Mechanical force can fine-tune redox potentials of disulfide bonds

Mechanical force applied along a disulfide bond alters its rate of reduction. We here aimed at quantifying the direct effect of force onto the chemical reactivity of a sulfur-sulfur bond in contrast to indirect, e.g., steric or mechanistic, influences. To this end, we evaluated the dependency of a disulfide bond's redox potential on a pulling force applied along the system. Our QM/MM simulations of cystine as a model system take conformational dynamics and explicit solvation into account and show that redox potentials increase over the whole range of forces probed here (30-3320 pN), and thus even in the absence of a significant disulfide bond elongation (<500 pN). Instead, at low forces, dihedrals and angles, as the softer degrees of freedom are stretched, contribute to the destabilization of the oxidized state. We find physiological forces to be likely to tune the disulfide's redox potentials to an extent similar to the tuning within proteins by point mutations.     

Synthesis, receptor binding affinities and conformational properties of cyclic methylenedithioether analogues of angiotensin II

Cyclic 12-, 13- and 14-membered ring angiotensin II analogues related to disulfides but encompassing methylene-dithioether bridges have been prepared. The affinity data from these derivatives were compared to those from the disulfides. The methylenedithioether analogues displayed good binding affinities to rat liver AT1 receptors although in most cases somewhat lower than their disulfide counterparts. One of the methylenedithioethers with a 13-membered ring system demonstrated the highest binding affinity among the thioethers. Theoretical conformational analysis of model compounds of the two series were performed suggesting a similarity between the disulfide and the corresponding methylenedithioether analogues and also between the ring size homologues. This analysis also suggested that some of the model compounds were prone to adopt inverse gamma-turn conformations, which was further supported by use of NMR spectroscopy of the 12-membered ring analogue in the series. The easily executed methylenedithioether cyclization should constitute a valuable complement to the common disulfide methodology for fine-tuning and for probing the bioactive conformation of peptides.     

Identification of Allosteric Disulfides from Prestress Analysis

Disulfide bonds serve to form physical cross-links between residues in protein structures, thereby stabilizing the protein fold. Apart from this purely structural role, they can also be chemically active, participating in redox reactions, and they may even potentially act as allosteric switches controlling protein functions. Specific types of disulfide bonds have been identified in static protein structures from their distinctive pattern of dihedral bond angles, and the allosteric function of such bonds is purported to be related to the torsional strain they store. Using all-atom molecular-dynamics simulations for ∼700 disulfide bonded proteins, we analyzed the intramolecular mechanical forces in 20 classes of disulfide bonds. We found that two particular classes, the −RHStaple and the −/+RHHook disulfides, are indeed more stressed than other disulfide bonds, but the stress is carried primarily by stretching of the S-S bond and bending of the neighboring bond angles, rather than by dihedral torsion. This stress corresponds to a tension force of magnitude ∼200 pN, which is balanced by repulsive van der Waals interactions between the cysteine Cα atoms. We confirm stretching of the S-S bond to be a general feature of the −RHStaples and the −/+RHHooks by analyzing ∼20,000 static protein structures. Given that forced stretching of S-S bonds is known to accelerate their cleavage, we propose that prestress of allosteric disulfide bonds has the potential to alter the reactivity of a disulfide, thereby allowing us to readily switch between functional states.     

Conformational changes in redox pairs of protein structures

Disulfides are conventionally viewed as structurally stabilizing elements in proteins but emerging evidence suggests two disulfide subproteomes exist. One group mediates the well known role of structural stabilization. A second redox‐active group are best known for their catalytic functions but are increasingly being recognized for their roles in regulation of protein function. Redox‐active disulfides are, by their very nature, more susceptible to reduction than structural disulfides; and conversely, the Cys pairs that form them are more susceptible to oxidation. In this study, we searched for potentially redox‐active Cys Pairs by scanning the Protein Data Bank for structures of proteins in alternate redox states. The PDB contains over 1134 unique redox pairs of proteins, many of which exhibit conformational differences between alternate redox states. Several classes of structural changes were observed, proteins that exhibit: disulfide oxidation following expulsion of metals such as zinc; major reorganisation of the polypeptide backbone in association with disulfide redox‐activity; order/disorder transitions; and changes in quaternary structure. Based on evidence gathered supporting disulfide redox activity, we propose disulfides present in alternate redox states are likely to have physiologically relevant redox activity.     

