Combination of two separate binding domains defines stoichiometry between type III secretion system chaperone IpgC and translocator protein IpaB

Type III secretion systems (TTSSs) utilized by enteropathogenic bacteria require the presence of small, acidic virulence-associated chaperones for effective host cell infection. We adopted a combination of biochemical and cellular techniques to define the chaperone binding domains (CBDs) in the translocators IpaB and IpaC associated with the chaperone IpgC from Shigella flexneri. We identified a novel CBD in IpaB and furthermore precisely mapped the boundaries of the CBDs in both translocator proteins. In IpaC a single binding domain associates with IpgC. In IpaB, we show that the binding of the newly characterized CBD is essential in maintaining the ternary arrangement of chaperone-translocator complex. This hitherto unknown function is reflected in the co-crystal structure as well, with an IpgC dimer bound to an IpaB fragment comprising both CBDs. Moreover, in the absence of this novel CBD the IpaB/IpgC complex aggregates. This dual-recognition of a domain in the protein by the chaperone in facilitating the correct chaperone-substrate organization describes a new function for the TTSS associated chaperone-substrate complexes.     

Outer membrane targeting of secretin PulD protein relies on disordered domain recognition by a dedicated chaperone

Interaction of bacterial outer membrane secretin PulD with its dedicated lipoprotein chaperone PulS relies on a disorder-to-order transition of the chaperone binding (S) domain near the PulD C terminus. PulS interacts with purified S domain to form a 1:1 complex. Circular dichroism, one-dimensional NMR, and hydrodynamic measurements indicate that the S domain is elongated and intrinsically disordered but gains secondary structure upon binding to PulS. Limited proteolysis and mass spectrometry identified the 28 C-terminal residues of the S domain as a minimal binding site with low nanomolar affinity for PulS in vitro that is sufficient for outer membrane targeting of PulD in vivo. The region upstream of this binding site is not required for targeting or multimerization and does not interact with PulS, but it is required for secretin function in type II secretion. Although other secretin chaperones differ substantially from PulS in sequence and secondary structure, they have all adopted at least superficially similar mechanisms of interaction with their cognate secretins, suggesting that intrinsically disordered regions facilitate rapid interaction between secretins and their chaperones.     

Molecular basis for the unique role of the AAA+ chaperone ClpV in type VI protein secretion

Ring-forming AAA(+) ATPases act in a plethora of cellular processes by remodeling macromolecules. The specificity of individual AAA(+) proteins is achieved by direct or adaptor-mediated association with substrates via distinct recognition domains. We investigated the molecular basis of substrate interaction for Vibrio cholerae ClpV, which disassembles tubular VipA/VipB complexes, an essential step of type VI protein secretion and bacterial virulence. We identified the ClpV recognition site within VipB, showed that productive ClpV-VipB interaction requires the oligomeric state of both proteins, solved the crystal structure of a ClpV N-domain-VipB peptide complex, and verified the interaction surface by mutant analysis. Our results show that the substrate is bound to a hydrophobic groove, which is formed by the addition of a single α-helix to the core N-domain. This helix is absent from homologous N-domains, explaining the unique substrate specificity of ClpV. A limited interaction surface between both proteins accounts for the dramatic increase in binding affinity upon ATP-driven ClpV hexamerization and VipA/VipB tubule assembly by coupling multiple weak interactions. This principle ensures ClpV selectivity toward the VipA/VipB macromolecular complex.     

Characterization of the Membrane-targeting C1 Domain in Pasteurella multocida Toxin

