Rational Design of Potent Peptide Inhibitors of the PD-1:PD-L1 Interaction for Cancer Immunotherapy.

Peptides have potential to be developed into immune checkpoint inhibitors, but the target interfaces are difficult to inhibit. Here, we explored an approach to mimic the binding surface of PD-1 to design inhibitors. Mimicking native PD-1 resulted in a mimetic with no activity. However, mimicking an affinity-optimized PD-1 resulted in the peptide mimetic MOPD-1 that displayed nanomolar affinity to PD-L1 and could inhibit PD-1:PD-L1 interactions in both protein- and cell-based assays. Mutagenesis and structural characterization using NMR spectroscopy and X-ray crystallography revealed that binding residues from the high affinity PD-1 are crucial for the bioactivity of MOPD-1. Furthermore, MOPD-1 was extremely stable in human serum and inhibited tumor growth in vivo, suggesting it has potential for use in cancer immunotherapy. The successful design of an inhibitor of PD-1:PD-L1 using the mimicry approach described herein illustrates the value of placing greater emphasis on optimizing the target interface before inhibitor design and is an approach that could have broader utility for the design of peptide inhibitors for other complex protein-protein interactions.

Enabling Efficient Folding and High-Resolution Crystallographic Analysis of Bracelet Cyclotides.

Cyclotides have attracted great interest as drug design scaffolds because of their unique cyclic cystine knotted topology. They are classified into three subfamilies, among which the bracelet subfamily represents the majority and comprises the most bioactive cyclotides, but are the most poorly utilized in drug design applications. A long-standing challenge has been the very low in vitro folding yields of bracelets, hampering efforts to characterize their structures and activities. Herein, we report substantial increases in bracelet folding yields enabled by a single point mutation of residue Ile-11 to Leu or Gly. We applied this discovery to synthesize mirror image enantiomers and used quasi-racemic crystallography to elucidate the first crystal structures of bracelet cyclotides. This study provides a facile strategy to produce bracelet cyclotides, leading to a general method to easily access their atomic resolution structures and providing a basis for development of biotechnological applications.

Application and Structural Analysis of Triazole-Bridged Disulfide Mimetics in Cyclic Peptides.

Ruthenium-catalysed azide-alkyne cycloaddition (RuAAC) provides access to 1,5-disubstituted 1,2,3-triazole motifs in peptide engineering applications. However, investigation of this motif as a disulfide mimetic in cyclic peptides has been limited, and the structural consequences remain to be studied. We report synthetic strategies to install various triazole linkages into cyclic peptides through backbone cyclisation and RuAAC cross-linking reactions. These linkages were evaluated in four serine protease inhibitors based on sunflower trypsin inhibitor-1. NMR and X-ray crystallography revealed exceptional consensus of bridging distance and backbone conformations (RMSD<0.5 Å) of the triazole linkages compared to the parent disulfide molecules. The triazole-bridged peptides also displayed superior half-lives in liver S9 stability assays compared to disulfide-bridged peptides. This work establishes a foundation for the application of 1,5-disubstituted 1,2,3-triazoles as disulfide mimetics.

Synthesis, Racemic X-ray Crystallographic, and Permeability Studies of Bioactive Orbitides from Jatropha Species.

Orbitides are small cyclic peptides with a diverse range of therapeutic bioactivities. They are produced by many plant species, including those of the Jatropha genus. Here, the objective was to provide new structural information on orbitides to complement the growing knowledge base on orbitide sequences and activities by focusing on three Jatropha orbitides: ribifolin (1), pohlianin C (7), and jatrophidin (12). To determine three-dimensional structures, racemic crystallography, an emerging structural technique that enables rapid crystallization of biomolecules by combining equal amounts of the two enantiomers, was used. The high-resolution structure of ribifolin (0.99 Å) was elucidated from its racemate and showed it was identical to the structure crystallized from its l-enantiomer only (1.35 Å). Racemic crystallography was also used to elucidate high-resolution structures of pohlianin C (1.20 Å) and jatrophidin (1.03 Å), for which there was difficulty forming crystals without using racemic mixtures. The structures were used to interpret membrane permeability data in PAMPA and a Caco-2 cell assay, showing they had poor permeability. Overall, the results show racemic crystallography can be used to obtain high-resolution structures of orbitides and is useful when enantiopure samples are difficult to crystallize or solution structures from NMR are of low resolution.

Mirror Images of Antimicrobial Peptides Provide Reflections on Their Functions and Amyloidogenic Properties.

