A Disulfide Bond-forming Machine Is Linked to the Sortase-mediated Pilus Assembly Pathway in the Gram-positive Bacterium Actinomyces oris

Export of cell surface pilins in Gram-positive bacteria likely occurs by the translocation of unfolded precursor polypeptides; however, how the unfolded pilins gain their native conformation is presently unknown. Here, we present physiological studies to demonstrate that the FimA pilin of Actinomyces oris contains two disulfide bonds. Alanine substitution of cysteine residues forming the C-terminal disulfide bridge abrogates pilus assembly, in turn eliminating biofilm formation and polymicrobial interaction. Transposon mutagenesis of A. oris yielded a mutant defective in adherence to Streptococcus oralis, and revealed the essential role of a vitamin K epoxide reductase (VKOR) gene in pilus assembly. Targeted deletion of vkor results in the same defects, which are rescued by ectopic expression of VKOR, but not a mutant containing an alanine substitution in its conserved CXXC motif. Depletion of mdbA, which encodes a membrane-bound thiol-disulfide oxidoreductase, abrogates pilus assembly and alters cell morphology. Remarkably, overexpression of MdbA or a counterpart from Corynebacterium diphtheriae, rescues the Δvkor mutant. By alkylation assays, we demonstrate that VKOR is required for MdbA reoxidation. Furthermore, crystallographic studies reveal that A. oris MdbA harbors a thioredoxin-like fold with the conserved CXXC active site. Consistently, each MdbA enzyme catalyzes proper disulfide bond formation within FimA in vitro that requires the catalytic CXXC motif. Because the majority of signal peptide-containing proteins encoded by A. oris possess multiple Cys residues, we propose that MdbA and VKOR constitute a major folding machine for the secretome of this organism. This oxidative protein folding pathway may be a common feature in Actinobacteria.     

Pilus Gene Pool Variation and the Virulence of Corynebacterium diphtheriae Clinical Isolates during Infection of a Nematode

Toxigenic Corynebacterium diphtheriae strains cause diphtheria in humans. The toxigenic C. diphtheriae isolate NCTC13129 produces three distinct heterotrimeric pili that contain SpaA, SpaD, and SpaH, making up the shaft structure. The SpaA pili are known to mediate bacterial adherence to pharyngeal epithelial cells. However, to date little is known about the expression of different pili in various clinical isolates and their importance in bacterial pathogenesis. Here, we characterized a large collection of C. diphtheriae clinical isolates for their pilin gene pool by PCR and for the expression of the respective pilins by immunoblotting with antibodies against Spa pilins. Consistent with the role of a virulence factor, the SpaA-type pili were found to be prevalent among the isolates, and most significantly, corynebacterial adherence to pharyngeal epithelial cells was strictly correlated with isolates that were positive for the SpaA pili. By comparison, the isolates were heterogeneous for the presence of SpaD- and SpaH-type pili. Importantly, using Caenorhabditis elegans as a model host for infection, we show here that strain NCTC13129 rapidly killed the nematodes, the phenotype similar to isolates that were positive for toxin and all pilus types. In contrast, isogenic mutants of NCTC13129 lacking SpaA-type pili or devoid of toxin and SpaA pili exhibited delayed killing of nematodes with similar kinetics. Consistently, nontoxigenic or toxigenic isolates that lack one, two, or all three pilus types were also attenuated in virulence. This work signifies the important role of pili in corynebacterial pathogenesis and provides a simple host model to identify additional virulence factors.     

Oxidative folding of synthetic polypeptides S‐protected as tert‐butylthio derivatives

A new method for oxidative folding of synthetic polypeptides assembled by stepwise solid phase synthesis is introduced. Folding is obtained in excellent yields by reacting S‐tert‐butylthiolated polypeptides with a 100‐fold molar excess of cysteine at 37 °C in a slightly alkaline buffer containing chaotropic salts, and in the presence of air‐oxygen. This novel protocol has been applied to the folding of S‐tert‐butylthiolated human thymus and activation‐regulated chemokine (hu‐TARC) derivatives as well as to larger segments of Plasmodium falciparum and Plasmodium berghei circumsporozoite proteins. Folded P. falciparum polypeptides have been used as substrates of endoproteinase Glu‐C (Glu‐C) and endoproteinase Asp‐N (Asp‐N) in an attempt to identify their disulfide connectivities. Particular practical advantages of the present method are (i) easy purification and storage of the S‐protected peptide derivatives, (ii) elimination of the risk of cysteine alkylation during the acidolytic cleavage deprotection and resin cleavage steps, (iii) possibility to precisely evaluate the extent of folding and disulfide bond formation by mass spectrometry, and (iv) facile recovery of the final folded product. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Analysis of the interacting surface of maurotoxin with the voltage‐gated Shaker B K+ channel