Pasteurella multocida toxin (PMT) is a virulence factor responsible for the pathogenesis of some forms of pasteurellosis. The toxin activates G_q- and G_12/13-dependent pathways through the deamidation of a glutamine residue in the α-subunit of heterotrimeric GTPases. We recently reported the crystal structure of the C terminus (residues 575–1285) of PMT (C-PMT), which is composed of three domains (C1, C2, and C3), and that the C1 domain is involved in the localization of C-PMT to the plasma membrane, and the C3 domain possesses a cysteine protease-like catalytic triad. In this study, we analyzed the membrane-targeting function of the C1 domain in detail. The C1 domain consists of seven helices of which the first four (residues 590–670), showing structural similarity to the N terminus of Clostridium difficile toxin B, were found to be involved in the recruitment of C-PMT to the plasma membrane. C-PMT lacking these helices (C-PMT ΔC1(4H)) neither localized to the plasma membrane nor stimulated the G_q/12/13-dependent signaling pathways. When the membrane-targeting property was complemented by a peptide tag with an N-myristoylation motif, C-PMT ΔC1(4H) recovered the PMT activity. Direct binding between the C1 domain and liposomes containing phospholipids was evidenced by surface plasmon resonance analyses. These results indicate that the C1 domain of C-PMT functions as a targeting signal for the plasma membrane.     

Structure and conservation of the periplasmic targeting factor Tic22 protein from plants and cyanobacteria

Mitochondria and chloroplasts are of endosymbiotic origin. Their integration into cells entailed the development of protein translocons, partially by recycling bacterial proteins. We demonstrate the evolutionary conservation of the translocon component Tic22 between cyanobacteria and chloroplasts. Tic22 in Anabaena sp. PCC 7120 is essential. The protein is localized in the thylakoids and in the periplasm and can be functionally replaced by a plant orthologue. Tic22 physically interacts with the outer envelope biogenesis factor Omp85 in vitro and in vivo, the latter exemplified by immunoprecipitation after chemical cross-linking. The physical interaction together with the phenotype of a tic22 mutant comparable with the one of the omp85 mutant indicates a concerted function of both proteins. The three-dimensional structure allows the definition of conserved hydrophobic pockets comparable with those of ClpS or BamB. The results presented suggest a function of Tic22 in outer membrane biogenesis.     

Structural evidence that colicin A protein binds to a novel binding site of TolA protein in Escherichia coli periplasm

The Tol assembly of proteins is an interacting network of proteins located in the Escherichia coli cell envelope that transduces energy and contributes to cell integrity. TolA is central to this network linking the inner and outer membranes by interactions with TolQ, TolR, TolB, and Pal. Group A colicins, such as ColA, parasitize the Tol network through interactions with TolA and/or TolB to facilitate translocation through the cell envelope to reach their cytotoxic site of action. We have determined the first structure of the C-terminal domain of TolA (TolAIII) bound to an N-terminal ColA polypeptide (TA(53-107)). The interface region of the TA(53-107)-TolAIII complex consists of polar contacts linking residues Arg-92 to Arg-96 of ColA with residues Leu-375-Pro-380 of TolA, which constitutes a β-strand addition commonly seen in more promiscuous protein-protein contacts. The interface region also includes three cation-π interactions (Tyr-58-Lys-368, Tyr-90-Lys-379, Phe-94-Lys-396), which have not been observed in any other colicin-Tol protein complex. Mutagenesis of the interface residues of ColA or TolA revealed that the effect on the interaction was cumulative; single mutations of either partner had no effect on ColA activity, whereas mutations of three or more residues significantly reduced ColA activity. Mutagenesis of the aromatic ring component of the cation-π interacting residues showed Tyr-58 of ColA to be essential for the stability of complex formation. TA(53-107) binds on the opposite side of TolAIII to that used by g3p, ColN, or TolB, illustrating the flexible nature of TolA as a periplasmic hub protein.     

Channel domain of colicin A modifies the dimeric organization of its immunity protein

Proteins conferring immunity against pore-forming colicins are localized in the Escherichia coli inner membrane. Their protective effects are mediated by direct interaction with the C-terminal domain of their cognate colicins. Cai, the immunity protein protecting E. coli against colicin A, contains four cysteine residues. We report cysteine cross-linking experiments showing that Cai forms homodimers. Cai contains four transmembrane segments (TMSs), and dimerization occurs via the third TMS. Furthermore, we observe the formation of intramolecular disulfide bonds that connect TMS2 with either TMS1 or TMS3. Co-expression of Cai with its target, the colicin A pore-forming domain (pfColA), in the inner membrane prevents the formation of intermolecular and intramolecular disulfide bonds, indicating that pfColA interacts with the dimer of Cai and modifies its conformation. Finally, we show that when Cai is locked by disulfide bonds, it is no longer able to protect cells against exogenous added colicin A.     

