Intersubunit Disulfide Research Articles

Conserved residues of the immunosuppressive domain of MLV are essential for regulating the fusion-critical SU-TM disulfide bond.

The Env protein of murine leukemia virus (MLV) is the prototype of a large clade of retroviral fusogens, collectively known as gamma-type Envs. Gamma-type Envs are found in retroviruses and endogenous retroviruses (ERVs) representing a broad range of vertebrate hosts. All gamma-type Envs contain a highly conserved stretch of 26-residues in the transmembrane subunit (TM) comprising two motifs, a putative immunosuppressive domain (ISD) and a CX6CC motif. Extraordinary conservation of the ISD and its invariant association with the CX6CC suggests a fundamental contribution to Env function. To investigate ISD function, we characterized several mutants with single amino acid substitutions at conserved positions in the MLV ISD. A majority abolished infectivity, although we did not observe a corresponding loss in intrinsic ability to mediate membrane fusion. Ratios of the surface subunit (SU) to capsid protein (CA) in virions were diminished for a majority of the ISD mutants, while TM:CA ratios were similar to wild type. Specific loss of SU reflected premature isomerization of the labile disulfide bond that links SU and TM prior to fusion. Indeed, all non-infectious mutants displayed significantly lower disulfide stability than wild-type Env. These results reveal a role for ISD positions 2, 3, 4, 7, and 10 in regulating a late step in entry after fusion peptide insertion but prior to creation of the fusion pore. This implies that the ISD is part of a larger domain, comprising the ISD and CX6CC motifs, that is critical for the formation and regulation of the metastable, intersubunit disulfide bond.IMPORTANCEThe gamma-type Env is a prevalent viral fusogen, found within retroviruses and endogenous retroviruses across vertebrate species and in filoviruses such as Ebolavirus. The fusion mechanism of gamma-type Envs is unique from other Class I fusogens such as those of influenza A virus and HIV-1. Gamma-type Envs contain a hallmark feature known as the immunosuppressive domain (ISD) that has been the subject of some controversy in the literature surrounding its putative immunosuppressive effects. Despite the distinctive conservation of the ISD, little has been done to investigate the role of this region for the function of this widespread fusogen. Our work demonstrates the importance of the ISD for the function of gamma-type Envs in infection, particularly in regulating the intermediate steps of membrane fusion. Understanding the fusion mechanism of gamma-type Envs has broad implications for understanding the entry of extant viruses and aspects of host biology connected to co-opted endogenous gamma-type Envs.

Physcomitrium patens flavodiiron proteins form heterotetrametric complexes

Flavodiiron proteins (FLVs) catalyze the reduction of oxygen to water by using electrons from Photosystem I (PSI). In several photosynthetic organisms such as cyanobacteria, green algae, mosses and gymnosperms, FLV-dependent electron flow protects PSI from over-reduction and consequent damage especially under fluctuating light conditions. In this work we investigated biochemical and structural properties of FLVA and FLVB from the model moss Physcomitrium patens. The two proteins, expressed and purified from Escherichia coli, bind both iron and flavin cofactors and show NAD(P)H oxidase activity as well as oxygen reductase capacities. Moreover, the co-expression of both FLVA and FLVB, coupled to a tandem affinity purification procedure with two different affinity tags, enabled the isolation of the stable and catalytically active FLVA/B hetero tetrameric protein complex with cooperative nature. The multimeric organization was shown to be stabilized by inter-subunit disulfide bonds. This investigation provides valuable new information on the biochemical properties of FLVs, with new insights into their in vivo activity.

Zinc transporter 1 functions in copper uptake and cuproptosis

Copper (Cu) is a co-factor for several essential metabolic enzymes. Disruption of Cu homeostasis results in genetic diseases such as Wilson's disease. Here, we show that the zinc transporter 1 (ZnT1), known to export zinc (Zn) out of the cell, also mediates Cu2+ entry into cells and is required for Cu2+-induced cell death, cuproptosis. Structural analysis and functional characterization indicate that Cu2+ and Zn2+ share the same primary binding site, allowing Zn2+ to compete for Cu2+ uptake. Among ZnT members, ZnT1 harbors a unique inter-subunit disulfide bond that stabilizes the outward-open conformations of both protomers to facilitate efficient Cu2+ transport. Specific knockout of the ZnT1 gene in the intestinal epithelium caused the loss of Lgr5+ stem cells due to Cu deficiency. ZnT1, therefore, functions as a dual Zn2+ and Cu2+ transporter and potentially serves as a target for using Zn2+ in the treatment of Wilson's disease caused by Cu overload.

