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The labile disulfides are characterized by high dihedral strain energy, elongation of the sulfur–sulfur bond distance and stretching of the neighbouring bond angles. (a) Angles and distances of the cystine residue. The values α1 and α2 represent the two relevant bending angles of the disulfide, and the five dihedral angles are χ¹, χ², χ³, χ2′ and χ1′. d is the sulfur–sulfur bond length. (b) Relative frequency of DSE ranging from 0 to 60 kJ mol⁻¹. The DSE was significantly increased for labile disulfides compared to all disulfide bonds in the PDB (p < 0.0001). (c) The relative frequency of the sulfur–sulfur bond distance ranging from 1.96 to 2.14 Å is shown. An increase in sulfur–sulfur bond distance is observed for labile disulfide bonds (p < 0.0001). (d) The average of both α angles was calculated for each disulfide bond. Shown is the relative frequency of the average angle ranging from 95 to 120°. Angles are increased for labile disulfide bonds (p < 0.0001). T-tests were used to compare total PDB to labile disulfides.
Source publication
Protein disulfide bonds link pairs of cysteine sulfur atoms and are either structural or functional motifs. The allosteric disulfides control the function of the protein in which they reside when cleaved or formed. Here, we identify potential allosteric disulfides in all Protein Data Bank X-ray structures from bonds that are present in some molecul...
Citations
... These have been categorized as redox switches or allosteric disulfides, see for instance [4,5]. Structurally, this group exhibits a narrow range of conformations of the disulfide bond characterized by high mean potential energy, high average torsional strain and stretching of the sulfur-sulfur bond [6,7]. Disulfides may also be formed between protein thiols and low molecular weight thiols such as the almost ubiquitously highly abundant tripeptide glutathione: γ-glutamyl-cysteinyl-glycine. ...
... For the identification of disulfide containing structures and their reduced counterparts, we have employed the strategy described in [7]. In brief, all X-ray structures released in the PDB with disulfide bonds present in any chains were identified by the presence of an SSBOND line in the PDB file. ...
... were recorded in about 20% of the disulfide-bonded and reduced protein pairs, some of which had been characterized before, for instance the catalytic disulfides of arsenate reductase [45], peroxiredoxins [46], and methionine sulfoxidases [47], as well as the allosteric disulfides of the intracellular chloride ion channel CLIC1 [48], and protein tyrosine phosphatases [49]. The median distances of the Cα atoms of the two Cys residues shorten by 0.82 Å from 6.37 to 5.55 Å and the two Sγ approach each other by 1.97 Å from 3.99 to 2.04 Å median distance upon disulfide formation (Fig. 1) [7]. Based on the disulfide conformations, a web interface was implemented to obtain the disulfide bond characteristics from PDB structures to identify potential functional disulfides [50]. ...
Redox modifications of specific cysteinyl and methionyl residues regulate key enzymes and signal-transducing proteins in various pathways. Here, we analyzed the effect of redox modifications on protein structure screening the RCSB protein data bank for oxidative modifications of proteins, i.e. protein disulfides, mixed disulfides with glutathione, cysteinyl sulfenic acids, cysteinyl S-nitrosylation, and methionyl sulfoxide residues. When available, these structures were compared to the structures of the same proteins in the reduced state with respect to both pre-requirements for the oxidative modifications as well as the structural consequences of the modifications. In general, the conformational changes induced by the redox modification are small, i.e. within the range of normal fluctuations. Some redox modifications, disulfides in particular, induces alterations in the electrostatic properties of the proteins. Solvent accessibility does not seem to be a strict pre-requirement for the redox modification of a particular residue. We identified an enrichment of certain other amino acid residues in the vicinity of the susceptible residues, for disulfide and sulfenic acid modifications, for instance, histidyl and tyrosyl residues. These motifs, as well as the specific features of the susceptible sulfur-containing amino acids, may become helpful for the prediction of redox modifications.
