High-resolution structures of Escherichia coli cDsbD in different redox states: A combined crystallographic, biochemical and computational study

Escherichia coli DsbD transports electrons from cytoplasmic thioredoxin to periplasmic target proteins. DsbD is composed of an N-terminal (nDsbD) and a C-terminal (cDsbD) periplasmic domain, connected by a central transmembrane domain. Each domain possesses two cysteine residues essential for electron transport. The transport proceeds via disulfide exchange reactions from cytoplasmic thioredoxin to the central transmembrane domain and via cDsbD to nDsbD, which then reduces the periplasmic target proteins. We determined four high-resolution structures of cDsbD: oxidized (1.65 A resolution), chemically reduced (1.3 A), photo-reduced (1.1 A) and chemically reduced at pH increased from 4.6 to 7. The latter structure was refined at 0.99 A resolution, the highest achieved so far for a thioredoxin superfamily member. The data reveal unprecedented structural details of cDsbD, demonstrating that the domain is very rigid and undergoes hardly any conformational change upon disulfide reduction or interaction with nDsbD. In full agreement with the crystallographic results, guanidinium chloride-induced unfolding and refolding experiments indicate that oxidized and reduced cDsbD are equally stable. We confirmed the structural rigidity of cDsbD by molecular dynamics simulations. A remarkable feature of cDsbD is the pKa of 9.3 for the active site Cys461: this value, determined using two different experimental methods, surprisingly was around 2.5 units higher than expected on the basis of the redox potential. Additionally, taking advantage of the very high quality of the cDsbD structures, we carried out pKa calculations, which gave results in agreement with the experimental findings. In conclusion, our wide-scope analysis of cDsbD, encompassing atomic-resolution crystallography, computational chemistry and biophysical measurements, highlighted two so far unrecognized key aspects of this domain: its unusual redox properties and extreme rigidity. Both are likely to be correlated to the role of cDsbD as a covalently linked electron shuttle between the membrane domain and the N-terminal periplasmic domain of DsbD.     

Structural basis and kinetics of inter- and intramolecular disulfide exchange in the redox catalyst DsbD

DsbD from Escherichia coli catalyzes the transport of electrons from cytoplasmic thioredoxin to the periplasmic disulfide isomerase DsbC. DsbD contains two periplasmically oriented domains at the N- and C-terminus (nDsbD and cDsbD) that are connected by a central transmembrane (TM) domain. Each domain contains a pair of cysteines that are essential for catalysis. Here, we show that Cys109 and Cys461 form a transient interdomain disulfide bond between nDsbD and cDsbD in the reaction cycle of DsbD. We solved the crystal structure of this catalytic intermediate at 2.85 A resolution, which revealed large relative domain movements in DsbD as a consequence of a strong overlap between the surface areas of nDsbD that interact with DsbC and cDsbD. In addition, we have measured the kinetics of all functional and nonfunctional disulfide exchange reactions between redox-active, periplasmic proteins and protein domains from the oxidative DsbA/B and the reductive DsbC/D pathway. We show that both pathways are separated by large kinetic barriers for nonfunctional disulfide exchange between components from different pathways.     

Thiol-disulfide exchange in an immunoglobulin-like fold: structure of the N-terminal domain of DsbD

Escherichia coli DsbD transports electrons across the plasma membrane, a pathway that leads to the reduction of protein disulfide bonds. Three secreted thioredoxin-like factors, DsbC, DsbE, and DsbG, reduce protein disulfide bonds whereby an active site C-X-X-C motif is oxidized to generate a disulfide bond. DsbD catalyzes the reduction of the disulfide of DsbC, DsbE, and DsbG but not of the thioredoxin-like oxidant DsbA. The reduction of DsbC, DsbE, and DsbG occurs by transport of electrons from cytoplasmic thioredoxin to the C-terminal thioredoxin-like domain of DsbD (DsbD(C)). The N-terminal domain of DsbD, DsbD(N), acts as a versatile adaptor in electron transport and is capable of forming disulfides with oxidized DsbC, DsbE, or DsbG as well as with reduced DsbD(C). Isolated DsbD(N) is functional in electron transport in vitro. Crystallized DsbD(N) assumes an immunoglobulin-like fold that encompasses two active site cysteines, C103 and C109, forming a disulfide bond between beta-strands. The disulfide of DsbD(N) is shielded from the environment and capped by a phenylalanine (F70). A model is discussed whereby the immunoglobulin fold of DsbD(N) may provide for the discriminating interaction with thioredoxin-like factors, thereby triggering movement of the phenylalanine cap followed by disulfide rearrangement.     