High torsional energy disulfides: relationship between cross-strand disulfides and right-handed staples

Redox-active disulfides are capable of being oxidized and reduced under physiological conditions. The enzymatic role of redox-active disulfides in thiol-disulfide reductases is well-known, but redox-active disulfides are also present in non-enzymatic protein structures where they may act as switches of protein function. Here, we examine disulfides linking adjacent beta-strands (cross-strand disulfides), which have been reported to be redox-active. Our previous work has established that these cross-strand disulfides have high torsional energies, a quantity likely to be related to the ease with which the disulfide is reduced. We examine the relationship between conformations of disulfides and their location in protein secondary structures. By identifying the overlap between cross-strand disulfides and various conformations, we wish to address whether the high torsional energy of a cross-strand disulfide is sufficient to confer redox activity or whether other factors, such as the presence of the cross-strand disulfide in a strained beta-sheet, are required.     

Cross‐strand disulfides in the hydrogen bonding site of antiparallel β‐sheet (aCSDhs): Forbidden disulfides that are highly strained,easily broken

Some disulfide bonds perform important structural roles in proteins, but another group has functional roles via redox reactions. Forbidden disulfides are stressed disulfides found in recognizable protein contexts, which currently constitute more than 10% of all disulfides in the PDB. They likely have functional redox roles and constitute a major subset of all redox‐active disulfides. The torsional strain of forbidden disulfides is typically higher than for structural disulfides, but not so high as to render them immediately susceptible to reduction under physionormal conditions. Previously we characterized the most abundant forbidden disulfide in the Protein Data Bank, the aCSDn: a canonical motif in which disulfide‐bonded cysteine residues are positioned directly opposite each other on adjacent anti‐parallel β‐strands such that the backbone hydrogen‐bonded moieties are directed away from each other. Here we perform a similar analysis for the aCSDh, a less common motif in which the opposed cysteine residues are backbone hydrogen bonded. Oxidation of two Cys in this context places significant strain on the protein system, with the β‐chains tilting toward each other to allow disulfide formation. Only left‐handed aCSDh conformations are compatible with the inherent right‐handed twist of β‐sheets. aCSDhs tend to be more highly strained than aCSDns, particularly when both hydrogen bonds are formed. We discuss characterized roles of aCSDh motifs in proteins of the dataset, which include catalytic disulfides in ribonucleotide reductase and ahpC peroxidase as well as a redox‐active disulfide in P1 lysozyme, involved in a major conformation change. The dataset also includes many binding proteins.     

Cholera toxin does not impair hormonal inhibition of adenylate cyclase and concomitant stimulation of a GTPase in adipocyte membranes

Factor H of the human complement system exhibits an unusual circular dichroism spectrum. The CD spectrum of Factor H exhibits a positive extreme at 230 nm and a negative extreme at 190 nm. No apparent alpha-helical or beta-sheet conformations were present in the native protein structure. However, when the disulfide bridges are reduced, followed either by reoxidation or alkylation, the structure of Factor H is modified so that it now exhibits conventional protein secondary structure as determined from its CD spectra in the far ultraviolet region. Factor H also fails to mediate its regulatory function of inhibiting the alternative pathway convertase once the disulfides have been ruptured and conformational rearrangement has occurred. CD studies indicate that minor conformational changes take place when Factor H and C3b associate in free solution.     

The disulfide-coupled folding pathway of apamin as derived from diselenide-quenched analogs and intermediates.