... Intriguingly, bridge n. 2 has been identified as labile in two group IIA sPLAs, the human one and the PLA2 homologue of Bothrops pirajai, piratoxin-I, as it is present in some but not all structures of the proteins. This suggests that it can be an allosteric bridge, i.e., a bridge that confers different activities to the protein depending on its state, open or closed [45]. A labile bridge was also identified in a group I sPLA2 of Naja atra (Uniprot entry P00598), the one corresponding to number 8 in Figure 1 [45]. ...
... This suggests that it can be an allosteric bridge, i.e., a bridge that confers different activities to the protein depending on its state, open or closed [45]. A labile bridge was also identified in a group I sPLA2 of Naja atra (Uniprot entry P00598), the one corresponding to number 8 in Figure 1 [45]. ...
Secreted phospholipases of type A2 (sPLA2s) are proteins of 14–16 kDa present in mammals in different forms and at different body sites. They are involved in lipid transformation processes, and consequently in various immune, inflammatory, and metabolic processes. sPLA2s are also major components of snake venoms, endowed with various toxic and pharmacological properties. The activity of sPLA2s is not limited to the enzymatic one but, through interaction with different types of molecules, they exert other activities that are still little known and explored, both outside and inside the cells, as they can be endocytosed. The aim of this review is to analyze three features of sPLA2s, yet under-explored, knowledge of which could be crucial to understanding the activity of these proteins. The first feature is their disulphide bridge pattern, which has always been considered immutable and necessary for their stability, but which might instead be modulable. The second characteristic is their ability to undergo various post-translational modifications that would control their interaction with other molecules. The third feature is their ability to participate in active molecular condensates both on the surface and within the cell. Finally, the implications of these features in the design of anti-inflammatory drugs are discussed.
... Samples were then separated by SDS-PAGE and visualised. As shown in the upper panel of Fig 2A, both enzymic and chemical reduction results in the appearance of bands corresponding to the proteins of interest, or an increase in their intensity, indicative of an increase in free cysteines; this confirmed we were able to reduce labile disulfides using both TCEP and thioredoxin and detect cysteine labelling in proteins previously reported to contain labile disulfide bonds, including factor (F) XI, von Willebrand factor (VWF), and beta glycoprotein I [13,21,[54][55][56][57]. Furthermore, in agreement with previous reports [26], we can also detect an increase in free cysteine labelling when recombinant B domain deleted (BDD) FVIII (BDD rFVIII) is incubated under chemical or enzymic reducing conditions (Fig 2B). ...
... Where suitable structures are available, some prediction can be made with regards to whether a disulfide bond may be redox labile or not. Using structural parameters like solvent accessibility or the distance between the alpha-carbons of the constituent cysteines and their 3D conformation [56], one can get an indication of how redox labile a given disulfide bond may be. Allosteric disulfide bonds tend to have high torsional bond strain as depicted by shorter alpha carbon distances; the −RHStaple and the −/+RHHook atomic arrangements are particularly prevalent conformations for allosteric disulfide bonds and more likely to undergo cleavage [57]. ...
Allosteric disulfide bonds permit highly responsive, transient ‘switch-like’ properties that are ideal for processes like coagulation and inflammation that require rapid and localised responses to damage or injury. Haemophilia A (HA) is a rare bleeding disorder managed with exogenous coagulation factor(F) VIII products. FVIII has eight disulfide bonds and is known to be redox labile, but it is not known how reduction/oxidation affects the structure-function relationship, or its immunogenicity—a serious complication for 30% severe HA patients. Understanding how redox-mediated changes influence FVIII can inform molecular engineering strategies aimed at improving activity and stability, and reducing immunogenicity. FVIII is a challenging molecule to work with owing to its poor expression and instability so, in a proof-of-concept study, we used molecular dynamics (MD) to identify which disulfide bonds were most likely to be reduced and how this would affect structure/function; results were then experimentally verified. MD identified Cys1899-Cys1903 disulfide as the most likely to undergo reduction based on energy and proximity criteria. Further MD suggested this reduction led to a more open conformation. Here we present our findings and highlight the value of MD approaches.
... However, such a disulfide bridge could be labile under many biological processes. 15 Therefore in the present work, we designed a tiger-17 analog, assisted by iAMPpred, 16 with head-to-tail cyclic property (c-KCWHCRPKPKP) to make the difference in the type of cyclic peptide. Nowadays, molecular dynamics simulation has become an accurate and reliable tool to investigate the behavior of lipid bilayers, including their behavior in the presence of AMPs. ...