Structural basis and kinetics of DsbD-dependent cytochrome c maturation

DsbD from Escherichia coli transports two electrons from cytoplasmic thioredoxin to the periplasmic substrate proteins DsbC, DsbG and CcmG. DsbD consists of an N-terminal periplasmic domain (nDsbD), a C-terminal periplasmic domain, and a central transmembrane domain. Each domain possesses two cysteines required for electron transport. Herein, we demonstrate fast (3.9 x 10(5) M(-1)s(-1)) and direct disulfide exchange between nDsbD and CcmG, a highly specific disulfide reductase essential for cytochrome c maturation. We determined the crystal structure of the disulfide-linked complex between nDsbD and the soluble part of CcmG at 1.94 A resolution. In contrast to the other two known complexes of nDsbD with target proteins, the N-terminal segment of nDsbD contributes to specific recognition of CcmG. This and other features, like the possibility of using an additional interaction surface, constitute the structural basis for the adaptability of nDsbD to different protein substrates.     

Active-site properties of the oxidized and reduced C-terminal domain of DsbD obtained by NMR spectroscopy

The periplasmic C-terminal domain of the Escherichia coli DsbD protein (cDsbD) has a thioredoxin fold. The two cysteine residues in the CXXC motif serve as the reductant for the disulfide bond of the N-terminal domain which can in turn act as a reductant for various periplasmic partners. The resulting disulfide bond in cDsbD is reduced via an unknown mechanism by the transmembrane helical domain of the protein. We show by NMR analysis of (13)C, (15)N-labelled cDsbD that the protein is rigid, is stable to extremes of pH and undergoes only localized conformational changes in the vicinity of the CXXC motif, and in adjacent regions of secondary structure, upon undergoing the reduced/oxidized transition. pK(a) values have been determined, using 2D NMR, for the N-terminal cysteine of the CXXC motif, Cys461, as well as for other active-site residues. It is demonstrated using site-directed mutagenesis that the negative charges of the side-chains of Asp455 and Glu468 in the active site contribute to the unusually high pK(a) value, 10.5, of Cys461. This value is higher than expected from knowledge of the reduction potential of cDsbD. In a double mutant of cDsbD, D455N/E468Q, the pK(a) value of Cys461 is lowered to 8.6, a value close to that expected for an unperturbed cysteine residue. The pK(a) value of the second cysteine in wild-type cDsbD, Cys464, is significantly higher than the maximum pH value that was studied (pH 12.2).     

Kinetics of the intramolecular disulfide exchange between the periplasmic domains of DsbD

DsbD from Escherichia coli catalyzes the transport of electrons from cytoplasmic thioredoxin to the periplasmic substrate proteins DsbC, DsbG and CcmG. DsbD consists of a periplasmic, N-terminal domain (nDsbD), a central transmembrane domain and a periplasmic, C-terminal domain (cDsbD). Each of these domains contains two essential cysteine residues that are required for intermolecular disulfide exchange between DsbD and substrates, and intramolecular disulfide exchange between the three DsbD domains. In order to determine the rate of intramolecular electron transfer from cDsbD to nDsbD, we constructed a redox-sensitive tryptophan variant of cDsbD (cDsbD(W)) that shows an approximately threefold increase in fluorescence upon reduction and has the same redox potential and reactivity as wild-type cDsbD. cDsbD(W) was then used for the construction of fusion proteins with nDsbD and cDsbD(W), connected via flexible linkers of different length. Using the DsbD substrate DsbC, which can only be reduced by nDsbD and does not react with cDsbD, we could directly measure the intramolecular electron transfer from cDsnD(W) to nDsbB in the fusion proteins. We show that the intramolecular disulfide exchange is significantly faster than the reaction between isolated nDsbD and cDsbD. Nevertheless, the effective concentration of 0.2 mM of the domains in the fusions is comaparably low. The rate of 23 s(-1) for the intramolecular disulfide exchange in the fusions was independent of the linker length and may represent the upper limit for the substrate turnover of full-length DsbD.     