The sequence of apamin, an 18 residue bee venom toxin, encloses all the information required for the correct disulfide-coupled folding into the cystine-stabilized alpha-helical motif. Three apamin analogs, each containing a pair of selenocysteine residues replacing the related cysteines, were synthesized to mimic the three possible apamin isomers with two crossed, parallel, or consecutive disulfides, respectively. Refolding experiments clearly revealed that the redox potential of selenocysteine prevails over the sequence encoded structural information for proper folding of apamin. Thus, selenocysteine can be used as a new device to generate productive and nonproductive folding intermediates of peptides and proteins. In fact, disulfides are selectively reduced in presence of the diselenide and the conformational features derived from these intermediates as well as from the three-dimensional (3D) structures of the selenocysteine-containing analogs with their nonnatural networks of diselenide/disulfide bridges allowed to gain further insight into the subtle driving forces for the correct folding of apamin that mainly derive from local conformational preferences.     

Synthesis, solution structure, binding activity, and cGMP activation of human guanylin and its disulfide isomer

Guanylin is a recently isolated peptide consisting of 15 amino acid residues with four cysteines, which may form two intramolecular disulfide bridges, and stimulates intestinal membrane guanylate cyclase. The position of the disulfide linkages of guanylin was predicted from its structural similarity to a heat stable enterotoxin which is thought to be responsible for secretory diarrhoea. Both guanylin, with disulfide positions 4–12 and 7–15, and its disulfide isomer, with disulfides positions 4–15 and 7–12, were chemically synthesized by the solid-phase method and purified. Two specific disulfides were selectively formed and confirmed by sequencing, mass spectrometry and high-performance liquid chromatography in combination with enzymatic cleavage. The structure of both isomers has been investigated in solution by ¹H nuclear magnetic resonance spectroscopy. Guanylin exists as a mixture of two stable conformations which have compact spiral structures, from comparison with literature data. In contrast, the disulfide isomer of guanylin shows only a single conformation with an elongated curved plate-like structure. Binding assays were performed using labelled guanylin with membranes obtained from rat jejunum. Both disulfide isomers were investigated by the cGMP assay. Both binding and cGMP assays indicated that the relevant form of disulfide bridges in the intact guanylin was as predicted.     

Chemistry on a single protein, vascular cell adhesion molecule-1, during forced unfolding

Proteins of many types experience tensile forces in their normal function, and vascular cell adhesion molecule-1 (VCAM-1) is typical in this. VCAM has seven Ig domains, and each has a disulfide bond (-S-S-) buried in its core that covalently stabilizes about half of each domain against unfolding. VCAM is extended here by single molecule atomic force microscopy in the presence or absence of reducing agents. In the absence of reducing agent, a sawtooth pattern of forced unfolding reveals an average period and total length consistent with disulfide locations in VCAM. With increasing reducing agent, accessible disulfides are specifically reduced (to SH); the average period for unfolding increases up to saturation together with additional metrics of unfolding. Steered molecular dynamics simulations of unfolding indeed show that the core disulfide bond is solvent-exposed in the very earliest stages of protein extension. Michaelis-Menten kinetics emerge with reduction catalyzed by force (tau(reduction) approximately 10(-4) s). The results establish single molecule reduction, one bond at a time, and show that mechanical forces can play a key role in modulating the redox state of cell adhesion proteins that are invariably stressed in cell adhesion.     

Steps in reductive activation of the disulfide-generating enzyme Ero1p

Ero1p is the primary catalyst of disulfide bond formation in the yeast endoplasmic reticulum (ER). Ero1p contains a pair of essential disulfide bonds that participate directly in the electron transfer pathway from substrate thiol groups to oxygen. Remarkably, elimination of certain other Ero1p disulfides by mutation enhances enzyme activity. In particular, the C150A/C295A Ero1p mutant exhibits increased thiol oxidation in vitro and in vivo and interferes with redox homeostasis in yeast cells by hyperoxidizing the ER. Inhibitory disulfides of Ero1p are thus important for enzyme regulation. To visualize the differences between de-regulated and wild-type Ero1p, we determined the crystal structure of Ero1p C150A/C295A. The structure revealed local changes compared to the wild-type enzyme around the sites of mutation, but no conformational transitions within 25 Å of the active site were observed. To determine how the C150—C295 disulfide nonetheless participates in redox regulation of Ero1p, we analyzed using mass spectrometry the changes in Ero1p disulfide connectivity as a function of time after encounter with reducing substrates. We found that the C150—C295 disulfide sets a physiologically appropriate threshold for enzyme activation by guarding a key neighboring disulfide from reduction. This study illustrates the diverse and interconnected roles that disulfides can play in redox regulation of protein activity.     