Structural basis for specific recognition of Rpt1p, an ATPase subunit of 26 S proteasome, by proteasome-dedicated chaperone Hsm3p

The 26 S proteasome is a 2.5-MDa molecular machine that degrades ubiquitinated proteins in eukaryotic cells. It consists of a proteolytic core particle and two 19 S regulatory particles (RPs) composed of 6 ATPase (Rpt) and 13 non-ATPase (Rpn) subunits. Multiple proteasome-dedicated chaperones facilitate the assembly of the proteasome, but little is known about the detailed mechanisms. Hsm3, a 19 S RP dedicated chaperone, transiently binds to the C-terminal domain of the Rpt1 subunit and forms a tetrameric complex, Hsm3-Rpt1-Rpt2-Rpn1, during maturation of the ATPase ring of 19 S RP. To elucidate the structural basis of Hsm3 function, we determined the crystal structures of Hsm3 and its complex with the C-terminal domain of the Rpt1 subunit (Rpt1C). Hsm3 has a C-shaped structure that consists of 11 HEAT repeats. The structure of the Hsm3-Rpt1C complex revealed that the interacting surface between Hsm3 and Rpt1 is a hydrophobic core and a complementary charged surface. Mutations in the Hsm3-Rpt1 surface resulted in the assembly defect of the 26 S proteasome. Furthermore, a structural model of the Hsm3-Rpt ring complex and an in vitro binding assay suggest that Hsm3 can bind Rpt2 in addition to Rpt1. Collectively, our results provide the structural basis of the molecular functions of Hsm3 for the RP assembly.     

Dynamic properties of the colicin E1 ion channel

Abstract The mechanism of channel formation and action of channel-forming colicins is a paradigm for the study of dynamic aspects of membrane-protein interactions. The following experimental results concerning interaction of the colicin E1 channel domain with target membranes, in vitro and in vivo, are discussed: (1) the nature of the translocation-competent state of the channel-forming domain; (2) unfolding of the colicin channel peptide during in vitro binding and anchoring of the channel to liposome membranes at acidic pH; (3) reversal of channel peptide binding to liposomes by an alkaline-directed pH shift; (4) voltage-driven translocation and gating of the ion channel, discussed in the context of a four-helix model for a monomeric channel; (5) rescue of colicin-treated cells by high levels of external K⁺; (6) trypsin rescue of cells depolarized by the colicin ion channel; and (7) interaction of the channel domain with its immunity protein.     

Human protein-disulfide isomerase is a redox-regulated chaperone activated by oxidation of domain a'

Protein-disulfide isomerase (PDI), with domains arranged as abb'xa'c, is a key enzyme and chaperone localized in the endoplasmic reticulum (ER) catalyzing oxidative folding and preventing misfolding/aggregation of proteins. It has been controversial whether the chaperone activity of PDI is redox-regulated, and the molecular basis is unclear. Here, we show that both the chaperone activity and the overall conformation of human PDI are redox-regulated. We further demonstrate that the conformational changes are triggered by the active site of domain a', and the minimum redox-regulated cassette is located in b'xa'. The structure of the reduced bb'xa' reveals for the first time that domain a' packs tightly with both domain b' and linker x to form one compact structural module. Oxidation of domain a' releases the compact conformation and exposes the shielded hydrophobic areas to facilitate its high chaperone activity. Thus, the study unequivocally provides mechanistic insights into the redox-regulated chaperone activity of human PDI.     