Enantiomeric forms of BTD-2, PG-1, and PM-1 were synthesized to delineate the structure and function of these ß-sheet antimicrobial peptides. Activity and lipid-binding assays confirm that these peptides act via a receptor-independent mechanism involving membrane interaction. The racemic crystal structure of BTD-2 solved at 1.45 Å revealed a novel oligomeric form of ß-sheet antimicrobial peptides within the unit cell: an antiparallel trimer, which we suggest might be related to its membrane-active form. The BTD-2 oligomer extends into a larger supramolecular state that spans the crystal lattice, featuring a steric-zipper motif that is common in structures of amyloid-forming peptides. The supramolecular structure of BTD-2 thus represents a new mode of fibril-like assembly not previously observed for antimicrobial peptides, providing structural evidence linking antimicrobial and amyloid peptides.

Structure of the Acinetobacter baumannii dithiol oxidase DsbA bound to elongation factor EF-Tu reveals a novel protein interaction site.

The multidrug resistant bacterium Acinetobacter baumannii is a significant cause of nosocomial infection. Biofilm formation, that requires both disulfide bond forming and chaperone-usher pathways, is a major virulence trait in this bacterium. Our biochemical characterizations show that the periplasmic A. baumannii DsbA (AbDsbA) enzyme has an oxidizing redox potential and dithiol oxidase activity. We found an unexpected non-covalent interaction between AbDsbA and the highly conserved prokaryotic elongation factor, EF-Tu. EF-Tu is a cytoplasmic protein but has been localized extracellularly in many bacterial pathogens. The crystal structure of this complex revealed that the EF-Tu switch I region binds to the non-catalytic surface of AbDsbA. Although the physiological and pathological significance of a DsbA/EF-Tu association is unknown, peptides derived from the EF-Tu switch I region bound to AbDsbA with submicromolar affinity. We also identified a seven-residue DsbB-derived peptide that bound to AbDsbA with low micromolar affinity. Further characterization confirmed that the EF-Tu- and DsbB-derived peptides bind at two distinct sites. These data point to the possibility that the non-catalytic surface of DsbA is a potential substrate or regulatory protein interaction site. The two peptides identified in this work together with the newly characterized interaction site provide a novel starting point for inhibitor design targeting AbDsbA.

Low-resolution solution structures of Munc18:Syntaxin protein complexes indicate an open binding mode driven by the Syntaxin N-peptide.

When nerve cells communicate, vesicles from one neuron fuse with the presynaptic membrane releasing chemicals that signal to the next. Similarly, when insulin binds its receptor on adipocytes or muscle, glucose transporter-4 vesicles fuse with the cell membrane, allowing glucose to be imported. These essential processes require the interaction of SNARE proteins on vesicle and cell membranes, as well as the enigmatic protein Munc18 that binds the SNARE protein Syntaxin. Here, we show that in solution the neuronal protein Syntaxin1a interacts with Munc18-1 whether or not the Syntaxin1a N-peptide is present. Conversely, the adipocyte protein Syntaxin4 does not bind its partner Munc18c unless the N-peptide is present. Solution-scattering data for the Munc18-1:Syntaxin1a complex in the absence of the N-peptide indicates that this complex adopts the inhibitory closed binding mode, exemplified by a crystal structure of the complex. However, when the N-peptide is present, the solution-scattering data indicate both Syntaxin1a and Syntaxin4 adopt extended conformations in complexes with their respective Munc18 partners. The low-resolution solution structure of the open Munc18:Syntaxin binding mode was modeled using data from cross-linking/mass spectrometry, small-angle X-ray scattering, and small-angle neutron scattering with contrast variation, indicating significant differences in Munc18:Syntaxin interactions compared with the closed binding mode. Overall, our results indicate that the neuronal Munc18-1:Syntaxin1a proteins can adopt two alternate and functionally distinct binding modes, closed and open, depending on the presence of the N-peptide, whereas Munc18c:Syntaxin4 adopts only the open binding mode.

The Arabidopsis B3 domain protein VERNALIZATION1 (VRN1) is involved in processes essential for development, with structural and mutational studies revealing its DNA-binding surface.

