An extended hydrophobic interactive surface of Yersinia pestis Caf1M chaperone is essential for subunit binding and F1 capsule assembly

A single polypeptide subunit, Caf1, polymerizes to form a dense, poorly defined structure (F1 capsule) on the surface of Yersinia pestis. The caf-encoded assembly components belong to the chaperone-usher protein family involved in the assembly of composite adhesive pili, but the Caf1M chaperone itself belongs to a distinct subfamily. One unique feature of this subfamily is the possession of a long, variable sequence between the F1 beta-strand and the G1 subunit binding beta-strand (FGL; F1 beta-strand to G1 beta-strand long). Deletion and insertion mutations confirmed that the FGL sequence was not essential for folding of the protein but was absolutely essential for function. Site-specific mutagenesis of individual residues identified Val-126, in particular, together with Val-128 as critical residues for the formation of a stable subunit-chaperone complex and the promotion of surface assembly. Differential effects on periplasmic polymerization of the subunit were also observed with different mutants. Together with the G1 strand, the FGL sequence has the potential to form an interactive surface of five alternating hydrophobic residues on Caf1M chaperone as well as in seven of the 10 other members of the FGL subfamily. Mutation of the absolutely conserved Arg-20 to Ser led to drastic reduction in Caf1 binding and surface assembled polymer. Thus, although Caf1M-Caf1 subunit binding almost certainly involves the basic principle of donor strand complementation elucidated for the PapD-PapK complex, a key feature unique to the chaperones of this subfamily would appear to be capping via high-affinity binding of an extended hydrophobic surface on the respective single subunits.     

Modelling of steric structure of a periplasmic molecular chaperone Caf1M of Yersinia pestis, a prototype member of a subfamily with characteristic structural and functional features

Abstract Steric structure of Caf1M, a periplasmic molecular chaperone of Yersinia pestis , was reconstructed by computer modelling based on a statistically significant primary structure homology between Caf1M and PapD protein from Escherichia coli , and using the known atomic coordinates obtained by the X-ray crystallography for PapD. In the three-dimensional model of Caf1M an accessory sequence between F1 and G1 β-strands (as compared to PapD) can form a strain-specific part of the binding pocket of surface organell subunits. This accessory sequence decreases the depth of the binding pocket. The characteristic structural feature of the subfamily of periplasmic molecular chaperones with the accessory sequence (Caf1M subfamily) is the existence of exposed to a solvent Cys residues in F1 and G1 β-strands which can form disulfide bond in the putative binding pocket. The characteristic functional feature of Caf1M subfamily is the chaperoning of more simple compositions of virulence-associated surface organells (in the case of Y. pestis a capsule consists of only F1 protein). Highly conserved R₈₂ and D₉₃, located at the domain surface remote from the putative subunit binding pocket, can participate in direct contacts with the conserved portion of molecular usher proteins.     

Donor strand complementation mechanism in the biogenesis of non-pilus systems

The F1 antigen of Yersinia pestis belongs to a class of non-pilus adhesins assembled via a classical chaperone-usher pathway. Such pathways consist of PapD-like chaperones that bind subunits and pilot them to the outer membrane usher, where they are assembled into surface structures. In a recombinant Escherichia coli model system, chaperone-subunit (Caf1M:Caf1n) complexes accumulate in the periplasm. Three independent methods showed that these complexes are rod- or coil-shaped linear arrays of Caf1 subunits capped at one end by a single copy of Caf1M chaperone. Deletion and point mutagenesis identified an N-terminal donor strand region of Caf1 that was essential for polymerization in vitro, in the periplasm and at the cell surface, but not for chaperone-subunit interaction. Partial protease digestion of periplasmic complexes revealed that this region becomes buried upon formation of Caf1:Caf1 contacts. These results show that, despite the capsule-like appearance of F1 antigen, the basic structure is assembled as a linear array of subunits held together by intersubunit donor strand complementation. This example shows that strikingly different architectures can be achieved by the same general principle of donor strand complementation and suggests that a similar basic polymer organization will be shared by all surface structures assembled by classical chaperone-usher pathways.     

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.     