Tiger-17 analog, a cyclo-undodecapeptide, is a promising antimicrobial peptide (AMP). This study evaluated the potency of the tiger-17 analog as an antimicrobial agent using molecular dynamics simulation. In this work, we employed 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) and zwitterionic 1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine (POPC). POPG is the component of an anionic bacterial-mimicking membrane, whereas POPC represents the human cell membrane. We analyzed the membrane disruption by visualizing the snapshots and calculated some parameters, including total hydrogen bonds, area per lipid, surface accessible surface area, radial distribution functions, electron density profile, and nonbonded interaction energies. Our study suggested that the tiger17 analog approaches the POPG membrane within 100 ns of the simulation. The peptide interacts stronger with the POPG than the POPC membrane during the simulation, supported by all analysis parameters calculated in this work. The tiger-17 analog exhibits a potential antimicrobial property with selective toxicity towards the bacterial-mimicking membrane, according to molecular dynamics simulation results. Therefore, further development of the tiger-17 analog into an antimicrobial agent is worth trying
... Previous studies based on the crystal structure of E suggested that the FL disulfide maintains the conformation of FL; however, USUV and recent cryo-EM structures reveal little conformational variations while displaying other sulfhydryl interactions at the site (SI Appendix, Fig. S8) (28,52). The FL disulfide site is fundamentally distinct from the other five sites: both Cys residues reside on a loop rather than a secondary structure like a β-strand, a feature of labile disulfide bonds, and the FL containing Cys105 is crucial for viral fusion and pathogenesis (53)(54)(55). Disulfides are particularly prone to radiation damage in X-ray diffraction and cryo-EM studies, which can be site-specific or preferential specific damage (56). However, to the best of our knowledge, a quantitative analysis of FL disulfide response to cryo-EM radiation dose has not been performed. ...
Significance
Usutu virus (USUV) was first identified in Africa in 1959; however, multiple, independent introductions via migratory birds and vector proliferation facilitated by climate change expanded the ecological niche of USUV across Europe. In humans, some African USUV lineages typically cause mild disease; however, the European strains, marked by their generally asymptomatic existence, possess the ability to cause meningoencephalitis and other neurological complications in some individuals. Treatment options for such infections are lacking. Our work provides the structural understanding into USUV biology and provides insight into pathogenesis for the development of therapies. The high-resolution structures highlight unique USUV characteristics as well as provide distinct details for the field of flavivirus biology, each with functional implications in the viral life cycle.
... We next inspected X-ray structures of the H1, H2, and H3 proteins for disulfide bonds that are present in some molecules of a protein crystal but absent in others. We hypothesized that such disulfides might naturally exist in unformed states (i.e., present as free thiols) in the native protein (41). ...
Aims:
Influenza A virus hemagglutinin (HA) binding to sialic acid on lung epithelial cells triggers membrane fusion and infection. Host thiol isomerases have been shown to play a role in Influenza A virus infection and we hypothesised this role involved manipulation of disulfide bonds in HA.
Results:
Analysis of HA crystal structures revealed that three of the six HA disulfides occur in high energy conformations and four of the six bonds can exist in unformed states, suggesting that the disulfide landscape of HA is generally strained and the bonds may be labile. We measured the redox state of Influenza A virus HA disulfide bonds and their susceptibility to cleavage by vascular thiol isomerases. Using differential cysteine alkylation and mass spectrometry, we show that all six HA disulfide bonds exist in unformed states in approximately one in ten recombinant and viral surface HA molecules. Four of the six H1 and H3 HA bonds are cleaved by the vascular thiol isomerases, thioredoxin and protein disulphide isomerase, in recombinant proteins, which correlated with surface exposure of the disulfides in crystal structures. In contrast, viral surface HA disulfide bonds are impervious to five different vascular thiol isomerases.
Innovation:
It has been assumed that the disulfide bonds in mature HA protein are intact and inert. We show that all six HA disulfide bonds can exist in unformed states.