The B3 DNA-binding domain is a plant-specific domain found throughout the plant kingdom from the alga Chlamydomonas to grasses and flowering plants. Over 100 B3 domain-containing proteins are found in the model plant Arabidopsis thaliana, and one of these is critical for accelerating flowering in response to prolonged cold treatment, an epigenetic process called vernalization. Despite the specific phenotype of genetic vrn1 mutants, the VERNALIZATION1 (VRN1) protein localizes throughout the nucleus and shows sequence-nonspecific binding in vitro. In this work, we used a dominant repressor tag that overcomes genetic redundancy to show that VRN1 is involved in processes beyond vernalization that are essential for Arabidopsis development. To understand its sequence-nonspecific binding, we crystallized VRN1(208-341) and solved its crystal structure to 1.6 Å resolution using selenium/single-wavelength anomalous diffraction methods. The crystallized construct comprises the second VRN1 B3 domain and a preceding region conserved among VRN1 orthologs but absent in other B3 domains. We established the DNA-binding face using NMR and then mutated positively charged residues on this surface with a series of 16 Ala and Glu substitutions, ensuring that the protein fold was not disturbed using heteronuclear single quantum correlation NMR spectra. The triple mutant R249E/R289E/R296E was almost completely incapable of DNA binding in vitro. Thus, we have revealed that although VRN1 is sequence-nonspecific in DNA binding, it has a defined DNA-binding surface.

Structural insights into the role of the cyclic backbone in a squash trypsin inhibitor.

MCoTI-II is a head-to-tail cyclic peptide with potent trypsin inhibitory activity and, on the basis of its exceptional proteolytic stability, is a valuable template for the design of novel drug leads. Insights into inhibitor dynamics and interactions with biological targets are critical for drug design studies, particularly for protease targets. Here, we show that the cyclization and active site loops of MCoTI-II are flexible in solution, but when bound to trypsin, the active site loop converges to a single well defined conformation. This finding of reduced flexibility on binding is in contrast to a recent study on the homologous peptide MCoTI-I, which suggested that regions of the peptide are more flexible upon binding to trypsin. We provide a possible explanation for this discrepancy based on degradation of the complex over time. Our study also unexpectedly shows that the cyclization loop, not present in acyclic homologues, facilitates potent trypsin inhibitory activity by engaging in direct binding interactions with trypsin.

Racemic and quasi-racemic X-ray structures of cyclic disulfide-rich peptide drug scaffolds.

Cyclic disulfide-rich peptides have exceptional stability and are promising frameworks for drug design. We were interested in obtaining X-ray structures of these peptides to assist in drug design applications, but disulfide-rich peptides can be notoriously difficult to crystallize. To overcome this limitation, we chemically synthesized the L- and D-forms of three prototypic cyclic disulfide-rich peptides: SFTI-1 (14-mer with one disulfide bond), cVc1.1 (22-mer with two disulfide bonds), and kB1 (29-mer with three disulfide bonds) for racemic crystallization studies. Facile crystal formation occurred from a racemic mixture of each peptide, giving structures solved at resolutions from 1.25 Što 1.9 Å. Additionally, we obtained the quasi-racemic structures of two mutants of kB1, [G6A]kB1, and [V25A]kB1, which were solved at a resolution of 1.25 Šand 2.3 Å, respectively. The racemic crystallography approach appears to have broad utility in the structural biology of cyclic peptides.

Membrane curvature protein exhibits interdomain flexibility and binds a small GTPase.

The APPL1 and APPL2 proteins (APPL (adaptor protein, phosphotyrosine interaction, pleckstrin homology (PH) domain, and leucine zipper-containing protein)) are localized to their own endosomal subcompartment and interact with a wide range of proteins and small molecules at the cell surface and in the nucleus. They play important roles in signal transduction through their ability to act as Rab effectors. (Rabs are a family of Ras GTPases involved in membrane trafficking.) Both APPL1 and APPL2 comprise an N-terminal membrane-curving BAR (Bin-amphiphysin-Rvs) domain linked to a PH domain and a C-terminal phosphotyrosine-binding domain. The structure and interactions of APPL1 are well characterized, but little is known about APPL2. Here, we report the crystal structure and low resolution solution structure of the BARPH domains of APPL2. We identify a previously undetected hinge site for rotation between the two domains and speculate that this motion may regulate APPL2 functions. We also identified Rab binding partners of APPL2 and show that these differ from those of APPL1, suggesting that APPL-Rab interaction partners have co-evolved over time. Isothermal titration calorimetry data reveal the interaction between APPL2 and Rab31 has a K(d) of 140 nM. Together with other biophysical data, we conclude the stoichiometry of the complex is 2:2.

