the CFA/I adhesin, incorporating a C-terminal extension comprising a flexible linker and 10-19 of the N-terminal residues of CfaB, the major subunit. Variants with a donor strand complement (dsc) of >or= 12 residues were recoverable from periplasmic fractions. Genetic disruption of the donor beta-strand reduced CfaE recovery. A hexahistidine-tagged variant of dsc19CfaE formed soluble monomers, folded into beta-sheet conformation, displayed adhesion characteristic of CFA/I, and elicited antibodies that inhibited mannose-resistant haemagglutination by ETEC expressing CFA/I, CS4 and CS14 fimbriae. Immunoelectron microscopy indicated that CfaE was confined to the distal fimbrial tip. Our findings provide the basis to elucidate structure and function of this class of fimbrial adhesins and assess the feasibility of an adhesin-based vaccine.     

Off‐pathway assembly of fimbria subunits is prevented by chaperone CfaA of CFA/I fimbriae from enterotoxigenic E. coli

The assembly of the class 5 colonization factor antigen I (CFA/I) fimbriae of enterotoxigenic E. coli was proposed to proceed via the alternate chaperone‐usher pathway. Here, we show that in the absence of the chaperone CfaA, CfaB, the major pilin subunit of CFA/I fimbriae, is able to spontaneously refold and polymerize into cyclic trimers. CfaA kinetically traps CfaB to form a metastable complex that can be stabilized by mutations. Crystal structure of the stabilized complex reveals distinctive interactions provided by CfaA to trap CfaB in an assembly competent state through donor‐strand complementation (DSC) and cleft‐mediated anchorage. Mutagenesis indicated that DSC controls the stability of the chaperone‐subunit complex and the cleft‐mediated anchorage of the subunit C‐terminus additionally assist in subunit refolding. Surprisingly, over‐stabilization of the chaperone‐subunit complex led to delayed fimbria assembly, whereas destabilizing the complex resulted in no fimbriation. Thus, CfaA acts predominantly as a kinetic trap by stabilizing subunit to avoid its off‐pathway self‐polymerization that results in energetically favorable trimers and could serve as a driving force for CFA/I pilus assembly, representing an energetic landscape unique to class 5 fimbria assembly.     

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.     

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.     

Infinite kinetic stability against dissociation of supramolecular protein complexes through donor strand complementation

Adhesive type 1 pili from uropathogenic Escherichia coli strains are heat and denaturant resistant, filamentous protein complexes. Individual pilus subunits associate through "donor strand complementation," whereby the incomplete immunoglobulin-like fold of each subunit is completed by the N-terminal extension of a neighboring subunit. We show that antiparallel donor strand insertion generally causes nonequilibrium behavior in protein folding and extreme activation energy barriers for dissociation of subunit-subunit complexes. We identify the most kinetically stable, noncovalent protein complex known to date. The complex between the pilus subunit FimG and the donor strand peptide of the subunit FimF shows an extrapolated dissociation half-life of 3 x 10(9) years. The 15 residue peptide forms ideal intermolecular beta sheet H-bonds with FimG over 10 residues, and its hydrophobic side chains strongly interact with the hydrophobic core of FimG. The results show that kinetic stability and nonequilibrium behavior in protein folding confers infinite stability against dissociation in extracellular protein complexes.     

Structural and functional roles of deamidation and/or truncation of N- or C-termini in human alpha A-crystallin