Conserved Hydrophobic Clusters on the Surface of the Caf1A Usher C-Terminal Domain Are Important for F1 Antigen Assembly

The outer membrane usher protein Caf1A of the plague pathogen Yersinia pestis is responsible for the assembly of a major surface antigen, the F1 capsule. The F1 capsule is mainly formed by thin linear polymers of Caf1 (capsular antigen fraction 1) protein subunits. The Caf1A usher promotes polymerization of subunits and secretion of growing polymers to the cell surface. The usher monomer (811 aa, 90.5 kDa) consists of a large transmembrane β-barrel that forms a secretion channel and three soluble domains. The periplasmic N-terminal domain binds chaperone-subunit complexes supplying new subunits for the growing fiber. The middle domain, which is structurally similar to Caf1 and other fimbrial subunits, serves as a plug that regulates the permeability of the usher. Here we describe the identification, characterization, and crystal structure of the Caf1A usher C-terminal domain (Caf1A_C). Caf1A_C is shown to be a periplasmic domain with a seven-stranded β-barrel fold. Analysis of C-terminal truncation mutants of Caf1A demonstrated that the presence of Caf1A_C is crucial for the function of the usher in vivo, but that it is not required for the initial binding of chaperone-subunit complexes to the usher. Two clusters of conserved hydrophobic residues on the surface of Caf1A_C were found to be essential for the efficient assembly of surface polymers. These clusters are conserved between the FGL family and the FGS family of chaperone-usher systems.     

Periplasmic chaperone recognition motif of subunits mediates quaternary interactions in the pilus.

The class of proteins collectively known as periplasmic immunoglobulin-like chaperones play an essential role in the assembly of a diverse set of adhesive organelles used by pathogenic strains of Gram-negative bacteria. Herein, we present a combination of genetic and structural data that sheds new light on chaperone-subunit and subunit-subunit interactions in the prototypical P pilus system, and provides new insights into how PapD controls pilus biogenesis. New crystallographic data of PapD with the C-terminal fragment of a subunit suggest a mechanism for how periplasmic chaperones mediate the extraction of pilus subunits from the inner membrane, a prerequisite step for subunit folding. In addition, the conserved N- and C-terminal regions of pilus subunits are shown to participate in the quaternary interactions of the mature pilus following their uncapping by the chaperone. By coupling the folding of subunit proteins to the capping of their nascent assembly surfaces, periplasmic chaperones are thereby able to protect pilus subunits from premature oligomerization until their delivery to the outer membrane assembly site.     

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.     

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 N-terminal -barrel domain of the Escherichia coli K88 periplasmic chaperone FaeE determines fimbrial subunit recognition and dimerization

The K88 periplasmic chaperone FaeE is a homodimer, whereas the K99 chaperone FanE is a monomer. The structural requirements for dimerization of the K88 fimbrial periplasmic chaperone and for fimbrial subunit-binding specificity were investigated by analysis of mutant chaperones. FaeE contains a C-terminal extension of 19 amino acid residues when compared to FanE and most other fimbrial chaperones. A C-terminal truncate of the K88 chaperone FaeE was constructed that lacked 19 C-terminal amino acid residues. Expression and complementation experiments revealed that this C-terminal shortened chaperone was still functional in binding the K88 major subunit FaeG and K88 biosynthesis. Two hybrid chaperones were constructed. Each hybrid protein contained one -barrel domain of FaeE and the other -barrel domain of FanE (Fae/FanE or Fan/FaeE, respectively). Expression and complementation experiments revealed that the Fae/FanE but not the Fan/FaeE hybrid chaperone was functional in the formation of K88 fimbriae. The Fan/FaeE hybrid chaperone was active in the biosynthesis of K99 fimbriae. The truncated FaeE mutant chaperone and the hybrid Fae/FanE chaperone were able to form stable periplasmic protein complexes with the K88 major fimbrial subunit FaeG. Cross-linking experiments suggested that the C-terminal shortened chaperone and the Fae/FanE hybrid chaperone were homodimers, as is the wild-type K88 chaperone. Altogether, the data suggested that the N-terminal -barrel domain of a fimbrial chaperone determines subunit specificity. In the case of the K88 periplasmic chaperone, this N-terminal domain also determines dimerization of the protein.     

Secretion of recombinant proteins via the chaperone/usher pathway in Escherichia coli

F1 antigen (Caf1) of Yersinia pestis is assembled via the Caf1M chaperone/Caf1A usher pathway. We investigated the ability of this assembly system to facilitate secretion of full-length heterologous proteins fused to the Caf1 subunit in Escherichia coli. Despite correct processing of a chimeric protein composed of a modified Caf1 signal peptide, mature human interleukin-1beta (hIL-1beta), and mature Caf1, the processed product (hIL-1beta:Caf1) remained insoluble. Coexpression of this chimera with a functional Caf1M chaperone led to the accumulation of soluble hIL-1beta:Caf1 in the periplasm. Soluble hIL-1beta:Caf1 reacted with monoclonal antibodies directed against structural epitopes of hIL-1beta. The results indicate that Caf1M-induced release of hIL-1beta:Caf1 from the inner membrane promotes folding of the hIL-1beta domain. Similar results were obtained with the fusion of Caf1 to hIL-1beta receptor antagonist or to human granulocyte-macrophage colony-stimulating factor. Following coexpression of the hIL-1beta:Caf1 precursor with both the Caf1M chaperone and Caf1A outer membrane protein, hIL-1beta:Caf1 could be detected on the cell surface of E. coli. These results demonstrate for the first time the potential application of the chaperone/usher secretion pathway in the transport of subunits with large heterogeneous N-terminal fusions. This represents a novel means for the delivery of correctly folded heterologous proteins to the periplasm and cell surface as either polymers or cleavable monomeric domains.     