Crystal structure and site-directed mutagenesis of circular bacteriocin plantacyclin B21AG reveals cationic and aromatic residues important for antimicrobial activity.

Plantacyclin B21AG is a circular bacteriocin produced by Lactiplantibacillus plantarum B21 which displays antimicrobial activity against various Gram-positive bacteria including foodborne pathogens, Listeria monocytogenes and Clostridium perfringens. It is a 58-amino acid cyclised antimicrobial peptide, with the N and C termini covalently linked together. The circular peptide backbone contributes to remarkable stability, conferring partial proteolytic resistance and structural integrity under a wide temperature and pH range. Here, we report the first crystal structure of a circular bacteriocin from a food grade Lactobacillus. The protein was crystallised using the hanging drop vapour diffusion method and the structure solved to a resolution of 1.8 Å. Sequence alignment against 18 previously characterised circular bacteriocins revealed the presence of conserved charged and aromatic residues. Alanine substitution mutagenesis validated the importance of these residues. Minimum inhibitory concentration analysis of these Ala mutants showed that Phe8Ala and Trp45Ala mutants displayed a 48- and 32-fold reduction in activity, compared to wild type. The Lys19Ala mutant displayed the weakest activity, with a 128-fold reduction. These experiments demonstrate the relative importance of aromatic and cationic residues for the antimicrobial activity of plantacyclin B21AG and by extension, other circular bacteriocins sharing these evolutionarily conserved residues.

The atypical thiol-disulfide exchange protein α-DsbA2 from Wolbachia pipientis is a homotrimeric disulfide isomerase.

Disulfide-bond-forming (DSB) oxidative folding enzymes are master regulators of virulence that are localized to the periplasm of many Gram-negative bacteria. The archetypal DSB machinery from Escherichia coli K-12 consists of a dithiol-oxidizing redox-relay pair (DsbA/B), a disulfide-isomerizing redox-relay pair (DsbC/D) and the specialist reducing enzymes DsbE and DsbG that also interact with DsbD. By contrast, the Gram-negative bacterium Wolbachia pipientis encodes just three DSB enzymes. Two of these, α-DsbA1 and α-DsbB, form a redox-relay pair analogous to DsbA/B from E. coli. The third enzyme, α-DsbA2, incorporates a DsbA-like sequence but does not interact with α-DsbB. In comparison to other DsbA enzymes, α-DsbA2 has ∼50 extra N-terminal residues (excluding the signal peptide). The crystal structure of α-DsbA2ΔN, an N-terminally truncated form in which these ∼50 residues are removed, confirms the DsbA-like nature of this domain. However, α-DsbA2 does not have DsbA-like activity: it is structurally and functionally different as a consequence of its N-terminal residues. Firstly, α-DsbA2 is a powerful disulfide isomerase and a poor dithiol oxidase: i.e. its role is to shuffle rather than to introduce disulfide bonds. Moreover, small-angle X-ray scattering (SAXS) of α-DsbA2 reveals a homotrimeric arrangement that differs from those of the other characterized bacterial disulfide isomerases DsbC from Escherichia coli (homodimeric) and ScsC from Proteus mirabilis (PmScsC; homotrimeric with a shape-shifter peptide). α-DsbA2 lacks the shape-shifter motif and SAXS data suggest that it is less flexible than PmScsC. These results allow conclusions to be drawn about the factors that are required for functionally equivalent disulfide isomerase enzymatic activity across structurally diverse protein architectures.

Studying Munc18:Syntaxin Interactions Using Small-Angle Scattering.

The interaction between the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein syntaxin (Sx) and regulatory partner Sec/Munc18 (SM) protein is a critical step in vesicle fusion. The exact role played by SM proteins, whether positive or negative, has been the topic of much debate. High-resolution structures of the SM:Sx complex have shown that SM proteins can bind syntaxin in a closed fusion incompetent state. However, in vitro and in vivo experiments also point to a positive regulatory role for SM proteins that is inconsistent with binding syntaxin in a closed conformation. Here we present protocols we used for the expression and purification of the SM proteins Munc18a and Munc18c and syntaxins 1 and 4 along with procedures used for small-angle X-ray and neutron scattering that showed that syntaxins can bind in an open conformation to SM proteins. We also describe methods for chemical cross-linking experiments and detail how this information can be combined with scattering data to obtain low-resolution structural models for SM:Sx protein complexes.

Identification of disulfide-containing chemical cross-links in proteins using MALDI-TOF/TOF-mass spectrometry.