Unlocking the gelling potential of oat protein: Synergistic effects of sonication and disulfide cleavage

The high thermal stability of oat globulin, attributed to inter-subunit disulfide bonds and strong hydrophobic interactions, poses a challenge to its gelling capacity. This study investigated the heat-induced gelling properties of oat protein isolate (OPI), focusing on the synergistic effect of physical (high-intensity ultrasound, HIU) and chemical (cysteine-assisted disulfide bond disruption) treatments. Creep-recovery and viscoelasticity (Gʹ/Gʹʹ) tests revealed decreased deformability and enhanced rigidity of OPI intermolecular structure upon cysteine treatment (100–400 mg/g protein), especially for HIU-treated protein. The HIU + cysteine process substantially increased the gel hardness (from 0.005 to 0.69 N) over the individual treatments with a synergy up to 432%. Correspondingly, cooking loss was reduced by 28–98% due to the move of free water into a restricted state (protein-bound protons) within the gel matrix as detected by 1H NMR. These findings suggest new possibilities for utilizing oat protein in gel-based food products.

Absence of calcium-sensing receptor basal activity due to inter-subunit disulfide bridges

G protein-coupled receptors naturally oscillate between inactive and active states, often resulting in receptor constitutive activity with important physiological consequences. Among the class C G protein-coupled receptors that typically sense amino-acids and their derivatives, the calcium sensing receptor (CaSR) tightly controls blood calcium levels. Its constitutive activity has not yet been studied. Here, we demonstrate the importance of the inter-subunit disulfide bridges in maintaining the inactive state of CaSR, resulting in undetectable constitutive activity, unlike the other class C receptors. Deletion of these disulfide bridges results in strong constitutive activity that is abolished by mutations preventing amino acid binding. It shows that this inter-subunit disulfide link is necessary to limit the agonist effect of amino acids on CaSR. Furthermore, human genetic mutations deleting these bridges and associated with hypocalcemia result in elevated CaSR constitutive activity. These results highlight the physiological importance of fine tuning the constitutive activity of G protein-coupled receptors.

Cysteines 1078 and 2991 cross-linking plays a critical role in redox regulation of cardiac ryanodine receptor (RyR)

The most common cardiac pathologies, such as myocardial infarction and heart failure, are associated with oxidative stress. Oxidation of the cardiac ryanodine receptor (RyR2) Ca2+ channel causes spontaneous oscillations of intracellular Ca2+, resulting in contractile dysfunction and arrhythmias. RyR2 oxidation promotes the formation of disulfide bonds between two cysteines on neighboring RyR2 subunits, known as intersubunit cross-linking. However, the large number of cysteines in RyR2 has been a major hurdle in identifying the specific cysteines involved in this pathology-linked post-translational modification of the channel. Through mutagenesis of human RyR2 and in-cell Ca2+ imaging, we identify that only two cysteines (out of 89) in each RyR2 subunit are responsible for half of the channel’s functional response to oxidative stress. Our results identify cysteines 1078 and 2991 as a redox-sensitive pair that forms an intersubunit disulfide bond between neighboring RyR2 subunits during oxidative stress, resulting in a pathological “leaky” RyR2 Ca2+ channel.

Up-regulated oxidized USP2a can increase Mdm2-p60-p53 to promote cell apoptosis

Mdm2 promotes the ubiquitination and degradation of p53, while Mdm2-p60 can bind to p53 and reduce the Mdm2-induced p53 ubiquitination to improve its stability. USP2a can deubiquitinate and stabilize Mdm2, whether USP2a can regulate Mdm2-p60 needs to be further confirmed and elucidated. We found that oxidative stress can up-regulate USP2a at the post-transcriptional level and induce USP2a to be oxidized by forming inter-subunit disulfide bonds. The oxidized USP2a is closely related with cell apoptosis. In apoptotic cells, oxidized USP2a has enhanced protein stability and further stabilizes Mdm2-p60 through deubiquitination, and the USP2a-Mdm2-p60-p53 axis plays a role in cell apoptosis. Altogether USP2a is oxygen sensitive, oxidized USP2a exerts apoptotic effects through the Mdm2-p60-p53 axis, which provides an experimental basis for regulating p53 apoptotic signaling by targeting USP2a.