Crystal structures of E. coli CcmG and its mutants reveal key roles of the N-terminal beta-sheet and the fingerprint region

CcmG, also designated DsbE, functions as a periplasmic protein thiol:disulfide oxidoreductase and is required for cytochrome c maturation. Here we report the crystal structures of Escherichia coli CcmG and its two mutants, P144A and the N-terminal fifty seven-residue deletion mutant, and two additional deletion mutants were studied by circular dichroism. Structural comparison of E. coli CcmG with its deletion mutants reveals that the N-terminal beta-sheet is essential for maintaining the folding topology and consequently maintaining the active-site structure of CcmG. Pro144 and Glu145 are key residues of the fingerprint region of CcmG. Pro144 is in cis-configuration, and it makes van der Waals interactions with the active-site disulfide Cys80-Cys83 and forms a C--H...O hydrogen bond with Thr82, helping stabilize the active-site structure. Glu145 forms a salt-bridge and hydrogen-bond network with other residues of the fingerprint region and with Arg158, further stabilizing the active-site structure. The cis-configuration of Pro144 makes the backbone nitrogen and oxygen of Ala143 exposed to solvent, favorable for interacting with binding partners. The key role of cis-Pro144 is verified by the P144A mutant, which contains trans-Ala144 and displays redox property changes. Structural comparison of E. coli CcmG with the recently reported structure of CcmG in complex with the N-terminal domain of DsbD reveals that Tyr141 undergoes conformational changes upon binding DsbD. A cis-proline located at the N-terminus of the first beta-strand of the betabetaalpha motif of the thioredoxin-like domain is a conserved structural feature of the thioredoxin superfamily.     

1H, 13C and 15N resonance assignments for the oxidized and reduced states of the N-terminal domain of DsbD from Escherichia coli

Viability and pathogenicity of Gram-negative bacteria is linked to the cytochrome c maturation and the oxidative protein folding systems in the periplasm. The transmembrane reductant conductor DsbD is a unique protein which provides the necessary reducing power to both systems through thiol-disulfide exchange reactions in a complex network of protein-protein interactions. The N-terminal domain of DsbD (nDsbD) is the delivery point of the reducing power originating from cytoplasmic thioredoxin to a variety of periplasmic partners. Here we report (1)H, (13)C and (15)N assignments for resonances of nDsbD in its oxidized and reduced states. These assignments provide the starting point for detailed investigations of the interactions of nDsbD with its protein partners.     

The disulfide bond isomerase DsbC is activated by an immunoglobulin-fold thiol oxidoreductase: crystal structure of the DsbC-DsbDalpha complex

The Escherichia coli disulfide bond isomerase DsbC rearranges incorrect disulfide bonds during oxidative protein folding. It is specifically activated by the periplasmic N-terminal domain (DsbDalpha) of the transmembrane electron transporter DsbD. An intermediate of the electron transport reaction was trapped, yielding a covalent DsbC-DsbDalpha complex. The 2.3 A crystal structure of the complex shows for the first time the specific interactions between two thiol oxidoreductases. DsbDalpha is a novel thiol oxidoreductase with the active site cysteines embedded in an immunoglobulin fold. It binds into the central cleft of the V-shaped DsbC dimer, which assumes a closed conformation on complex formation. Comparison of the complex with oxidized DsbDalpha reveals major conformational changes in a cap structure that regulates the accessibility of the DsbDalpha active site. Our results explain how DsbC is selectively activated by DsbD using electrons derived from the cytoplasm.     