Conclusion:
These findings indicate that Influenza A virus HA disulfides are naturally labile but not substrates for thiol isomerases when expressed on the viral surface.
... Because the disulfide bond is very weak [20], careful attention is required to obtain maximal efficacy at the same dose when saline reconstitution is used. When reconstituting BoNT-A with normal saline, one should not shake the vial vigorously. ...
Botulinum toxin type A (BoNT-A), onabotulinumtoxinA (Botox) was approved by the United States Food and Drug Administration for temporary improvement of glabellar lines in patients 65 years and younger in 2002, and has also been used widely for aesthetic purposes such as hyperhidrosis, body shape contouring, and other noninvasive facial procedures. BoNT-A inhibits presynaptic exocytosis of acetylcholine (ACh)-containing vesicles into the neuromuscular junction at cholinergic nerve endings of the peripheral nervous system, thereby paralyzing skeletal muscles. ACh is the most broadly used neurotransmitter in the somatic nervous system, preganglionic and postganglionic fibers of parasympathetic nerves, and preganglionic fibers or postganglionic sudomotor nerves of sympathetic nerves. The scientific basis for using BoNT-A in various cosmetic procedures is that its function goes beyond the dual role of muscle paralysis and neuromodulation by inhibiting the secretion of ACh. Although the major target organs for aesthetic procedures are facial expression muscles, skeletal body muscles, salivary glands, and sweat glands, which are innervated by the somatic or autonomic nerves of the peripheral cholinergic nerve system, few studies have attempted to directly explain the anatomy of the areas targeted for injection by addressing the neural physiology and rationale for specific aesthetic applications of BoNT-A therapy. In this article, we classify the various cosmetic uses of BoNT-A according to the relevant component of the peripheral nervous system, and describe scientific theories regarding the anatomy and physiology of the cholinergic nervous system. We also review critical physiological factors and conditions influencing the efficacy of BoNT-A for the rational aesthetic use of BoNT-A. We hope that this comprehensive review helps promote management policies to support long-term, safe, successful practice. Furthermore, based on this, we look forward to developing and expanding new advanced indications for the aesthetic use of BoNT-A in the future.
... In practice, the mean distance and angle in 13,030 types of proteins are 104.7 • and 2.046Å, respectively. Additionally, SS bonds are rendered labile by increased conformational stress of the secondary structure if the angle is stretched or distance is elongated (Pijning et al., 2018). Our analyses, conducted using 3D models of SA, reveal that the distances between sulfur atoms in all SS bonds were appropriate and similar between HSA and DSA-HM. ...
... Sequence propriety was confirmed by N-terminus sequencing, which showed that mature DSA was obtained after the removal of the N-terminal pro-peptide (six residues) and signal peptide (18 residues) that are common to HSA (Peters, 1995). However, in DSA-HM, the angle between the sulfur and β-carbon atoms showed increased deviation, in which two SS bonds ( 123 Cys-168 Cys with 106.20 • and 108.28 • , and 277 Cys-288 Cys with 108.72 • and 106.61 • , respectively, Supplementary Table S2) had stretching angles of over 106.01 • in both cysteine residues; this is the mean value of labile SS bonds in 551 types of proteins (Pijning et al., 2018). Conversely, in 559 Cys, 106.01 • was the greatest stretching angle in the SS bonds of HSA. ...
Serum albumin (SA), the most abundant protein in circulation, functions as a carrier protein, osmoregulator, and antioxidant. Generally, SA exerts its antioxidative effects by scavenging reactive oxygen species. Because marine mammals are superior divers, they are intermittently exposed to oxidative stress induced by rapid reperfusion of oxygen to ischemic tissues after the dive. Although several antioxidants in marine mammals have been described, SA activity remains largely uncharacterized. In this study, we investigated the antioxidative activity of SA in marine mammals by comparing features of the primary and steric structures, biochemical properties, and antioxidative activities of common bottlenose dolphin SA (DSA) and human SA (HSA). Our results revealed that DSA lacked free cysteine at position 34 that is important for the antioxidative activity of HSA; however, the antioxidative capacity and thiol activity of DSA were stronger than those of HSA. Circular dichroism spectra showed different patterns in DSA and HSA. Ultraviolet fluorescence intensities of DSA were higher than those of HSA, suggesting lower surface hydrophobicity of DSA. Additionally, DSA showed higher excess heat capacity than HSA. We then compared a homology model of DSA with a 3D model of HSA. Our results indicate that DSA was more unstable than HSA at least in the body-temperature range, probably due to the mode of molecules involved in the disulfide bonds and/or the lower surface hydrophobicity, and it may be related to the equivalent or stronger antioxidant potency of DSA. These data show that DSA is an effective antioxidant in the circulation of the dolphin.