Decoding toxicity: deducing the sequence requirements of IbsC, a type I toxin in Escherichia coli

Bacterial genomes encode a collection of small peptides that are deleterious to their hosts when overexpressed. The physiological relevance of the majority of these peptides is unknown at present, although many of them have been implicated in regulatory processes important for cell survival and adaptability. One peptide that is of particular interest to us is a 19-amino acid proteic toxin, coined IbsC, whose production is repressed by SibC, an RNA antitoxin. Together, IbsC and SibC constitute a type I toxin-antitoxin (TA) pair. To better understand the function of IbsC and to decipher the sequence determinants for its toxic phenotype, we carried out extensive sequence analyses of the peptide. We generated a series of truncation and single amino acid deletion mutants to determine the minimal sequence required for toxicity. We further probed into functionally relevant amino acids with a comprehensive set of IbsC mutants produced using a systematic sequence randomization strategy. We found that IbsC remained toxic in the presence of multiple deletions and single amino acid substitutions, despite being well-conserved in Escherichia coli and across other Gram-negative bacteria. The toxicity of this peptide was determined to be dependent on a stretch of highly hydrophobic residues near its center. Our results defined sequence-function relationship of IbsC and offered additional insights into properties common to membrane-targeting type I toxins in E. coli and related species.     

Electron cryomicroscopy structure of a membrane-anchored mitochondrial AAA protease

FtsH-related AAA proteases are conserved membrane-anchored, ATP-dependent molecular machines, which mediate the processing and turnover of soluble and membrane-embedded proteins in eubacteria, mitochondria, and chloroplasts. Homo- and hetero-oligomeric proteolytic complexes exist, which are composed of homologous subunits harboring an ATPase domain of the AAA family and an H41 metallopeptidase domain. Mutations in subunits of mitochondrial m-AAA proteases have been associated with different neurodegenerative disorders in human, raising questions on the functional differences between homo- and hetero-oligomeric AAA proteases. Here, we have analyzed the hetero-oligomeric yeast m-AAA protease composed of homologous Yta10 and Yta12 subunits. We combined genetic and structural approaches to define the molecular determinants for oligomer assembly and to assess functional similarities between Yta10 and Yta12. We demonstrate that replacement of only two amino acid residues within the metallopeptidase domain of Yta12 allows its assembly into homo-oligomeric complexes. To provide a molecular explanation, we determined the 12 Å resolution structure of the intact yeast m-AAA protease with its transmembrane domains by electron cryomicroscopy (cryo-EM) and atomic structure fitting. The full-length m-AAA protease has a bipartite structure and is a hexamer in solution. We found that residues in Yta12, which facilitate homo-oligomerization when mutated, are located at the interface between neighboring protomers in the hexamer ring. Notably, the transmembrane and intermembrane space domains are separated from the main body, creating a passage on the matrix side, which is wide enough to accommodate unfolded but not folded polypeptides. These results suggest a mechanism regarding how proteins are recognized and degraded by m-AAA proteases.     

The Influence of Natural Lipid Asymmetry upon the Conformation of a Membrane-inserted Protein (Perfringolysin O)

Eukaryotic membrane proteins generally reside in membrane bilayers that have lipid asymmetry. However, in vitro studies of the impact of lipids upon membrane proteins are generally carried out in model membrane vesicles that lack lipid asymmetry. Our recently developed method to prepare lipid vesicles with asymmetry similar to that in plasma membranes and with controlled amounts of cholesterol was used to investigate the influence of lipid composition and lipid asymmetry upon the conformational behavior of the pore-forming, cholesterol-dependent cytolysin perfringolysin O (PFO). PFO conformational behavior in asymmetric vesicles was found to be distinct both from that in symmetric vesicles with the same lipid composition as the asymmetric vesicles and from that in vesicles containing either only the inner leaflet lipids from the asymmetric vesicles or only the outer leaflet lipids from the asymmetric vesicles. The presence of phosphatidylcholine in the outer leaflet increased the cholesterol concentration required to induce PFO binding, whereas phosphatidylethanolamine and phosphatidylserine in the inner leaflet of asymmetric vesicles stabilized the formation of a novel deeply inserted conformation that does not form pores, even though it contains transmembrane segments. This conformation may represent an important intermediate stage in PFO pore formation. These studies show that lipid asymmetry can strongly influence the behavior of membrane-inserted proteins.     