TrbB from conjugative plasmid F is a structurally distinct disulfide isomerase that requires DsbD for redox state maintenance

TrbB, a periplasmic protein encoded by the conjugative plasmid F, has a predicted thioredoxin-like fold and possesses a C-X-X-C redox active site motif. TrbB may function in the conjugative process by serving as a disulfide bond isomerase, facilitating proper folding of a subset of F-plasmid-encoded proteins in the periplasm. Previous studies have demonstrated that a ΔtrbB F plasmid in Escherichia coli lacking DsbC(E.coli), its native disulfide bond isomerase, experiences a 10-fold decrease in mating efficiency but have not provided direct evidence for disulfide bond isomerase activity. Here we demonstrate that trbB can partially restore transfer of a variant of the distantly related R27 plasmid when both chromosomal and plasmid genes encoding disulfide bond isomerases have been disrupted. In addition, we show that TrbB displays both disulfide bond isomerase and reductase activities on substrates not involved in the conjugative process. Unlike canonical members of the disulfide bond isomerase family, secondary structure predictions suggest that TrbB lacks both an N-terminal dimerization domain and an α-helical domain found in other disulfide bond isomerases. Phylogenetic analyses support the conclusion that TrbB belongs to a unique family of plasmid-based disulfide isomerases. Interestingly, although TrbB diverges structurally from other disulfide bond isomerases, we show that like those isomerases, TrbB relies on DsbD from E. coli for maintenance of its C-X-X-C redox active site motif.     

Solution structure and dynamics of the reduced and oxidized forms of the N-terminal domain of PilB from Neisseria meningitidis

The secreted form of the PilB protein was proposed to be involved in pathogen survival fighting against the defensive host's oxidative burst. PilB protein is composed of three domains. The central and the C-terminal domains display methionine sulfoxide reductase A and B activities, respectively. The N-terminal domain, which possesses a CXXC motif, was recently shown to regenerate in vitro the reduced forms of the methionine sulfoxide reductase domains of PilB from their oxidized forms, as does the thioredoxin 1 from E. coli, via a disulfide bond exchange. The thioredoxin-like N-terminal domain belongs to the cytochrome maturation protein structural family, but it possesses a unique additional segment (99)FLHE (102) localized in a loop. This segment covers one edge of the active site in the crystal structure of the reduced form of the N-terminal domain of PilB. We have determined the solution structure and the dynamics of the N-terminal domain from Neisseria meningitidis, in its reduced and oxidized forms. The FLHE loop adopts, in both redox states, a well-defined conformation. Subtle conformational and dynamic changes upon oxidation are highlighted around the active site, as well as in the FLHE loop. The functional consequences of the cytochrome maturation protein topology and those of the presence of FLHE loop are discussed in relation to the enzymatic properties of the N-terminal domain.     

Evolutionary domain fusion expanded the substrate specificity of the transmembrane electron transporter DsbD

Modular organization of proteins has been postulated as a widely used strategy for protein evolution. The multidomain transmembrane protein DsbD catalyzes the transfer of electrons from the cytoplasm to the periplasm of Escherichia coli. Most bacterial species do not have DsbD, but instead their genomes encode a much smaller protein, CcdA, which resembles the central hydrophobic domain of DsbD. We used reciprocal heterologous complementation assays between E.coli and Rhodobacter capsulatus to show that, despite their differences in size and structure, DsbD and CcdA are functional homologs. While DsbD transfers reducing potential to periplasmic protein disulfide bond isomerases and to the cytochrome c thioreduction pathway, CcdA appears to be involved only in cytochrome c biogenesis. Our findings strongly suggest that, by the acquisition of additional thiol-redox active domains, DsbD expanded its substrate specificity.     