The purpose of the study was to compare the effects of deamidation alone, truncation alone, or both truncation and deamidation on structural and functional properties of human lens alphaA-crystallin. Specifically, the study investigated whether deamidation of one or two sites in alphaA-crystallin (i.e., alphaA-N101D, alphaA-N123D, alphaA-N101/123D) and/or truncation of the N-terminal domain (residues 1-63) or C-terminal extension (residues 140-173) affected the structural and functional properties relative to wild-type (WT) alphaA. Human WT-alphaA and human deamidated alphaA (alphaA-N101D, alphaA-N123D, alphaA-N101/123D) were used as templates to generate the following eight N-terminal domain (residues 1-63) deleted or C-terminal extension (residues 140-173) deleted alphaA mutants and deamidated plus N-terminal domain or C-terminal extension deleted mutants: (i) alphaA-NT (NT, N-terminal domain deleted), (ii) alphaA-N101D-NT, (iii) alphaA-N123D-NT, (iv) alphaA-N101/123D-NT, (v) alphaA-CT (CT, C-terminal extension deleted), (vi) alphaA-N101D-CT, (vii) alphaA-N123D-CT, and (viii) alphaA-N101/123D-CT. All of the proteins were purified and their structural and functional (chaperone activity) properties determined. The desired deletions in the alphaA-crystallin mutants were confirmed by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometric analysis. Relative to WT-alphaA homomers, the mutant proteins exhibited major structural and functional changes. The maximum decrease in chaperone activity in homomers occurred on deamidation of N123 residue, but it was substantially restored after N- or C-terminal truncations in this mutant protein. Far-UV circular dichroism (CD) spectral analyses generally showed an increase in the beta-contents in alphaA mutants with deletions of N-terminal domain or C-terminal extension and also with deamidation plus above N- or C-terminal deletions. Intrinsic tryptophan (Trp) and total fluorescence spectral studies suggested altered microenvironments in the alphaA mutant proteins. Similarly, the ANS (8-anilino-1-naphthalenesulfate) binding showed generally increased fluorescence with blue shift on deletion of the N-terminal domain in the deamidated mutant proteins, but opposite effects were observed on deletion of the C-terminal extension. Molecular mass, polydispersity of homomers, and the rate of subunit exchange with WT-alphaB-crystallin increased on deletion of the C-terminal extension in the deamidated alphaA mutants, but on N-terminal domain deletion these values showed variable results based on the deamidation site. In summary, the data suggested that the deamidation alone showed greater effect on chaperone activity than the deletion of N-terminal domain or C-terminal extension of alphaA-crystallin. The N123 residue of alphaA-crystallin plays a crucial role in maintaining its chaperone function. However, both the N-terminal domain and C-terminal extension are also important for the chaperone activity of alphaA-crystallin because the activity was partially or fully recovered following either deletion in the alphaA-N123D mutant. The results of subunit exchange rates among alphaA mutants and WT-alphaB suggested that such exchange is an important determinant in maintenance of chaperone activity following deamidation and/or deletion of the N-terminal domain or C-terminal extension in alphaA-crystallin.     

Structural Insight into Archaic and Alternative Chaperone-Usher Pathways Reveals a Novel Mechanism of Pilus Biogenesis

Gram-negative pathogens express fibrous adhesive organelles that mediate targeting to sites of infection. The major class of these organelles is assembled via the classical, alternative and archaic chaperone-usher pathways. Although non-classical systems share a wider phylogenetic distribution and are associated with a range of diseases, little is known about their assembly mechanisms. Here we report atomic-resolution insight into the structure and biogenesis of Acinetobacter baumannii Csu and Escherichia coli ECP biofilm-mediating pili. We show that the two non-classical systems are structurally related, but their assembly mechanism is strikingly different from the classical assembly pathway. Non-classical chaperones, unlike their classical counterparts, maintain subunits in a substantially disordered conformational state, akin to a molten globule. This is achieved by a unique binding mechanism involving the register-shifted donor strand complementation and a different subunit carboxylate anchor. The subunit lacks the classical pre-folded initiation site for donor strand exchange, suggesting that recognition of its exposed hydrophobic core starts the assembly process and provides fresh inspiration for the design of inhibitors targeting chaperone-usher systems.     

A novel self-capping mechanism controls aggregation of periplasmic chaperone Caf1M

The chaperone Caf1M belongs to a family of ATP-independent periplasmic chaperones that together with outer membrane ushers assemble and secrete filamentous adhesion organelles in Gram-negative pathogens. It assists in folding and transport of Caf1 subunits of the F1 capsular antigen of Yersinia pestis, the microbe causing bubonic plague. In the periplasm, Caf1M prevents subunit aggregation by capping the extensive hydrophobic surface of activated Caf1. We found that subunit-free Caf1M exists predominantly as a tetramer [K(d) = (2-30) x 10(-14) M(3) in the 12-37 degrees C interval]. A 2.9 A resolution crystal structure of the Caf1M tetramer reveals that each of the four molecules contribute its subunit binding sequences (the A(1) and G(1) strands) to form an eight-stranded hetero-sandwich with a well-packed phenylalanine-rich hydrophobic core. Tetramerization protects chaperone molecules against enzymatic proteolysis. Deletions in the subunit binding motifs completely abolish tetramer assembly, suggesting that the hetero-sandwich is the main structural feature holding the tetramer together. Arresting tetramer assembly by a deletion of the N-terminal binding motif, while leaving the major subunit binding motif VGVFVQFAI (G(1) strand) intact, results in accumulation of unspecific aggregates. Deletions in the VGVFVQFAI motif abolish both tetramer assembly and aggregation, consistent with the predicted high beta-aggregation propensity for this motif. These results suggest that the packing of the aggregation-prone subunit binding sequences into the hetero-domain is a novel molecular mechanism preventing unspecific aggregation of the free chaperone.