The reactivity of the disulfide bonds of human serum haemopexin

The reactivity of the disulfide bonds of the specific haeme-binding plasma protein-human haemopexin has been studied with 2-mercaptoethanol. A molecule of haemopexin has six intrachain disulfide bridges (Takahashi et al., 1985) or which four are reactive while the remaining two can be reduced in the presence of greater than or equal to 4M urea. Disruption of the four reactive disulfide bonds in apohaemopexin abolishes the haeme binding ability. In equimolar haeme-haemopexin complex only one disulfide is reactive which suggests a large change in the tertiary structure of this protein on haeme binding.     

Structures of the alpha L I domain and its complex with ICAM-1 reveal a shape-shifting pathway for integrin regulation

The structure of the I domain of integrin alpha L beta 2 bound to the Ig superfamily ligand ICAM-1 reveals the open ligand binding conformation and the first example of an integrin-IgSF interface. The I domain Mg2+ directly coordinates Glu-34 of ICAM-1, and a dramatic swing of I domain residue Glu-241 enables a critical salt bridge. Liganded and unliganded structures for both high- and intermediate-affinity mutant I domains reveal that ligand binding can induce conformational change in the alpha L I domain and that allosteric signals can convert the closed conformation to intermediate or open conformations without ligand binding. Pulling down on the C-terminal alpha 7 helix with introduced disulfide bonds ratchets the beta 6-alpha 7 loop into three different positions in the closed, intermediate, and open conformations, with a progressive increase in affinity.     

Identification of disulfide-bridged substructures within human von Willebrand factor

In the course of identifying substructural domains within the homooligomeric protein von Willebrand factor [270 kilodaltons (kDa) per polypeptide chain], seven large fragments of 8-90 kDa have been generated by limited proteolysis. A monomeric fragment that binds coagulation factor VIIIc is identified as residues 1-272. A fragment that binds platelet glycoprotein Ib is identified as a homodimer containing two pairs of identical chains, i.e., residues 273-511 and 674-728. Disulfide bonds have been identified by several methods, including direct observation of the phenylthiohydantoin of cystine during Edman degradation of isolated peptides. Among half-cystine residues in the amino-terminal 1365-residue region, 52 have been paired. They place structural constraints on folding possibilities within three structural domains. Additional clusters of disulfide bonds are evident. It has been shown that at least 35 disulfides must form intrachain bridges, specifically the cystines among residues 1-272 and 906-1492. Intersubunit disulfide bonds are partially localized in an interior region (residues 283-695) and a carboxyl-terminal region (residues 1908-2050). Each of these regions appears to be linked to a corresponding region of a neighboring subunit in the network of interconnected chains. The difficulties of pairing all 169 half-cystines (per chain) and of distinguishing intrachain from interchain disulfides are evaluated.     

Conformational preferences in diglycosyl disulfides: NMR and molecular modeling studies

The conformations of several β1→β1′ diglycosyl disulfides were investigated by NMR and computational methods. Experimental data, such as NOEs, proton–proton and proton–carbon-13 coupling constants, measured for solutions in DMSO, are in good agreement with values obtained by MD simulations in explicit DMSO. The disulfide torsion angles (C1–S–S–C1′) preferentially sample values close to either +90° or −90° (+g or −g) and appear as the main metric that determines the conformational behavior of these glycomimetics. There is more conformational freedom around the C1–S and C1′–S′ bonds (Φ and Ω torsions, respectively) and population cluster analysis allowed to identify up to four allowed conformational regions for each of the +g or −g forms. Population analysis of the hydroxylic group rotamers, based on proton–proton and proton–carbon-13 couplings as well as on calculated hydrogen bonding statistics, did not reveal any significant intramolecular hydrogen bonds in DMSO solution.     