Structure of CfaA Suggests a New Family of Chaperones Essential for Assembly of Class 5 Fimbriae

Adhesive pili on the surface of pathogenic bacteria comprise polymerized pilin subunits and are essential for initiation of infections. Pili assembled by the chaperone-usher pathway (CUP) require periplasmic chaperones that assist subunit folding, maintain their stability, and escort them to the site of bioassembly. Until now, CUP chaperones have been classified into two families, FGS and FGL, based on the short and long length of the subunit-interacting loops between its F1 and G1 β-strands, respectively. CfaA is the chaperone for assembly of colonization factor antigen I (CFA/I) pili of enterotoxigenic E. coli (ETEC), a cause of diarrhea in travelers and young children. Here, the crystal structure of CfaA along with sequence analyses reveals some unique structural and functional features, leading us to propose a separate family for CfaA and closely related chaperones. Phenotypic changes resulting from mutations in regions unique to this chaperone family provide insight into their function, consistent with involvement of these regions in interactions with cognate subunits and usher proteins during pilus assembly.     

Chaperone Caf1M stabilizes hybrid proteins containing sequences of F1 antigen subunit from Yersinia pestis

The Yersinia pestis (causative agent of plague) capsule antigen is a homopolymer of Caf1 protein. Export of the subunits is mediated by the periplasmic chaperone Caf1M. To study the mechanism of Caf1M activity, two hybrid genes including coding sequences for the Caf1 signal peptide, human granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-1 (IL-1) receptor antagonist, and mature Caf1 were constructed and expressed in Escherichia coli. We have shown that in the absence of Caf1M the majority of Caf1 moieties within the hybrid proteins undergo proteolysis in the periplasmic space, presumably by the DegP protease. The coexpression of a gene for chaperone Caf1M significantly increased the amount of full-size hybrid proteins in the periplasm, probably as a result of stabilization of the subunits spatial structure within the hybrid. This effect was not observed in JCB571 cells, which lack periplasmic disulfide isomerase DsbA, essential for Caf1M activity.     

Chaperone Caf1M Stabilizes Hybrid Proteins Containing Sequences of F1 Antigen Subunit from Yersinia pestis

The Yersinia pestis(causative agent of plague) capsule antigen is a homopolymer of Caf1 protein. Export of the subunits is mediated by the periplasmic chaperone Caf1M. To study the mechanism of Caf1M activity, two hybrid genes including coding sequences for the Caf1 signal peptide, human granulocyte–macrophage colony-stimulating factor (GM-CSF) or interleukin-1 (IL-1) receptor antagonist, and mature Caf1 were constructed and expressed in Escherichia coli.We have shown that in the absence of Caf1M the majority of Caf1 moieties within the hybrid proteins undergo proteolysis in the periplasmic space, presumably by the DegP protease. The coexpression of a gene for chaperone Caf1M significantly increased the amount of full-size hybrid proteins in the periplasm, probably as a result of stabilization of the subunit's spatial structure within the hybrid. This effect was not observed in JCB571 cells, which lack periplasmic disulfide isomerase DsbA, essential for Caf1M activity.     

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.     

The usher N terminus is the initial targeting site for chaperone-subunit complexes and participates in subsequent pilus biogenesis events

Pilus biogenesis on the surface of uropathogenic Escherichia coli requires the chaperone/usher pathway, a terminal branch of the general secretory pathway. In this pathway, periplasmic chaperone-subunit complexes target an outer membrane (OM) usher for subunit assembly into pili and secretion to the cell surface. The molecular mechanisms of protein secretion across the OM are not well understood. Mutagenesis of the P pilus usher PapC and the type 1 pilus usher FimD was undertaken to elucidate the initial stages of pilus biogenesis at the OM. Deletion of residues 2 to 11 of the mature PapC N terminus abolished the targeting of the usher by chaperone-subunit complexes and rendered PapC nonfunctional for pilus biogenesis. Similarly, an intact FimD N terminus was required for chaperone-subunit binding and pilus biogenesis. Analysis of PapC-FimD chimeras and N-terminal fragments of PapC localized the chaperone-subunit targeting domain to the first 124 residues of PapC. Single alanine substitution mutations were made in this domain that blocked pilus biogenesis but did not affect targeting of chaperone-subunit complexes. Thus, the usher N terminus does not function simply as a static binding site for chaperone-subunit complexes but also participates in subsequent pilus assembly events.     