Maurotoxin (MTX) is a 34‐residue toxin that was isolated initially from the venom of the scorpion Scorpio maurus palmatus. Unlike the other toxins of the α‐KTx6 family (Pi1, Pi4, Pi7, and HsTx1), MTX exhibits a unique disulfide bridge organization of the type C₁? C₅, C₂? C₆, C₃? C₄, and C₇? C₈ (instead of the conventional C₁? C₅, C₂? C₆, C₃? C₇, and C₄? C₈, herein referred to as Pi1‐like) that does not prevent its folding along the classic α/β scaffold of scorpion toxins. MTX_Pi1 is an MTX variant with a conventional pattern of disulfide bridging without any primary structure alteration of the toxin. Here, using MTX and/or MTX_Pi1 as models, we investigated how the type of folding influences toxin recognition of the Shaker B potassium channel. Amino acid residues of MTX that were studied for Shaker B recognition were selected on the basis of their homologous position in charybdotoxin, a three disulfide‐bridged scorpion toxin also active on this channel type. These residues favored either an MTX‐ or MTX_Pi1‐like folding. Our data indicate clearly that Lys²³ and Tyr³² (two out of ten amino acid residues studied) are the most important residues for Shaker B channel blockage by MTX. For activity on SKCa channels, the same amino acid residues also affect, directly or indirectly, the recognition of SK channels. The molecular modeling technique and computed docking indicate the existence of a correlation between the half cystine pairings of the mutated analogs and their activity on the Shaker B K⁺ channel. Overall, mutations in MTX could, or could not, change the reorganization of disulfide bridges of this molecule without affecting its α/β scaffold. However, changing of the peptide backbone (cross linking disulfide bridges from MTX‐like type vs MTX_Pi1‐like type) appears to have less impact on the molecule activity than mutation of certain key amino acids such as Lys²³ and Tyr³² in this toxin. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Disulfide-Bond-Forming Pathways in Gram-Positive Bacteria

Disulfide bonds are important for the stability and function of many secreted proteins. In Gram-negative bacteria, these linkages are catalyzed by thiol-disulfide oxidoreductases (Dsb) in the periplasm. Protein oxidation has been well studied in these organisms, but it has not fully been explored in Gram-positive bacteria, which lack traditional periplasmic compartments. Recent bioinformatics analyses have suggested that the high-GC-content bacteria (i.e., actinobacteria) rely on disulfide-bond-forming pathways. In support of this, Dsb-like proteins have been identified in Mycobacterium tuberculosis, but their functions are not known. Actinomyces oris and Corynebacterium diphtheriae have recently emerged as models to study disulfide bond formation in actinobacteria. In both organisms, disulfide bonds are catalyzed by the membrane-bound oxidoreductase MdbA. Remarkably, unlike known Dsb proteins, MdbA is important for pathogenesis and growth, which makes it a potential target for new antibacterial drugs. This review will discuss disulfide-bond-forming pathways in bacteria, with a special focus on Gram-positive bacteria.     

Methods to identify the substrates of thiol‐disulfide oxidoreductases

The formation of a disulfide bond is a critical step in the folding of numerous secretory and membrane proteins and catalyzed in vivo. A variety of mechanisms and protein structures have evolved to catalyze oxidative protein folding. Those enzymes that directly interact with a folding protein to accelerate its oxidative folding are mostly thiol‐disulfide oxidoreductases that belong to the thioredoxin superfamily. The enzymes of this class often use a CXXC active‐site motif embedded in their thioredoxin‐like fold to promote formation, isomerization, and reduction of a disulfide bond in their target proteins. Over the past decade or so, an increasing number of substrates of the thiol‐disulfide oxidoreductases that are present in the ER of mammalian cells have been discovered, revealing that the enzymes play unexpectedly diverse physiological functions. However, functions of some of these enzymes still remain unclear due to the lack of information on their substrates. Here, we review the methods used by researchers to identify the substrates of these enzymes and provide data that show the importance of using trichloroacetic acid in sample preparation for the substrate identification, hoping to aid future studies. We particularly focus on successful studies that have uncovered physiological substrates and functions of the enzymes in the periplasm of Gram‐negative bacteria and the endoplasmic reticulum of mammalian cells. Similar approaches should be applicable to enzymes in other cellular compartments or in other organisms.     