Oxidized and synchrotron cleaved structures of the disulfide redox center in the N-terminal domain of Salmonella typhimurium AhpF

The flavoprotein component (AhpF) of Salmonella typhimurium alkyl hydroperoxide reductase contains an N-terminal domain (NTD) with two contiguous thioredoxin folds but only one redox-active disulfide (within the sequence -Cys129-His-Asn-Cys132-). This active site is responsible for mediating the transfer of electrons from the thioredoxin reductase-like segment of AhpF to AhpC, the peroxiredoxin component of the two-protein peroxidase system. The previously reported crystal structure of AhpF possessed a reduced NTD active site, although fully oxidized protein was used for crystallization. To further investigate this active site, we crystallized an isolated recombinant NTD (rNTD); using diffraction data sets collected first at our in-house X-ray source and subsequently at a synchrotron, we showed that the active site disulfide bond (Cys129-Cys132) is oxidized in the native crystals but becomes reduced during synchrotron data collection. The NTD disulfide bond is apparently particularly sensitive to radiation cleavage compared with other protein disulfides. The two data sets provide the first view of an oxidized (disulfide) form of NTD and show that the changes in conformation upon reduction of the disulfide are localized and small. Furthermore, we report the apparent pKa of the active site thiol to be approximately 5.1, a relatively low pKa given its redox potential (approximately 265 mV) compared with most members of the thioredoxin family.     

Crystal structure of DsbDgamma reveals the mechanism of redox potential shift and substrate specificity(1)

The Escherichia coli transmembrane protein DsbD transfers electrons from the cytoplasm to the periplasm through a cascade of thiol-disulfide exchange reactions. In this process, the C-terminal periplasmic domain of DsbD (DsbDgamma) shuttles the reducing potential from the membrane domain (DsbDbeta) to the N-terminal periplasmic domain (DsbDalpha). The crystal structure of DsbDgamma determined at 1.9 A resolution reveals that the domain has a thioredoxin fold with an extended N-terminal stretch. In comparison to thioredoxin, the DsbDgamma structure exhibits the stabilized active site conformation and the extended active site alpha2 helix that explain the domain's substrate specificity and the redox potential shift, respectively. The hypothetical model of the DsbDgamma:DsbDalpha complex based on the DsbDgamma structure and previous structural studies indicates that the conserved hydrophobic residue in the C-X-X-C motif of DsbDgamma may be important in the specific recognition of DsbDalpha.     

Conserved role of the linker alpha-helix of the bacterial disulfide isomerase DsbC in the avoidance of misoxidation by DsbB

In the bacterial periplasm the co-existence of a catalyst of disulfide bond formation (DsbA) that is maintained in an oxidized state and of a reduced enzyme that catalyzes the rearrangement of mispaired cysteine residues (DsbC) is important for the folding of proteins containing multiple disulfide bonds. The kinetic partitioning of the DsbA/DsbB and DsbC/DsbD pathways partly depends on the ability of DsbB to oxidize DsbA at rates >1000 times greater than DsbC. We show that the resistance of DsbC to oxidation by DsbB is abolished by deletions of one or more amino acids within the alpha-helix that connects the N-terminal dimerization domain with the C-terminal thioredoxin domain. As a result, mutant DsbC carrying alpha-helix deletions could catalyze disulfide bond formation and complemented the phenotypes of dsbA cells. Examination of DsbC homologues from Haemophilus influenzae, Pseudomonas aeruginosa, Erwinia chrysanthemi, Yersinia pseudotuberculosis, Vibrio cholerae (30-70% sequence identity with the Escherichia coli enzyme) revealed that the mechanism responsible for avoiding oxidation by DsbB is a general property of DsbC family enzymes. In addition we found that deletions in the linker region reduced, but did not abolish, the ability of DsbC to assist the formation of active vtPA and phytase in vivo, in a DsbD-dependent manner, revealing that interactions between DsbD and DsbC are also conserved.     