Bacterial outer membrane ushers contain distinct targeting and assembly domains for pilus biogenesis

Biogenesis of a superfamily of surface structures by gram-negative bacteria requires the chaperone/usher pathway, a terminal branch of the general secretory pathway. In this pathway a periplasmic chaperone works together with an outer membrane usher to direct substrate folding, assembly, and secretion to the cell surface. We analyzed the structure and function of the PapC usher required for P pilus biogenesis by uropathogenic Escherichia coli. Structural analysis indicated PapC folds as a beta-barrel with short extracellular loops and extensive periplasmic domains. Several periplasmic regions were localized, including two domains containing conserved cysteine pairs. Functional analysis of deletion mutants revealed that the PapC C terminus was not required for insertion of the usher into the outer membrane or for proper folding. The usher C terminus was not necessary for interaction with chaperone-subunit complexes in vitro but was required for pilus biogenesis in vivo. Interestingly, coexpression of PapC C-terminal truncation mutants with the chromosomal fim gene cluster coding for type 1 pili allowed P pilus biogenesis in vivo. These studies suggest that chaperone-subunit complexes target an N-terminal domain of the usher and that subunit assembly into pili depends on a subsequent function provided by the usher C terminus.     

Structure and biogenesis of the capsular F1 antigen from Yersinia pestis: preserved folding energy drives fiber formation

Most gram-negative pathogens express fibrous adhesive virulence organelles that mediate targeting to the sites of infection. The F1 capsular antigen from the plague pathogen Yersinia pestis consists of linear fibers of a single subunit (Caf1) and serves as a prototype for nonpilus organelles assembled via the chaperone/usher pathway. Genetic data together with high-resolution X-ray structures corresponding to snapshots of the assembly process reveal the structural basis of fiber formation. Comparison of chaperone bound Caf1 subunit with the subunit in the fiber reveals a novel type of conformational change involving the entire hydrophobic core of the protein. The observed conformational change suggests that the chaperone traps a high-energy folding intermediate of Caf1. A model is proposed in which release of the subunit allows folding to be completed, driving fiber formation.     

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.     

Secretion of Recombinant Proteins via the Chaperone/Usher Pathway in Escherichia coli

F1 antigen (Caf1) of Yersinia pestis is assembled via the Caf1M chaperone/Caf1A usher pathway. We investigated the ability of this assembly system to facilitate secretion of full-length heterologous proteins fused to the Caf1 subunit in Escherichia coli. Despite correct processing of a chimeric protein composed of a modified Caf1 signal peptide, mature human interleukin-1β (hIL-1β), and mature Caf1, the processed product (hIL-1β:Caf1) remained insoluble. Coexpression of this chimera with a functional Caf1M chaperone led to the accumulation of soluble hIL-1β:Caf1 in the periplasm. Soluble hIL-1β:Caf1 reacted with monoclonal antibodies directed against structural epitopes of hIL-1β. The results indicate that Caf1M-induced release of hIL-1β:Caf1 from the inner membrane promotes folding of the hIL-1β domain. Similar results were obtained with the fusion of Caf1 to hIL-1β receptor antagonist or to human granulocyte-macrophage colony-stimulating factor. Following coexpression of the hIL-1β:Caf1 precursor with both the Caf1M chaperone and Caf1A outer membrane protein, hIL-1β:Caf1 could be detected on the cell surface of E. coli. These results demonstrate for the first time the potential application of the chaperone/usher secretion pathway in the transport of subunits with large heterogeneous N-terminal fusions. This represents a novel means for the delivery of correctly folded heterologous proteins to the periplasm and cell surface as either polymers or cleavable monomeric domains.     

Identification and characterization of the chaperone-subunit complex-binding domain from the type 1 pilus assembly platform FimD

The outer membrane protein FimD represents the assembly platform of adhesive type 1 pili from Escherichia coli. FimD forms ring-shaped oligomers of 91.4 kDa subunits that recognize complexes between the pilus chaperone FimC and individual pilus subunits in the periplasm and mediate subunit translocation through the outer membrane. Here, we have identified a periplasmic domain of FimD (FimD(N)) comprising the N-terminal 139 residues of FimD. Purified FimD(N) is a monomeric, soluble protein that specifically recognizes complexes between FimC and individual type 1 pilus subunits, but does not bind the isolated chaperone, or isolated subunits. In addition, FimD(N) retains the ability of FimD to recognize different chaperone-subunit complexes with different affinities, and has the highest affinity towards the FimC-FimH complex. Overexpression of FimD(N) in the periplasm of wild-type E.coli cells diminished incorporation of FimH at the tip of type 1 pili, while pilus assembly itself was not affected. The identification of FimD(N) and its ternary complexes with FimC and individual pilus subunits opens the avenue to structural characterization of critical type 1 pilus assembly intermediates.