Cross-linking can be used to identify spatial relationships between amino acids in proteins or protein complexes. A rapid and sensitive method for identifying the site of protein cross-linking using dithiobis(sulfosuccinimidyl propionate) (DTSSP) is presented and illustrated with experiments using murine cortactin, actin and acyl-CoA thioesterase. A characteristic 66 Da doublet, which arises from the asymmetric fragmentation of the disulfide of DTSSP-modified peptides, is observed in the mass spectra obtained under MALDI-TOF/TOF-MS conditions and allows rapid assignment of cross-links in modified proteins. This doublet is observed not only for linear cross-linked peptides but also in the mass spectra of cyclic cross-linked peptides when simultaneous fragmentation of the disulfide and the peptide backbone occurs. We suggest a likely mechanism for this fragmentation. We use guanidinylation of the cross-linked peptides with O-methyl isourea to extend the coverage of cross-linked peptides observed in this MALDI-MS technique. The methodology we report is robust and amenable to automation, and permits the analysis of native cystines along with those introduced by disulfide-containing cross-linkers.

Cloning, expression, purification and characterization of a DsbA-like protein from Wolbachia pipientis.

Wolbachia pipientis are obligate endosymbionts that infect a wide range of insect and other arthropod species. They act as reproductive parasites by manipulating the host reproduction machinery to enhance their own transmission. This unusual phenotype is thought to be a consequence of the actions of secreted Wolbachia proteins that are likely to contain disulfide bonds to stabilize the protein structure. In bacteria, the introduction or isomerization of disulfide bonds in proteins is catalyzed by Dsb proteins. The Wolbachia genome encodes two proteins, alpha-DsbA1 and alpha-DsbA2, that might catalyze these steps. In this work we focussed on the 234 residue protein alpha-DsbA1; the gene was cloned and expressed in Escherichia coli, the protein was purified and its identity confirmed by mass spectrometry. The sequence identity of alpha-DsbA1 for both dithiol oxidants (E. coli DsbA, 12%) and disulfide isomerases (E. coli DsbC, 14%) is similar. We therefore sought to establish whether alpha-DsbA1 is an oxidant or an isomerase based on functional activity. The purified alpha-DsbA1 was active in an oxidoreductase assay but had little isomerase activity, indicating that alpha-DsbA1 is DsbA-like rather than DsbC-like. This work represents the first successful example of the characterization of a recombinant Wolbachia protein. Purified alpha-DsbA1 will now be used in further functional studies to identify protein substrates that could help explain the molecular basis for the unusual Wolbachia phenotypes, and in structural studies to explore its relationship to other disulfide oxidoreductase proteins.

Revisiting interaction specificity reveals neuronal and adipocyte Munc18 membrane fusion regulatory proteins differ in their binding interactions with partner SNARE Syntaxins.

The efficient delivery of cellular cargo relies on the fusion of cargo-carrying vesicles with the correct membrane at the correct time. These spatiotemporal fusion events occur when SNARE proteins on the vesicle interact with cognate SNARE proteins on the target membrane. Regulatory Munc18 proteins are thought to contribute to SNARE interaction specificity through interaction with the SNARE protein Syntaxin. Neuronal Munc18a interacts with Syntaxin1 but not Syntaxin4, and adipocyte Munc18c interacts with Syntaxin4 but not Syntaxin1. Here we show that this accepted view of specificity needs revision. We find that Munc18c interacts with both Syntaxin4 and Syntaxin1, and appears to bind "non-cognate" Syntaxin1 a little more tightly than Syntaxin4. Munc18a binds Syntaxin1 and Syntaxin4, though it interacts with its cognate Syntaxin1 much more tightly. We also observed that when bound to non-cognate Munc18c, Syntaxin1 captures its neuronal SNARE partners SNAP25 and VAMP2, and Munc18c can bind to pre-formed neuronal SNARE ternary complex. These findings reveal that Munc18a and Munc18c bind Syntaxins differently. Munc18c relies principally on the Syntaxin N-peptide interaction for binding Syntaxin4 or Syntaxin1, whereas Munc18a can bind Syntaxin1 tightly whether or not the Syntaxin1 N-peptide is present. We conclude that Munc18a and Munc18c differ in their binding interactions with Syntaxins: Munc18a has two tight binding modes/sites for Syntaxins as defined previously but Munc18c has just one that requires the N-peptide. These results indicate that the interactions between Munc18 and Syntaxin proteins, and the consequences for in vivo function, are more complex than can be accounted for by binding specificity alone.