Intersubunit cross-linking is the key mechanism of cardiac ryanodine receptor dysfunction during oxidative stress: The role of cysteines 1078 and 2991.

The type 2 ryanodine receptor (RyR2) plays a key role in regulating cardiac intracellular Ca2+. We have previously shown that oxidative stress can regulate RyR2 by promoting the formation of disulfide bonds between neighboring RyR2 subunits (intersubunit cross-linking). However, the functional significance of this redox modification and cysteines involved in cross-linking remain unknown. Here, we used site-directed mutagenesis to replace cysteines 1078 and 2991 with serines in human RyR2 (hRyR2). Obtained constructs were expressed in HEK293 cells and treated with diamide to induce oxidative stress. Western blot analysis revealed that both mutations substantially protected against hRyR2 cross-linking. Analysis of the ER lumen [Ca2+] ([Ca2+]ER) dynamics with the ER Ca2+ sensor R-CEPIA1er in HEK293 cells expressing hRyR2 revealed that the mutation of either 1078 or 2991 cysteines prevented the depletion of ER Ca2+ load caused by RyR2 oxidation (by >40%). The simultaneous mutation of both cysteines produced the similar protection against hRyR2 cross-linking and [Ca2+]ER depletion, indicating a common mechanism of redox regulation involving both residues. Given the structural localization of both cysteines at the neighboring RyR2 subunits, we suggest that they form an intersubunit disulfide bond during channel oxidation, followed by the formation of the pathologically “leaky” channel. Although the N-terminal region (NTR) of RyR2 plays an important role in the channel's tetramer formation and functional regulation, the individual cysteines in this region are not directly involved in hRyR2 cross-linking. However, the structural integrity of the NTR has an allosteric effect on hRyR2 complex re-arrangements required for the channel's cross-linking. In conclusion, the results of this study revealed two critical cysteines within hRyR2 that are involved in the intersubunit disulfide cross-linking and the channel's functional response to oxidative stress.

Molecular and cellular mechanisms underlying tissue-specific MICU-dimer assembly in the mitochondrial calcium uniporter complex.

The mitochondria calcium uniporter is a multi-subunit Ca2+ channel that mediates mitochondrial Ca2+ uptake. It regulates multiple physiological processes including oxidative phosphorylation, intracellular Ca2+ signaling, and apoptosis. The uniporter is activated by Ca2+—It remains closed at resting cellular [Ca2+] and becomes open when stimulated by intracellular Ca2+ signals. Previous work suggests that in non-neuronal cells, such Ca2+ activation is exclusively mediated by a MICU1-2 heterodimer. Here, we find that kidney and skeletal muscle uniporters also complex with a MICU1-1 homodimer. Kinetic analyses show that the uniporter is more readily activated by Ca2+ when complexed with MICU1-1 (K0.5=0.6 µM) than MICU1-2 (K0.5=1.2 µM). Thus, tissues can create specific mitochondrial Ca2+ uptake properties by varying the population of MICU1-1 and MICU1-2. Here, we demonstrate two findings to establish the mechanisms by which cells control the relative abundance of these MICU dimers. Firstly, MICU1 has a >50-fold tendency to dimerize with MICU1 than MICU2; Secondly, mitochondria import MICU2 faster than MICU1 to maintain an excess of MICU2 monomers while the MICU1 monomer is depleted. In several types of cells, due to this MICU2 excess in mitochondria, an imported MICU1 monomer can only encounter MICU2 to form MICU1-2 heterodimers. Other types of cells can produce MICU1-1 by importing MICU1 slightly faster so MICU1 could form homodimers due to its higher tendency to homodimerize. Contradicting previous studies, we show that a conserved MICU intersubunit disulfide is not required for the MICUs to regulate uniporter transport. Instead, this disulfide functions to protect properly assembled MICU dimers from proteolytic degradation by YME1L1. These results shed light on the molecular and cellular mechanisms by which different types of cells can produce unique mitochondrial Ca2+ uptake properties to fulfill their unique physiological functions.