The basal and major pilins in the Corynebacterium diphtheriae SpaA pilus adopt similar structures that competitively react with the pilin polymerase

Many species of pathogenic gram-positive bacteria display covalently crosslinked protein polymers (called pili or fimbriae) that mediate microbial adhesion to host tissues. These structures are assembled by pilus-specific sortase enzymes that join the pilin components together via lysine-isopeptide bonds. The archetypal SpaA pilus from Corynebacterium diphtheriae is built by the ^CdSrtA pilus-specific sortase, which crosslinks lysine residues within the SpaA and SpaB pilins to build the shaft and base of the pilus, respectively. Here, we show that ^CdSrtA crosslinks SpaB to SpaA via a K139(SpaB)-T494(SpaA) lysine-isopeptide bond. Despite sharing only limited sequence homology, an NMR structure of SpaB reveals striking similarities with the N-terminal domain of SpaA (^NSpaA) that is also crosslinked by ^CdSrtA. In particular, both pilins contain similarly positioned reactive lysine residues and adjacent disordered AB loops that are predicted to be involved in the recently proposed “latch” mechanism of isopeptide bond formation. Competition experiments using an inactive SpaB variant and additional NMR studies suggest that SpaB terminates SpaA polymerization by outcompeting ^NSpaA for access to a shared thioester enzyme–substrate reaction intermediate.     

The essential cell division protein FtsN contains a critical disulfide bond in a non‐essential domain

Disulfide bonds are found in many proteins associated with the cell wall of Escherichia coli, and for some of these proteins the disulfide bond is critical to their stability and function. One protein found to contain a disulfide bond is the essential cell division protein FtsN, but the importance of this bond to the protein's structural integrity is unclear. While it evidently plays a role in the proper folding of the SPOR domain of FtsN, this domain is non‐essential, suggesting that the disulfide bond might also be dispensable. However, we find that FtsN mutants lacking cysteines give rise to filamentous growth. Furthermore, FtsN protein levels in strains expressing these mutants were significantly lower than in a strain expressing the wild‐type allele, as were FtsN levels in strains incapable of making disulfide bonds (dsb^‐) exposed to anaerobic conditions. These results strongly suggest that FtsN lacking a disulfide bond is unstable, thereby making this disulfide critical for function. We have previously found that dsb^‐ strains fail to grow anaerobically, and the results presented here suggest that this growth defect may be due in part to misfolded FtsN. Thus, proper cell division in E. coli is dependent upon disulfide bond formation.     

The disulfide oxidoreductase SdbA is active in Streptococcus gordonii using a single C‐terminal cysteine of the CXXC motif

Recently, we identified a novel disulfide oxidoreductase, SdbA, in the oral bacterium Streptococcus gordonii. Disulfide oxidoreductases form disulfide bonds in nascent proteins using a CXXC catalytic motif. Typically, the N‐terminal cysteine interacts with substrates, whereas the C‐terminal cysteine is buried and only reacts with the first cysteine of the motif. In this study, we investigated the SdbA C⁸⁶P⁸⁷D⁸⁸C⁸⁹ catalytic motif. In vitro, SdbA single cysteine variants at the N or C‐terminal position (SdbA_C86P and SdbA_C89A) were active but displayed different susceptibility to oxidation, and N‐terminal cysteine was prone to sulfenylation. In S. gordonii, mutants with a single N‐terminal cysteine were inactive and formed unstable disulfide adducts with other proteins. Activity was partially restored by inactivation of pyruvate oxidase, a hydrogen peroxide generator. Presence of the C‐terminal cysteine alone (in the SdbA_C86P variant) could complement the ΔsdbA mutant and restore disulfide bond formation in recombinant and natural protein substrates. These results provide evidence that certain disulfide oxidoreductases can catalyze disulfide bond formation using a single cysteine of the CXXC motif, including the buried C‐terminal cysteine.     

Molecular cloning,expression profile,odorant affinity,and stability of two odorant‐binding proteins in Macrocentrus cingulum Brischke (Hymenoptera: Braconidae)