Conformations of cysteine disulfides of peptide toxins: Advantage of differentiating forward and reverse asymmetric disulfide conformers

Conformations of cysteine disulfides were analyzed in X-ray, nuclear magnetic resonance (NMR), and co-crystal structures of peptide toxins retrieved from Protein Data Bank. The parameters side chain torsional angles, disulfide strain energy, interatomic Cα/Cβ distances, and Ramachandran angles were used as probes to derive conformational features of cysteine disulfides. Schmidt, Ho, and Hogg (2006 Schmidt, B., Ho, L., &; Hogg, P. J. (2006). Allosteric disulfide bonds. Biochemistry, 45, 7429–7433.[Crossref], [PubMed], [Web of Science ®] , [Google Scholar]) Allosteric disulfide bonds. Biochemistry, 45, 7429–7433 scheme was adapted to classify the disulfide conformations of peptide toxins. Anomalies were observed while treating “forward” and “reverse” asymmetric disulfide conformers as same disulfide conformation in peptide toxins. Thus, new scheme was proposed to classify “forward” and “reverse” asymmetric disulfide conformers separately. Total available conformers space for classification of toxins disulfides is 32. Interestingly, all 32 disulfide conformations are observed in peptide toxins. –LHSpiral is predominant disulfide conformation of peptide toxins. Significant variations were observed in population of occurrence of disulfide conformers, disulfide strain energy, and distribution of D_Cα-Cα and D_Cβ-Cβ values between X-ray, NMR, and co-crystal structures of peptide toxins. The observed differences in conformations of disulfides of same peptide toxins between different states were used as platform to demonstrate advantage of differentiating forward and reverse asymmetric disulfide conformers. Newly proposed scheme allows accurate representation of true conformational diversity of disulfides between X-ray and NMR structures of same peptide toxins. Newly proposed scheme also permits to derive additional structural information from nomenclature which was illustrated by comparing conformations of disulfides between unbound and bound form of toxin with channel/receptor. The results will be of interest for growing field of structural venomics and conformational analysis of peptide/protein disulfides.

Communicated by Ramaswamy H. Sarma     

Identification of Allosteric Disulfides from Prestress Analysis

Disulfide bonds serve to form physical cross-links between residues in protein structures, thereby stabilizing the protein fold. Apart from this purely structural role, they can also be chemically active, participating in redox reactions, and they may even potentially act as allosteric switches controlling protein functions. Specific types of disulfide bonds have been identified in static protein structures from their distinctive pattern of dihedral bond angles, and the allosteric function of such bonds is purported to be related to the torsional strain they store. Using all-atom molecular-dynamics simulations for ∼700 disulfide bonded proteins, we analyzed the intramolecular mechanical forces in 20 classes of disulfide bonds. We found that two particular classes, the −RHStaple and the −/+RHHook disulfides, are indeed more stressed than other disulfide bonds, but the stress is carried primarily by stretching of the S-S bond and bending of the neighboring bond angles, rather than by dihedral torsion. This stress corresponds to a tension force of magnitude ∼200 pN, which is balanced by repulsive van der Waals interactions between the cysteine Cα atoms. We confirm stretching of the S-S bond to be a general feature of the −RHStaples and the −/+RHHooks by analyzing ∼20,000 static protein structures. Given that forced stretching of S-S bonds is known to accelerate their cleavage, we propose that prestress of allosteric disulfide bonds has the potential to alter the reactivity of a disulfide, thereby allowing us to readily switch between functional states.     