Microgravity environment grown crystal structure information based engineering of direct electron transfer type glucose dehydrogenase

The heterotrimeric flavin adenine dinucleotide dependent glucose dehydrogenase is a promising enzyme for direct electron transfer (DET) principle-based glucose sensors within continuous glucose monitoring systems. We elucidate the structure of the subunit interface of this enzyme by preparing heterotrimer complex protein crystals grown under a space microgravity environment. Based on the proposed structure, we introduce inter-subunit disulfide bonds between the small and electron transfer subunits (5 pairs), as well as the catalytic and the electron transfer subunits (9 pairs). Without compromising the enzyme’s catalytic efficiency, a mutant enzyme harboring Pro205Cys in the catalytic subunit, Asp383Cys and Tyr349Cys in the electron transfer subunit, and Lys155Cys in the small subunit, is determined to be the most stable of the variants. The developed engineered enzyme demonstrate a higher catalytic activity and DET ability than the wild type. This mutant retains its full activity below 70 °C as well as after incubation at 75 °C for 15 min – much higher temperatures than the current gold standard enzyme, glucose oxidase, is capable of withstanding.

The MerR-family regulator NmlR is involved in the defense against oxidative stress in Streptococcus pneumoniae.

Streptococcus pneumoniae has to cope with the strong oxidant hypochlorous acid (HOCl), during host-pathogen interactions. Thus, we analyzed the global gene expression profile of S. pneumoniae D39 towards HOCl stress. In the RNA-seq transcriptome, the NmlR, SifR, CtsR, HrcA, SczA and CopY regulons and the etrx1-ccdA1-msrAB2 operon were most strongly induced under HOCl stress, which participate in the oxidative, electrophile and metal stress response in S. pneumoniae. The MerR-family regulator NmlR harbors a conserved Cys52 and controls the alcohol dehydrogenase-encoding adhC gene under carbonyl and NO stress. We demonstrated that NmlR senses also HOCl stress to activate transcription of the nmlR-adhC operon. HOCl-induced transcription of adhC required Cys52 of NmlR in vivo. Using mass spectrometry, NmlR was shown to be oxidized to intersubunit disulfides or S-glutathionylated under oxidative stress in vitro. A broccoli-FLAP-based assay further showed that both NmlR disulfides significantly increased transcription initiation at the nmlR promoter by RNAP in vitro, which depends on Cys52. Phenotype analyses revealed that NmlR functions in the defense against oxidative stress and promotes survival of S. pneumoniae during macrophage infections. In conclusion, NmlR was characterized as HOCl-sensing transcriptional regulator, which activates transcription of adhC under oxidative stress by thiol switches in S. pneumoniae.

Crystal structure of the collagen prolyl 4-hydroxylase (C-P4H) catalytic domain complexed with PDI: Toward a model of the C-P4H α2β2 tetramer

Collagen prolyl 4-hydroxylases (C-P4H) are α2β2 tetramers, which catalyze the prolyl 4-hydroxylation of procollagen, allowing for the formation of the stable triple-helical collagen structure in the endoplasmic reticulum. The C-P4H α-subunit provides the N-terminal dimerization domain, the middle peptide-substrate-binding (PSB) domain, and the C-terminal catalytic (CAT) domain, whereas the β-subunit is identical to the enzyme protein disulfide isomerase (PDI). The structure of the N-terminal part of the α-subunit (N-terminal region and PSB domain) is known, but the structures of the PSB-CAT linker region and the CAT domain as well as its mode of assembly with the β/PDI subunit, are unknown. Here, we report the crystal structure of the CAT domain of human C-P4H-II complexed with the intact β/PDI subunit, at 3.8Å resolution. The CAT domain interacts with the a, b', and a' domains of the β/PDI subunit, such that the CAT active site is facing bulk solvent. The structure also shows that the C-P4H-II CAT domain has a unique N-terminal extension, consisting of α-helices and a β-strand, which is the edge strand of its major antiparallel β-sheet. This extra region of the CAT domain interacts tightly with the β/PDI subunit, showing that the CAT-PDI interface includes an intersubunit disulfide bridge with the a' domain and tight hydrophobic interactions with the b' domain. Using this new information, the structure of the mature C-P4H-II α2β2 tetramer is predicted. The model suggests that the CAT active-site properties are modulated by α-helices of the N-terminal dimerization domains of both subunits of the α2-dimer.