The polyembryonic endoparasitoid wasp Macrocentrus cingulum Brischke (Hymenoptera: Braconidae) is deployed successfully as a biocontrol agent for corn pest insects from the Lepidopteran genus Ostrinia in Europe and throughout Asia, including Japan, Korea, and China. The odorants are recognized, bound, and solubilized by odorant‐binding protein (OBP) in the initial biochemical recognition steps in olfaction that transport them across the sensillum lymph to initiate behavioral response. In the present study, we examine the odorant‐binding effects on thermal stability of McinOBP2, McinOBP3, and their mutant form that lacks the third disulfide bonds. Real‐time PCR experiments indicate that these two are expressed mainly in adult antennae, with expression levels differing by sex. Odorant‐binding affinities of aldehydes, terpenoids, and aliphatic alcohols were measured with circular dichroism spectroscopy based on changes in the thermal stability of the proteins upon their affinities to odorants. The obtained results reveal higher affinity of trans‐caryophelle, farnesene, and cis‐3‐Hexen‐1‐ol exhibits to both wild and mutant McinOBP2 and McinOBP3. Although conformational flexibility of the mutants and shape of binding cavity make differences in odorant affinity between the wild‐type and mutant, it suggested that lacking the third disulfide bond in mutant proteins may have chance to incorrect folded structures that reduced the affinity to these odorants. In addition, CD spectra clearly indicate proteins enriched with α‐helical content.     

Analysis of tryptophan‐rich region in Clostridium septicum alpha‐toxin involved with binding to glycosylphosphatidylinositol‐anchored proteins

Clostridium septicum alpha‐toxin has a unique tryptophan‐rich region (³⁰²NGYSEWDWKWV³¹²) that consists of 11 amino acid residues near the C‐terminus. Using mutant toxins, the contribution of individual amino acids in the tryptophan‐rich region to cytotoxicity and binding to glycosylphosphatidylinositol (GPI)‐anchored proteins was examined. For retention of maximum cytotoxic activity, W307 and W311 are essential residues and residue 309 has to be hydrophobic and possess an aromatic side chain, such as tryptophan or phenylalanine. When residue 308, which lies between tryptophans (W307 and W309) is changed from an acidic to a basic amino acid, the cytotoxic activity of the mutant is reduced to less than that of the wild type. It was shown by a toxin overlay assay that the cytotoxic activity of each mutant toxin correlates closely with affinity to GPI‐anchored proteins. These findings indicate that the WDW_W sequence in the tryptophan‐rich region plays an important role in the cytotoxic mechanism of alpha‐toxin, especially in the binding to GPI‐anchored proteins as cell receptors.     

Disulfide bond of Mycoplasma pneumoniae community‐acquired respiratory distress syndrome toxin is essential to maintain the ADP‐ribosylating and vacuolating activities

Mycoplasma pneumoniae is the leading cause of bacterial community‐acquired pneumonia among hospitalised children in United States and worldwide. Community‐acquired respiratory distress syndrome (CARDS) toxin is a key virulence determinant of M. pneumoniae. The N‐terminus of CARDS toxin exhibits ADP‐ribosyltransferase (ADPRT) activity, and the C‐terminus possesses binding and vacuolating activities. Thiol‐trapping experiments of wild‐type (WT) and cysteine‐to‐serine‐mutated CARDS toxins with alkylating agents identified disulfide bond formation at the amino terminal cysteine residues C230 and C247. Compared with WT and other mutant toxins, C247S was unstable and unusable for comparative studies. Although there were no significant variations in binding, entry, and retrograde trafficking patterns of WT and mutated toxins, C230S did not elicit vacuole formation in intoxicated cells. In addition, the ADPRT domain of C230S was more sensitive to all tested proteases when compared with WT toxin. Despite its in vitro ADPRT activity, the reduction of C230S CARDS toxin‐mediated ADPRT activity‐associated IL‐1β production in U937 cells and the recovery of vacuolating activity in the protease‐released carboxy region of C230S indicated that the disulfide bond was essential not only to maintain the conformational stability of CARDS toxin but also to properly execute its cytopathic effects.     

Cysteine‐free rop: A four‐helix bundle core mutant has wild‐type stability and structure but dramatically different unfolding kinetics

Cysteine residues can complicate the folding and storage of proteins due to improper formation of disulfide bonds or oxidation of residues that are natively reduced. Wild‐type Rop is a homodimeric four‐helix bundle protein and an important model for protein design in the understanding of protein stability, structure and folding kinetics. In the native state, Rop has two buried, reduced cysteine residues in its core, but these are prone to oxidation in destabilized variants, particularly upon extended storage. To circumvent this problem, we designed and characterized a Cys‐free variant of Rop, including solving the 2.3 Å X‐ray crystal structure. We show that the C38A C52V variant has similar structure, stability and in vivo activity to wild‐type Rop, but that it has dramatically faster unfolding kinetics like virtually every other mutant of Rop that has been characterized. This cysteine‐free Rop has already proven useful for studies on solution topology and on the relationship of core mutations to stability. It also suggests a general strategy for removal of pairs of Cys residues in proteins, both to make them more experimentally tractable and to improve their storage properties for therapeutic or industrial purposes.     