The X-ray structure of the N-terminal domain of PILB from Neisseria meningitidis reveals a thioredoxin-fold

The secreted form of the PilB protein was recently shown to be bound to the outer membrane of Neisseria gonorrhoeae and proposed to be involved in survival of the pathogen to the host's oxidative burst. PilB is composed of three domains. The central and the C-terminal domains display methionine sulfoxide reductase (Msr) A and B activities respectively, i.e. the ability to reduce specifically the S and the R enantiomers of the sulfoxide function of the methionine sulfoxides, which are easily formed upon oxidation of methionine residues. The N-terminal domain of PilB (Dom1(PILB)) of N.meningitidis, which possesses a CXXC motif, was recently shown to recycle the oxidized forms of the PilB Msr domains in vitro, as the Escherichia coli thioredoxin (Trx) 1 does. The X-ray structure of Dom1(PILB) of N.meningitidis determined here shows a Trx-fold, in agreement with the biochemical properties of Dom1(PILB). However, substantial structural differences with E.coli Trx1 exist. Dom1(PILB) displays more structural homologies with the periplasmic disulfide oxidoreductases involved in cytochrome maturation pathways in bacteria. The active site of the reduced form of Dom1(PILB) reveals a high level of stabilization of the N-terminal catalytic cysteine residue and a hydrophobic environment of the C-terminal recycling cysteine in the CXXC motif, consistent with the pK(app) values measured for Cys67 (<6) and Cys70 (9.3), respectively. Compared to cytochrome maturation disulfide oxidoreductases and to Trx1, one edge of the active site is covered by four additional residues (99)FLHE(102). The putative role of the resulting protuberance is discussed in relation to the disulfide reductase properties of Dom1(PILB).     

Respiratory chain strongly oxidizes the CXXC motif of DsbB in the Escherichia coli disulfide bond formation pathway

Escherichia coli DsbB has four essential cysteine residues, among which Cys41 and Cys44 form a CXXC redox active site motif and the Cys104-Cys130 disulfide bond oxidizes the active site cysteines of DsbA, the disulfide bond formation factor in the periplasm. Functional respiratory chain is required for the cell to keep DsbA oxidized. In this study, we characterized the roles of essential cysteines of DsbB in the coupling with the respiratory chain. Cys104 was found to form the inactive complex with DsbA under respiration-defective conditions. While DsbB, under normal aerobic conditions, is in the oxidized state, having two intramolecular disulfide bonds, oxidation of Cys104 and Cys130 requires the presence of Cys41-Cys44. Remarkably, the Cys41-Cys44 disulfide bond is refractory to reduction by a high concentration of dithiothreitol, unless the membrane is solubilized with a detergent. This reductant resistance requires both the respiratory function and oxygen, since Cys41-Cys44 became sensitive to the reducing agent when membrane was prepared from quinone- or heme-depleted cells or when a membrane sample was deaerated. Thus, the Cys41-Val-Leu-Cys44 motif of DsbB is kept both strongly oxidized and strongly oxidizing when DsbB is integrated into the membrane with the normal set of respiratory components.     

Disulfide bonding arrangements in active forms of the somatomedin B domain of human vitronectin

The N-terminal cysteine-rich somatomedin B (SMB) domain (residues 1-44) of the human glycoprotein vitronectin contains the high-affinity binding sites for plasminogen activator inhibitor-1 (PAI-1) and the urokinase receptor (uPAR). We previously showed that the eight cysteine residues of recombinant SMB (rSMB) are organized into four disulfide bonds in a linear uncrossed pattern (Cys(5)-Cys(9), Cys(19)-Cys(21), Cys(25)-Cys(31), and Cys(32)-Cys(39)). In the present study, we use an alternative method to show that this disulfide bond arrangement remains a major preferred one in solution, and we determine the solution structure of the domain using NMR analysis. The solution structure shows that the four disulfide bonds are tightly packed in the center of the domain, replacing the traditional hydrophobic core expected for a globular protein. The few noncysteine hydrophobic side chains form a cluster on the outside of the domain, providing a distinctive binding surface for the physiological partners PAI-1 and uPAR. The hydrophobic surface consists mainly of side chains from the loop formed by the Cys(25)-Cys(31) disulfide bond, and is surrounded by conserved acidic and basic side chains, which are likely to contribute to the specificity of the intermolecular interactions of this domain. Interestingly, the overall fold of the molecule is compatible with several arrangements of the disulfide bonds. A number of different disulfide bond arrangements were able to satisfy the NMR restraints, and an extensive series of conformational energy calculations performed in explicit solvent confirmed that several disulfide bond arrangements have comparable stabilization energies. An experimental demonstration of the presence of alternative disulfide conformations in active rSMB is provided by the behavior of a mutant in which Asn(14) is replaced by Met. This mutant has the same PAI-1 binding activity as rVN1-51, but its fragmentation pattern following cyanogen bromide treatment is incompatible with the linear uncrossed disulfide arrangement. These results suggest that active forms of the SMB domain may have a number of allowed disulfide bond arrangements as long as the Cys(25)-Cys(31) disulfide bond is preserved.     