Mechanisms and significance of tissue-specific MICU regulation of the mitochondrial calcium uniporter complex

Mitochondrial Ca2+ uptake, mediated by the mitochondrial Ca2+ uniporter, regulates oxidative phosphorylation, apoptosis, and intracellular Ca2+ signaling. Previous studies suggest that non-neuronal uniporters are exclusively regulated by a MICU1-MICU2 heterodimer. Here, we show that skeletal-muscle and kidney uniporters also complex with a MICU1-MICU1 homodimer and that human/mouse cardiac uniporters are largely devoid of MICUs. Cells employ protein-importation machineries to fine-tune the relative abundance of MICU1 homo- and heterodimers and utilize a conserved MICU intersubunit disulfide to protect properly assembled dimers from proteolysis by YME1L1. Using the MICU1 homodimer or removing MICU1 allows mitochondria to more readily take up Ca2+ so that cells can produce more ATP in response to intracellular Ca2+ transients. However, the trade-off is elevated ROS, impaired basal metabolism, and higher susceptibility to death. These results provide mechanistic insights into how tissues can manipulate mitochondrial Ca2+ uptake properties to support their unique physiological functions.

Gastrointestinal Tract Stabilized Protein Delivery Using Disulfide Thermostable Exoshell System.

Thermostable exoshells (tES) are engineered proteinaceous nanoparticles used for the rapid encapsulation of therapeutic proteins/enzymes, whereby the nanoplatform protects the payload from proteases and other denaturants. Given the significance of oral delivery as the preferred model for drug administration, we structurally improved the stability of tES through multiple inter-subunit disulfide linkages that were initially absent in the parent molecule. The disulfide-linked tES, as compared to tES, significantly stabilized the activity of encapsulated horseradish peroxidase (HRP) at acidic pH and against the primary human digestive enzymes, pepsin, and trypsin. Furthermore, the disulfide-linked tES (DS-tES) exhibited significant intestinal permeability as evaluated using Caco2 cells. In vivo bioluminescence assay showed that encapsulated Renilla luciferase (rluc) was ~3 times more stable in mice compared to the free enzyme. DS-tES collected mice feces had ~100 times more active enzyme in comparison to the control (free enzyme) after 24 h of oral administration, demonstrating strong intestinal stability. Taken together, the in vitro and in vivo results demonstrate the potential of DS-tES for intraluminal and systemic oral drug delivery applications.

Biochemical and structural studies of two tetrameric nucleoside 2′-deoxyribosyltransferases from psychrophilic and mesophilic bacteria: Insights into cold-adaptation

Nucleoside 2′-deoxyribosyltransferases (NDTs) catalyze the cleavage of glycosidic bonds of 2′-deoxynucleosides and the following transfer of the 2′-deoxyribose moiety to acceptor nucleobases. Here, we report the crystal structures and biochemical properties of the first tetrameric NDTs: the type I NDT from the mesophilic bacterium Enterococcus faecalis V583 (EfPDT) and the type II NDT from the bacterium Desulfotalea psychrophila (DpNDT), the first psychrophilic NDT. This novel structural and biochemical data permitted an exhaustive comparative analysis aimed to shed light into the basis of the high global stability of the psychrophilic DpNDT, which has a higher melting temperature than EfPDT (58.5 °C versus 54.4 °C) or other mesophilic NDTs. DpNDT possesses a combination of unusual structural motifs not present neither in EfPDT nor any other NDT that most probably contribute to its global stability, in particular, a large aliphatic isoleucine-leucine-valine (ILV) bundle accompanied by a vicinal disulfide bridge and also an intersubunit disulfide bridge, the first described for an NDT. The functional and structural features of DpNDT do not fit the standard features of psychrophilic enzymes, which lead us to consider the implication of (sub)cellular levels together with the protein level in the adaptation of enzymatic activity to low temperatures.

Structural Investigations of Human A2M Identify a Hollow Native Conformation That Underlies Its Distinctive Protease-Trapping Mechanism