A comparative structure‐function analysis of active‐site inhibitors of Vibrio cholerae cholix toxin

Cholix toxin from Vibrio cholerae is a novel mono‐ADP‐ribosyltransferase (mART) toxin that shares structural and functional properties with Pseudomonas aeruginosa exotoxin A and Corynebacterium diphtheriae diphtheria toxin. Herein, we have used the high‐resolution X‐ray structure of full‐length cholix toxin in the apo form, NAD⁺ bound, and 10 structures of the cholix catalytic domain (C‐domain) complexed with several strong inhibitors of toxin enzyme activity (NAP, PJ34, and the P‐series) to study the binding mode of the ligands. A pharmacophore model based on the active pose of NAD⁺ was compared with the active conformation of the inhibitors, which revealed a cationic feature in the side chain of the inhibitors that may determine the active pose. Moreover, a conformational search was conducted for the missing coordinates of one of the main active‐site loops (R‐loop). The resulting structural models were used to evaluate the interaction energies and for 3D‐QSAR modeling. Implications for a rational drug design approach for mART toxins were derived. Copyright © 2015 John Wiley & Sons, Ltd.     

<Emphasis Type="Italic">Corynebacterium diphtheriae</Emphasis> invasion-associated protein (DIP1281) is involved in cell surface organization,adhesion and internalization in epithelial cells

Background  

Corynebacterium diphtheriae, the causative agent of diphtheria, is well-investigated in respect to toxin production, while little is known about C. diphtheriae factors crucial for colonization of the host. In this study, we investigated the function of surface-associated protein DIP1281, previously annotated as hypothetical invasion-associated protein.     

Sequence‐dependent folding and unfolding of ligand‐bound purine riboswitches

Riboswitch regulation of gene expression requires ligand‐mediated RNA folding. From the fluorescence lifetime distribution of bound 2‐aminopurine ligand, we resolve three RNA conformers (C_o, C_i, C_c) of the liganded G‐ and A‐sensing riboswitches from Bacillus subtilis. The ligand binding affinities, and sensitivity to Mg²⁺, together with results from mutagenesis, suggest that C_o and C_i are partially unfolded species compromised in key loop‐loop interactions present in the fully folded C_c. These data verify that the ligand‐bound riboswitches may dynamically fold and unfold in solution, and reveal differences in the distribution of folded states between two structurally homologous purine riboswitches: Ligand‐mediated folding of the G‐sensing riboswitch is more effective, less dependent on Mg²⁺, and less debilitated by mutation, than the A‐sensing riboswitch, which remains more unfolded in its liganded state. We propose that these sequence‐dependent RNA dynamics, which adjust the balance of ligand‐mediated folding and unfolding, enable different degrees of kinetic discrimination in ligand binding, and fine‐tuning of gene regulatory mechanisms. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 953–965, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

One short cysteine‐rich sequence pattern – two different disulfide‐bonded structures – a molecular dynamics simulation study

The nematocyst walls of Hydra are formed by proteins containing small cysteine‐rich domains (CRDs) of ~25 amino acids. The first CRD of nematocyst outer all antigen (NW1) and the C‐terminal CRD of minicollagen‐1 (Mcol1C) contain six cysteines at identical sequence positions, however adopt different disulfide bonded structures. NW1 shows the disulfide connectivities C2‐C14/C6‐C19/C10‐C18 and Mcol1C C2‐C18/C6‐C14/C10‐C19. To analyze if both show structural preferences in the open, non‐disulfide bonded form, which explain the formation of either disulfide connectivity pattern, molecular dynamics (MD) simulations at different temperatures were performed. NW1 maintained in the 100‐ns MD simulations at 283 K a rather compact fold that is stabilized by specific hydrogen bonds. The Mcol1C structure fluctuated overall more, however stayed most of the time also rather compact. The analysis of the backbone Φ/ψ angles indicated different turn propensities for NW1 and Mcol1C, which mostly can be explained based on published data about the influence of different amino acid side chains on the local backbone conformation. Whereas a folded precursor mechanism may be considered for NW1, Mcol1C may fold according to the quasi‐stochastic folding model involving disulfide bond reshuffling and conformational changes, locking the native disulfide conformations. The study further demonstrates the power of MD simulations to detect local structural preferences in rather dynamic systems such as the open, non‐disulfide bonded forms of NW1 and Mcol1C, which complement published information from NMR backbone residual dipolar couplings. Because the backbone structural preferences encoded by the amino acid sequence embedding the cysteines influence which disulfide connectivities are formed, the data are generally interesting for a better understanding of oxidative folding and the design of disulfide stabilized therapeutics. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.