The nature of the Syntaxin4 C-terminus affects Munc18c-supported SNARE assembly.

Vesicular transport of cellular cargo requires targeted membrane fusion and formation of a SNARE protein complex that draws the two apposing fusing membranes together. Insulin-regulated delivery and fusion of glucose transporter-4 storage vesicles at the cell surface is dependent on two key proteins: the SNARE integral membrane protein Syntaxin4 (Sx4) and the soluble regulatory protein Munc18c. Many reported in vitro studies of Munc18c:Sx4 interactions and of SNARE complex formation have used soluble Sx4 constructs lacking the native transmembrane domain. As a consequence, the importance of the Sx4 C-terminal anchor remains poorly understood. Here we show that soluble C-terminally truncated Sx4 dissociates more rapidly from Munc18c than Sx4 where the C-terminal transmembrane domain is replaced with a T4-lysozyme fusion. We also show that Munc18c appears to inhibit SNARE complex formation when soluble C-terminally truncated Sx4 is used but does not inhibit SNARE complex formation when Sx4 is C-terminally anchored (by a C-terminal His-tag bound to resin, by a C-terminal T4L fusion or by the native C-terminal transmembrane domain in detergent micelles). We conclude that the C-terminus of Sx4 is critical for its interaction with Munc18c, and that the reported inhibitory role of Munc18c may be an artifact of experimental design. These results support the notion that a primary role of Munc18c is to support SNARE complex formation and membrane fusion.

Milligram quantities of homogeneous recombinant full-length mouse Munc18c from Escherichia coli cultures.

Vesicle fusion is an indispensable cellular process required for eukaryotic cargo delivery. The Sec/Munc18 protein Munc18c is essential for insulin-regulated trafficking of glucose transporter4 (GLUT4) vesicles to the cell surface in muscle and adipose tissue. Previously, our biophysical and structural studies have used Munc18c expressed in SF9 insect cells. However to maximize efficiency, minimize cost and negate any possible effects of post-translational modifications of Munc18c, we investigated the use of Escherichia coli as an expression host for Munc18c. We were encouraged by previous reports describing Munc18c production in E. coli cultures for use in in vitro fusion assay, pulldown assays and immunoprecipitations. Our approach differs from the previously reported method in that it uses a codon-optimized gene, lower temperature expression and autoinduction media. Three N-terminal His-tagged constructs were engineered, two with a tobacco etch virus (TEV) or thrombin protease cleavage site to enable removal of the fusion tag. The optimized protocol generated 1-2 mg of purified Munc18c per L of culture at much reduced cost compared to Munc18c generated using insect cell culture. The purified recombinant Munc18c protein expressed in bacteria was monodisperse, monomeric, and functional. In summary, we developed methods that decrease the cost and time required to generate functional Munc18c compared with previous insect cell protocols, and which generates sufficient purified protein for structural and biophysical studies.

Structural and functional characterization of ScsC, a periplasmic thioredoxin-like protein from Salmonella enterica serovar Typhimurium.

AIMS: The prototypical protein disulfide bond (Dsb) formation and protein refolding pathways in the bacterial periplasm involving Dsb proteins have been most comprehensively defined in Escherichia coli. However, genomic analysis has revealed several distinct Dsb-like systems in bacteria, including the pathogen Salmonella enterica serovar Typhimurium. This includes the scsABCD locus, which encodes a system that has been shown via genetic analysis to confer copper tolerance, but whose biochemical properties at the protein level are not defined. The aim of this study was to provide functional insights into the soluble ScsC protein through structural, biochemical, and genetic analyses. RESULTS: Here we describe the structural and biochemical characterization of ScsC, the soluble DsbA-like component of this system. Our crystal structure of ScsC reveals a similar overall fold to DsbA, although the topology of ß-sheets and α-helices in the thioredoxin domains differ. The midpoint reduction potential of the CXXC active site in ScsC was determined to be -132 mV versus normal hydrogen electrode. The reactive site cysteine has a low pKa, typical of the nucleophilic cysteines found in DsbA-like proteins. Deletion of scsC from S. Typhimurium elicits sensitivity to copper (II) ions, suggesting a potential involvement for ScsC in disulfide folding under conditions of copper stress. INNOVATION AND CONCLUSION: ScsC is a novel disulfide oxidoreductase involved in protection against copper ion toxicity.