... ACTG2 WT and ACTG2 R257C have similar allowed and favorable dihedral angles and minor differences in outlier residues with unusual dihedral angles. Finally, unlike R257, C257 might interact with C218 (Supplemental Figure 3), but thermodynamics of potential disulfide bond formation between these cysteine residues are unknown and distance is approximately 3.2-fold longer than a disulfide bond in these models (26). Collectively, these observations suggest that the effects of ACTG2 R257C on ACTG2 secondary structure are not dramatic, although molecular dynamics simulations or modeling with F-actin-binding proteins, such as tropomyosin, might show additional problems. ...
Actin γ 2, smooth muscle (ACTG2) R257C mutation is the most common genetic cause of visceral myopathy. Individuals with ACTG2 mutations endure prolonged hospitalizations and surgical interventions, become dependent on intravenous nutrition and bladder catheterization, and often die in childhood. Currently, we understand little about how ACTG2 mutations cause disease, and there are no mechanism-based treatments. Our goal was to characterize the effects of ACTG2R257C on actin organization and function in visceral smooth muscle cells. We overexpressed ACTG2WT or ACTG2R257C in primary human intestinal smooth muscle cells (HISMCs) and performed detailed quantitative analyses to examine effects of ACTG2R257C on (a) actin filament formation and subcellular localization, (b) actin-dependent HISMC functions, and (c) smooth muscle contractile gene expression. ACTG2R257C resulted in 41% fewer, 13% thinner, 33% shorter, and 40% less branched ACTG2 filament bundles compared with ACTG2WT. Curiously, total F-actin probed by phalloidin and a pan-actin antibody was unchanged between ACTG2WT- and ACTG2R257C-expressing HISMCs, as was ultrastructural F-actin organization. ACTG2R257C-expressing HISMCs contracted collagen gels similar to ACTG2WT-expressing HISMCs but spread 21% more and were 11% more migratory. In conclusion, ACTG2R257C profoundly affects ACTG2 filament bundle structure, without altering global actin cytoskeleton in HISMCs.
... We downloaded the dataset of culled crystal structures with unique disulfides from (64) and selected structures with a resolution of less than or equal to 1.5 Å to plot the histogram in Fig. 4B. ...
Aberrant regulation of metabolic kinases by altered redox homeostasis substantially contributes to aging and various diseases, such as diabetes. We found that the catalytic activity of a conserved family of fructosamine-3-kinases (FN3Ks), which are evolutionarily related to eukaryotic protein kinases, is regulated by redox-sensitive cysteine residues in the kinase domain. The crystal structure of the FN3K homolog from Arabidopsis thaliana revealed that it forms an unexpected strand-exchange dimer in which the ATP-binding P-loop and adjoining β strands are swapped between two chains in the dimer. This dimeric configuration is characterized by strained interchain disulfide bonds that stabilize the P-loop in an extended conformation. Mutational analysis and solution studies confirmed that the strained disulfides function as redox “switches” to reversibly regulate the activity and dimerization of FN3K. Human FN3K, which contains an equivalent P-loop Cys, was also redox sensitive, whereas ancestral bacterial FN3K homologs, which lack a P-loop Cys, were not. Furthermore, CRISPR-mediated knockout of FN3K in human liver cancer cells altered the abundance of redox metabolites, including an increase in glutathione. We propose that redox regulation evolved in FN3K homologs in response to changing cellular redox conditions. Our findings provide insights into the origin and evolution of redox regulation in the protein kinase superfamily and may open new avenues for targeting human FN3K in diabetic complications.