Conformational selection and folding-upon-binding of intrinsically disordered protein CP12 regulate photosynthetic enzymes assembly

Carbon assimilation in plants is regulated by the reduction of specific protein disulfides by light and their re-oxidation in the dark. The redox switch CP12 is an intrinsically disordered protein that can form two disulfide bridges. In the dark oxidized CP12 forms an inactive supramolecular complex with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase, two enzymes of the carbon assimilation cycle. Here we show that binding of CP12 to GAPDH, the first step of ternary complex formation, follows an integrated mechanism that combines conformational selection with induced folding steps. Initially, a CP12 conformation characterized by a circular structural motif including the C-terminal disulfide is selected by GAPDH. Subsequently, the induced folding of the flexible C-terminal tail of CP12 in the active site of GAPDH stabilizes the binary complex. Formation of several hydrogen bonds compensates the entropic cost of CP12 fixation and terminates the interaction mechanism that contributes to carbon assimilation control.     

Effects of Macromolecular Crowding on Protein Conformational Changes

Many protein functions can be directly linked to conformational changes. Inside cells, the equilibria and transition rates between different conformations may be affected by macromolecular crowding. We have recently developed a new approach for modeling crowding effects, which enables an atomistic representation of “test” proteins. Here this approach is applied to study how crowding affects the equilibria and transition rates between open and closed conformations of seven proteins: yeast protein disulfide isomerase (yPDI), adenylate kinase (AdK), orotidine phosphate decarboxylase (ODCase), Trp repressor (TrpR), hemoglobin, DNA β-glucosyltransferase, and Ap₄A hydrolase. For each protein, molecular dynamics simulations of the open and closed states are separately run. Representative open and closed conformations are then used to calculate the crowding-induced changes in chemical potential for the two states. The difference in chemical-potential change between the two states finally predicts the effects of crowding on the population ratio of the two states. Crowding is found to reduce the open population to various extents. In the presence of crowders with a 15 Å radius and occupying 35% of volume, the open-to-closed population ratios of yPDI, AdK, ODCase and TrpR are reduced by 79%, 78%, 62% and 55%, respectively. The reductions for the remaining three proteins are 20–44%. As expected, the four proteins experiencing the stronger crowding effects are those with larger conformational changes between open and closed states (e.g., as measured by the change in radius of gyration). Larger proteins also tend to experience stronger crowding effects than smaller ones [e.g., comparing yPDI (480 residues) and TrpR (98 residues)]. The potentials of mean force along the open-closed reaction coordinate of apo and ligand-bound ODCase are altered by crowding, suggesting that transition rates are also affected. These quantitative results and qualitative trends will serve as valuable guides for expected crowding effects on protein conformation changes inside cells.     

Disulfide conformation and design at helix N‐termini

To understand structural and thermodynamic features of disulfides within an α‐helix, a non‐redundant dataset comprising of 5025 polypeptide chains containing 2311 disulfides was examined. Thirty‐five examples were found of intrahelical disulfides involving a CXXC motif between the N‐Cap and third helical positions. GLY and PRO were the most common amino acids at positions 1 and 2, respectively. The N‐Cap residue for disulfide bonded CXXC motifs had average (?,ψ) values of (?112 ± 25.2°, 106 ± 25.4°). To further explore conformational requirements for intrahelical disulfides, CYS pairs were introduced at positions N‐Cap‐3; 1,4; 7,10 in two helices of an Escherichia coli thioredoxin mutant lacking its active site disulfide (nSS Trx). In both helices, disulfides formed spontaneously during purification only at positions N‐Cap‐3. Mutant stabilities were characterized by chemical denaturation studies (in both oxidized and reduced states) and differential scanning calorimetry (oxidized state only). All oxidized as well as reduced mutants were destabilized relative to nSS Trx. All mutants were redox active, but showed decreased activity relative to wild‐type thioredoxin. Such engineered disulfides can be used to probe helix start sites in proteins of unknown structure and to introduce redox activity into proteins. Conversely, a protein with CYS residues at positions N‐Cap and 3 of an α‐helix is likely to have redox activity. Proteins 2010. © 2009 Wiley‐Liss, Inc.