Structural Basis of Chaperone Recognition of Type III Secretion System Minor Translocator Proteins

The type III secretion system (T3SS) is a complex nanomachine employed by many Gram-negative pathogens, including the nosocomial agent Pseudomonas aeruginosa, to inject toxins directly into the cytoplasm of eukaryotic cells. A key component of all T3SS is the translocon, a proteinaceous channel that is inserted into the target membrane, which allows passage of toxins into target cells. In most bacterial species, two distinct membrane proteins (the “translocators”) are involved in translocon formation, whereas in the bacterial cytoplasm, however, they remain associated to a common chaperone. To date, the strategy employed by a single chaperone to recognize two distinct translocators is unknown. Here, we report the crystal structure of a complex between the Pseudomonas translocator chaperone PcrH and a short region from the minor translocator PopD. PcrH displays a 7-helical tetratricopeptide repeat fold that harbors the PopD peptide within its concave region, originally believed to be involved in recognition of the major translocator, PopB. Point mutations introduced into the PcrH-interacting region of PopD impede translocator-chaperone recognition in vitro and lead to impairment of bacterial cytotoxicity toward macrophages in vivo. These results indicate that T3SS translocator chaperones form binary complexes with their partner molecules, and the stability of their interaction regions must be strictly maintained to guarantee bacterial infectivity. The PcrH-PopD complex displays homologs among a number of pathogenic strains and could represent a novel, potential target for antibiotic development.     

YidC Protein,a Molecular Chaperone for LacY Protein Folding via the SecYEG Protein Machinery

To understand how YidC and SecYEG function together in membrane protein topogenesis, insertion and folding of the lactose permease of Escherichia coli (LacY), a 12-transmembrane helix protein LacY that catalyzes symport of a galactoside and an H⁺, was studied. Although both the SecYEG machinery and signal recognition particle are required for insertion of LacY into the membrane, YidC is not required for translocation of the six periplasmic loops in LacY. Rather, YidC acts as a chaperone, facilitating LacY folding. Upon YidC depletion, the conformation of LacY is perturbed, as judged by monoclonal antibody binding studies and by in vivo cross-linking between introduced Cys pairs. Disulfide cross-linking also demonstrates that YidC interacts with multiple transmembrane segments of LacY during membrane biogenesis. Moreover, YidC is strictly required for insertion of M13 procoat protein fused into the middle cytoplasmic loop of LacY. In contrast, the loops preceding and following the inserted procoat domain are dependent on SecYEG for insertion. These studies demonstrate close cooperation between the two complexes in membrane biogenesis and that YidC functions primarily as a foldase for LacY.     

The structure and interactions of SpoIISA and SpoIISB, a toxin-antitoxin system in Bacillus subtilis

Spore formation in Bacillus subtilis begins with an asymmetric cell division, following which differential gene expression is established by alternative compartment-specific RNA polymerase σ factors. The spoIISAB operon of B. subtilis was identified as a locus whose mutation leads to increased activity of the first sporulation-specific sigma factor, σ^F. Inappropriate spoIISA expression causes lysis of vegetatively growing B. subtilis cells and Escherichia coli cells when expressed heterologously, effects that are countered by co-expression of spoIISB, identifying SpoIISA-SpoIISB as a toxin-antitoxin system. SpoIISA has three putative membrane-spanning segments and a cytoplasmic domain. Here, the crystal structure of a cytoplasmic fragment of SpoIISA (CSpoIISA) in complex with SpoIISB has been determined by selenomethionine-multiwavelength anomalous dispersion phasing to 2.5 Å spacing, revealing a CSpoIISA₂·SpoIISB₂ heterotetramer. CSpoIISA has a single domain α/β structure resembling a GAF domain with an extended α-helix at its N terminus. The two CSpoIISA protomers form extensive interactions through an intermolecular four-helix bundle. Each SpoIISB chain is highly extended and lacking tertiary structure. The SpoIISB chains wrap around the CSpoIISA dimer, forming extensive interactions with both CSpoIISA protomers. CD spectroscopy experiments indicate that SpoIISB is a natively disordered protein that adopts structure only in the presence of CSpoIISA, whereas surface plasmon resonance experiments revealed that the CSpoIISA·SpoIISB complex is stable with a dissociation constant in the nanomolar range. The results are interpreted in relation to sequence conservation and mutational data, and possible mechanisms of cell killing by SpoIISA are discussed.     