Human α2-macroglobulin (A2M) is the most characterized protease inhibitor in the alpha-macroglobulin (αM) superfamily, but the structure of its native conformation has not been determined. Here, we combined negative stain electron microscopy (EM), small-angle X-ray scattering (SAXS), and cross-linking–mass spectrometry (XL-MS) to investigate native A2M and its collapsed conformations that are obtained through aminolysis of its thiol ester by methylamine or cleavage of its bait region by trypsin. The combined interpretation of these data resulted in a model of the native A2M tetramer and its conformational changes. Native A2M consists of two crescent-shaped disulfide-bridged subunit dimers, which face toward each other and surround a central hollow space. In native A2M, interactions across the disulfide-bridged dimers are minimal, with a single major interface between the linker (LNK) regions of oppositely positioned subunits. Bait region cleavage induces both intrasubunit domain repositioning and an altered configuration of the disulfide-bridged dimer. These changes collapse the tetramer into a more compact conformation, which encloses an interior protease-trapping cavity. A recombinant A2M with a modified bait region was used to map the bait region’s position in native A2M by XL-MS. A second recombinant A2M introduced an intersubunit disulfide into the LNK region, demonstrating the predicted interactions between these regions in native A2M. Altogether, our native A2M model provides a structural foundation for understanding A2M’s protease-trapping mechanism, its conformation-dependent receptor interactions, and the dissociation of native A2M into dimers due to inflammatory oxidative stress.

Construction of Super-Stabilized Direct Electron Transfer Type Glucose Dehydrogenase for Long Term Continuous Glucose Sensing System

Introduction: The development of super-stabilized flavin adenine dinucleotide glucose dehydrogenase (FADGDH) was achieved based on the rational biomolecular engineering.The FADGDH from Burkholderia cepacia (BcGDH) is the ideal enzyme for continuous glucose monitoring (CGM) sensor, because of its inherent DET ability (Yamazaki 2008, Lee 2018). BcGDH is composed of three subunits, which are the catalytic subunit harboring FAD and iron-sulfur (3Fe-4S) cluster, the electron transfer subunit harboring three heme c, and the small subunit which functions as a “hitchhiker” protein. We recently reported the BcGDH catalytic subunit complexed with small subunit (Yoshida et al., 2019, PDB 6a2u), thereby elucidated the intra-molecular electron transfer pathway, and inherent disulfide bond between catalytic subunit and small subunit.BcGDH shows two distinct temperature profiles due to variations in its quaternary structure (Sode 1996, Yamazaki 1999). BcGDH composed of three subunits showed an optimum reaction temperature of approximately 45 ̊C. By increasing temperature, enzyme activity gradually decreases, due to the dissociation of electron transfer subunit. However, at temperature above 60 ̊C, enzyme activity increases again with the increase the temperature and shows second peak at around 70 ̊C, which is derived from the complex composed of catalytic subunit and small subunit. This phenomenon suggested that the dissociation of the electron transfer subunit dominates the stability of BcGDH three subunit complex, which is the essential structure to show DET ability. Namely, to stabilize BcGDH enzyme complex, the stabilization of quaternary structure is the most rational strategy for this enzyme.We recently succeeded in the elucidation of crystal structure of BcGDH complex together with the electron transfer subunit. The availability of quaternary structure of this enzyme enabled us to design inter-subunit disulfide bond in BcGDH. In this study, the designing and characterization of super-stabilized BcGDH is presented. We also characterize the DET ability of the engineered BcGDH, which is the mandatory characteristic of this enzyme for the development of the future innovative biosensing systems. Methods: Amino acid substitution to cysteine residues were carried out at the residues existing on the surface of each subunit, to form inter-subunit disulfide bonds. Recombinantly prepared BcGDH mutant complexes were purified by affinity chromatography. The catalytic activity and thermal stability were investigated. Then DET-type sensors were constructed by immobilization on the gold electrode with self-assembled monolayer (SAM) layer, and investigated their stability by continuous operation. Results and Discussion: Based on the 3D structure of BcGDH complex, disulfide bonds between electron transfer subunit and either catalytic subunit or small subunit were designed. By the screening of the cystaine residue substituted mutants, some mutants showed remarkable high thermal stability as BcGDH complex without the decrease of the catalytic activity. Furthermore, multiple inter-subunit disulfide bonds made the enzyme as super-stabilized which showed high catalytic activity even by the incubation at 70 ̊C. Thus developed engineered enzyme revealed to show DET ability with electrode, indistinguishable to the wild type one. This super-stabilized enzyme will contribute the improvement of the operational stability of enzyme sensors. References Lee et al., Bioelectrochemistry. 121 (2018) 1–6Sode et al., Enzyme Microb. Technol. 19 (1996) 82–85Yamazaki et al., Appl. Biochem. Biotechnol. 77 (1999) 325–336Yamazaki et al., Anal. Lett. 41 (2008) 2363-2373.Yoshida et al., Acta Crystallogr. Sect. D Struct. Biol. 75 (2019) 841–851

Unusual quaternary structure of a homodimeric synergistic-type toxin from mamba snake venom defines its molecular evolution.