Structure of intact AhpF reveals a mirrored thioredoxin-like active site and implies large domain rotations during catalysis

AhpF, a homodimer of 57 kDa subunits, is a flavoenzyme which catalyzes the NADH-dependent reduction of redox-active disulfide bonds in the peroxidase AhpC, a member of the recently identified peroxiredoxin class of antioxidant enzymes. The structure of AhpF from Salmonella typhimurium at 2.0 A resolution, determined using multiwavelength anomalous dispersion, shows that the C-terminal portion of AhpF (residues 210-521) is structurally like Escherichia coli thioredoxin reductase. In addition, AhpF has an N-terminal domain (residues 1-196) formed from two contiguous thioredoxin folds, but containing just a single redox-active disulfide (Cys129-Cys132). A flexible linker (residues 197-209) connects the domains, consistent with experiments showing that the N-terminal domain acts as an appended substrate, first being reduced by the C-terminal portion of AhpF, and subsequently reducing AhpC. Modeling studies imply that an intrasubunit electron transfer accounts for the reduction of the N-terminal domain in dimeric AhpF. Furthermore, comparing the N-terminal domain with protein disulfide oxidoreductase from Pyrococcus furiosis, we describe a new class of protein disulfide oxidoreductases based on a novel mirror-image active site arrangement, with a distinct carboxylate (Glu86) being functionally equivalent to the key acid (Asp26) of E. coli thioredoxin. A final fortuitous result is that the N-terminal redox center is reduced and provides a high-resolution view of the thiol-thiolate hydrogen bond that has been predicted to stabilize the attacking thiolate in thioredoxin-like proteins.     

One-Way Allosteric Communication between the Two Disulfide Bonds in Tissue Factor

Tissue factor (TF) is a transmembrane glycoprotein that plays distinct roles in the initiation of extrinsic coagulation cascade and thrombosis. TF contains two disulfide bonds, one each in the N-terminal and C-terminal extracellular domains. The C-domain disulfide, Cys186-Cys209, has a ?RHStaple configuration in crystal structures, suggesting that this disulfide carries high pre-stress. The redox state of this disulfide has been proposed to regulate TF encryption/decryption. Ablating the N-domain Cys49-Cys57 disulfide bond was found to increase the redox potential of the Cys186-Cys209 bond, implying an allosteric communication between the domains. Using molecular dynamics simulations, we observed that the Cys186-Cys209 disulfide bond retained the ?RHStaple configuration, whereas the Cys49-Cys57 disulfide bond fluctuated widely. The Cys186-Cys209 bond featured the typical ?RHStaple disulfide properties, such as a longer S-S bond length, larger C-S-S angles, and higher bonded prestress, in comparison to the Cys49-Cys57 bond. Force distribution analysis was used to sense the subtle structural changes upon ablating the disulfide bonds, and allowed us to identify a one-way allosteric communication mechanism from the N-terminal to the C-terminal domain. We propose a force propagation pathway using a shortest-pathway algorithm, which we suggest is a useful method for searching allosteric signal transduction pathways in proteins. As a possible explanation for the pathway being one-way, we identified a pronounced lower degree of conformational fluctuation, or effectively higher stiffness, in the N-terminal domain. Thus, the changes of the rigid domain (N-terminal domain) can induce mechanical force propagation to the soft domain (C-terminal domain), but not vice versa.