Stimulating the substrate folding activity of a single ring GroEL variant by modulating the cochaperonin GroES

In mediating protein folding, chaperonin GroEL and cochaperonin GroES form an enclosed chamber for substrate proteins in an ATP-dependent manner. The essential role of the double ring assembly of GroEL is demonstrated by the functional deficiency of the single ring GroEL(SR). The GroEL(SR)-GroES is highly stable with minimal ATPase activity. To restore the ATP cycle and the turnover of the folding chamber, we sought to weaken the GroEL(SR)-GroES interaction systematically by concatenating seven copies of groES to generate groES(7). GroES Ile-25, Val-26, and Leu-27, residues on the GroEL-GroES interface, were substituted with Asp on different groES modules of groES(7). GroES(7) variants activate ATP activity of GroEL(SR), but only some restore the substrate folding function of GroEL(SR), indicating a direct role of GroES in facilitating substrate folding through its dynamics with GroEL. Active GroEL(SR)-GroES(7) systems may resemble mammalian mitochondrial chaperonin systems.     

Processing and maturation of the pilin of the type IV secretion system encoded within the gonococcal genetic island

The type IV secretion system (T4SS) encoded within the gonococcal genetic island (GGI) of Neisseria gonorrhoeae has homology to the T4SS encoded on the F plasmid. The GGI encodes the putative pilin protein TraA and a serine protease TrbI, which is homologous to the TraF protein of the RP4 plasmid involved in circularization of pilin subunits of P-type pili. TraA was processed to a 68-amino acid long circular peptide by leader peptidase and TrbI. Processing occurred after co-translational membrane insertion and was independent of other proteins. Circularization occurred after removal of three C-terminal amino acids. Mutational analysis of TraA revealed limited flexibility at the cleavage and joining sites. Mutagenesis of TrbI showed that the conserved Lys-93 and Asp-155 are essential, whereas mutagenesis of Ser-52, the putative catalytic serine did not influence circularization. Further mutagenesis of other serine residues did not identify a catalytic serine, indicating that TrbI either contains redundant catalytic serine residues or does not function via a serine-lysine dyad mechanism. In vitro studies revealed that circularization occurs via a covalent intermediate between the C terminus of TraA and TrbI. The intermediate is processed to the circular form after cleavage of the N-terminal signal sequence. This is the first demonstration of a covalent intermediate in the circularization mechanism of conjugative pili.     

Synthesis, maturation, and trafficking of human Na+-dicarboxylate cotransporter NaDC1 requires the chaperone activity of cyclophilin B

Renal excretion of citrate, an inhibitor of calcium stone formation, is controlled mainly by reabsorption via the apical Na⁺-dicarboxylate cotransporter NaDC1 (SLC13A2) in the proximal tubule. Recently, it has been shown that the protein phosphatase calcineurin inhibitors cyclosporin A (CsA) and FK-506 induce hypocitraturia, a risk factor for nephrolithiasis in kidney transplant patients, but apparently through urine acidification. This suggests that these agents up-regulate NaDC1 activity. Using the Xenopus lævis oocyte and HEK293 cell expression systems, we examined first the effect of both anti-calcineurins on NaDC1 activity and expression. While FK-506 had no effect, CsA reduced NaDC1-mediated citrate transport by lowering heterologous carrier expression (as well as endogenous carrier expression in HEK293 cells), indicating that calcineurin is not involved. Given that CsA also binds specifically to cyclophilins, we determined next whether such proteins could account for the observed changes by examining the effect of selected cyclophilin wild types and mutants on NaDC1 activity and cyclophilin-specific siRNA. Interestingly, our data show that the cyclophilin isoform B is likely responsible for down-regulation of carrier expression by CsA and that it does so via its chaperone activity on NaDC1 (by direct interaction) rather than its rotamase activity. We have thus identified for the first time a regulatory partner for NaDC1, and have gained novel mechanistic insight into the effect of CsA on renal citrate transport and kidney stone disease, as well as into the regulation of membrane transporters in general.