Snake venoms are complex mixtures of enzymes and nonenzymatic proteins that have evolved to immobilize and kill prey animals or deter predators. Among them, three-finger toxins (3FTxs) belong to the largest superfamily of nonenzymatic proteins. They share a common structure of three β-stranded loops extending like fingers from a central core containing all four conserved disulfide bonds. Most 3FTxs are monomers and through subtle changes in their amino acid sequences, they interact with different receptors, ion channels and enzymes to exhibit a wide variety of biological effects. The 3FTxs have further expanded their pharmacological space through covalent or noncovalent dimerization. Synergistic-type toxins (SynTxs) isolated from the deadly mamba venoms, although nontoxic, have been known to enhance the toxicity of other venom proteins. However, the details of three-dimensional structure and molecular mechanism of activity of this unusual class of 3FTxs are unclear. We determined the first three-dimensional structure of a SynTx isolated from Dendroaspis jamesoni jamesoni (Jameson's mamba) venom. The SynTx forms a unique homodimer that is held together by an interchain disulfide bond. The dimeric interface is elaborate and encompasses loops II and III. In addition to the inter-subunit disulfide bond, the hydrogen bonds and hydrophobic interactions between the monomers contribute to the dimer formation. Besides, two sulfate ions that mediate interactions between the monomers. This unique quaternary structure is evolved through noncovalent homodimers such as κ-bungarotoxins. This novel dimerization further enhances the diversity in structure and function of 3FTxs.

Probing the Dynamics of AcrB Through Disulfide Bond Formation.

The resistant-nodulation-division (RND) superfamily member tripartite AcrA-AcrB-TolC efflux pump is a major contributor to the multidrug resistance in Escherichia coli. AcrB is the inner membrane protein of the efflux complex and is responsible for the recognition and binding of compounds before their transportation out of the cell. Understanding the dynamics of AcrB during functional rotation in the process of drug efflux is the focus of this study. For this purpose, we introduced six inter-subunit disulfide bonds into the periplasmic domain of AcrB using site-directed mutagenesis to study the importance of the relative flexibility at the inter-subunit interface. Western blot analysis revealed the formation of disulfide bond-linked AcrB oligomers, which were reduced into monomers under reducing conditions. The impact of mutation and formation of disulfide bond on efflux were evaluated via comparison of the minimum inhibitory concentration (MIC) of an acrB knockout strain expressing different mutants. The double Cys mutants tested led to equal or higher susceptibility to AcrB substrates compared to their corresponding single mutants. To determine if the reduction of activity in a double mutant is due to restriction on conformational changes by the disulfide bond formation, ethidium bromide accumulation assays were conducted utilizing dithiothreitol (DTT) as the reducing agent. In two cases, the activities of the double Cys mutants were partially restored by DTT reduction, confirming the importance of relative movement in the respective location for function. These findings provide new insights into the dynamics of the AcrAB-TolC efflux pump in E. coli.

Real-time monitoring of peroxiredoxin oligomerization dynamics in living cells

Peroxiredoxins are central to cellular redox homeostasis and signaling. They serve as peroxide scavengers, sensors, signal transducers, and chaperones, depending on conditions and context. Typical 2-Cys peroxiredoxins are known to switch between different oligomeric states, depending on redox state, pH, posttranslational modifications, and other factors. Quaternary states and their changes are closely connected to peroxiredoxin activity and function but so far have been studied, almost exclusively, outside the context of the living cell. Here we introduce the use of homo-FRET (Förster resonance energy transfer between identical fluorophores) fluorescence polarization to monitor dynamic changes in peroxiredoxin quaternary structure inside the crowded environment of living cells. Using the approach, we confirm peroxide- and thioredoxin-related quaternary transitions to take place in cellulo and observe that the relationship between dimer-decamer transitions and intersubunit disulfide bond formation is more complex than previously thought. Furthermore, we demonstrate the use of the approach to compare different peroxiredoxin isoforms and to identify mutations and small molecules affecting the oligomeric state inside cells. Mutagenesis experiments reveal that the dimer-decamer equilibrium is delicately balanced and can be shifted by single-atom structural changes. We show how to use this insight to improve the design of peroxiredoxin-based redox biosensors.