Disulfide Pairs Research Articles

Co-translational formation of disulfides guides folding of the SARS-CoV-2 receptor binding domain

Many secreted proteins, including viral proteins, contain multiple disulfide bonds. How disulfide formation is coupled to protein folding in the cell remains poorly understood at the molecular level. Here, we combine experiment and simulation to address this question as it pertains to the SARS-CoV-2 receptor binding domain (RBD). We show that the RBD can only refold reversibly if its native disulfides are present before folding. But in their absence, the RBD spontaneously misfolds into a nonnative, molten-globule-like state that is structurally incompatible with complete disulfide formation and that is highly prone to aggregation. Thus, the RBD native structure represents a metastable state on the protein’s energy landscape with reduced disulfides, indicating that nonequilibrium mechanisms are needed to ensure native disulfides form before folding. Our atomistic simulations suggest that this may be achieved via co-translational folding during RBD secretion into the endoplasmic reticulum. Namely, at intermediate translation lengths, native disulfide pairs are predicted to come together with high probability, and thus, under suitable kinetic conditions, this process may lock the protein into its native state and circumvent highly aggregation-prone nonnative intermediates. This detailed molecular picture of the RBD folding landscape may shed light on SARS-CoV-2 pathology and molecular constraints governing SARS-CoV-2 evolution.

The role of glutathione in periplasmic redox homeostasis and oxidative protein folding in Escherichia coli

The thiol redox balance in the periplasm of E. coli depends on the DsbA/B pair for oxidative power and the DsbC/D system as its complement for isomerization of non-native disulfides. While the standard redox potentials of those systems are known, the in vivo “steady state” redox potential imposed onto protein thiol disulfide pairs in the periplasm remains unknown. Here, we used genetically encoded redox probes (roGFP2 and roGFP-iL), targeted to the periplasm, to directly probe the thiol redox homeostasis in this compartment. These probes contain two cysteine residues that are virtually completely reduced in the cytoplasm, but once exported into the periplasm, can form a disulfide bond, a process that can be monitored by fluorescence spectroscopy. Even in the absence of DsbA, roGFP2, exported to the periplasm, was almost fully oxidized, suggesting the presence of an alternative system for the introduction of disulfide bonds into exported proteins. However, the absence of DsbA shifted the steady state periplasmic thiol-redox potential from −228 mV to a more reducing −243 mV and the capacity to re-oxidize periplasmic roGFP2 after a reductive pulse was significantly decreased. Re-oxidation in a DsbA strain could be fully restored by exogenous oxidized glutathione (GSSG), while reduced GSH accelerated re-oxidation of roGFP2 in the WT. In line, a strain devoid of endogenous glutathione showed a more reducing periplasm, and was significantly worse in oxidatively folding PhoA, a native periplasmic protein and substrate of the oxidative folding machinery. PhoA oxidative folding could be enhanced by the addition of exogenous GSSG in the WT and fully restored in a ΔdsbA mutant. Taken together this suggests the presence of an auxiliary, glutathione-dependent thiol-oxidation system in the bacterial periplasm.

La4Ga2S8O3: A Rare-Earth Gallium Oxysulfide with Disulfide Ions.

Band gap engineering using multiple anions is an established approach to novel photocatalysts that exhibit suitable band gap energies for water splitting and high photocorrosion resistance. However, few studies have been conducted on photocatalysts with polyanions, including polychalcogenide ions. Here, we present a new quaternary gallium oxysulfide with disulfide pairs (S2)2-, La4Ga2S8O3, grown out of a KI molten salt. Single-crystal X-ray diffraction analysis revealed that the oxysulfide crystallizes in the orthorhombic space group Pbcn with lattice constants of a = 18.3330(6) Å, b = 13.0590(5) Å, and c = 5.9022(3) Å. In the crystal structure, the GaS4-based zigzag chains and OLa4-based fluorite-like strips are independently arranged in two dimensions, which alternately stack via the disulfide pairs along the third direction. The oxysulfide is a direct-type semiconductor with a band gap of 2.45 eV. First-principles calculations combined with X-ray photoemission spectroscopy measurements show that S 3p states derived from the disulfide pairs dominate the valence band maximum and conduction band minimum, and these band-edge positions are suitable for the oxidation and reduction of water. Our comprehensive study based on the electronic structure suggests that the disulfide pairs make La4Ga2S8O3 a potential photocatalyst for water splitting under visible-light irradiation.

A search for the resting conformation of the bacterial mechanosensitive channel MSCs.

Structure determination for membrane proteins remains not only technically challenging but is further complicated by potentially non-native conformations resulting from removal of stabilizing lipids. This is especially true for mechanosensitive channels, which are designed to undergo gating transitions under membrane tension that negates part of the bilayer's lateral pressure. At tensions near its activation threshold (5-7 mN/m), the MscS channel slowly transitions from the resting to the inactivated state, which is tension insensitive. This interconversion between the two non-conductive states is accompanied by an 8 nm2 lateral expansion, revealing that the resting state must be more compact. We attempt to relate these two functional states with existing MscS structures experimentally and computationally. Molecular dynamics simulations used to verify experimental conductance and in-plane area expansion revealed that the many MscS structures are non-conductive or semi-conductive. These non-conductive structures share a splayed conformation of lipid-facing TM1-TM2 helical pairs, deep crevices between TM2 and TM3, and a de-coupling of the TM3a gate from the TM1-TM2 tension-transmitting domains. Yet, functional studies demonstrate the necessity of close hydrophobic TM2-TM3 contact for the resting to open transition; hydrophilic mutations lead to immediate inactivation instead of opening. This prompted the modeling of a “compact” resting state characterized by “upright” rather than splayed TM1-TM2 pairs and tighter TM2-TM3 contact that would reconnect the gate to TM1-TM2 helices. Simulated transitions from structures with splayed TM1-TM2 pairs to this compact state revealed two conserved stabilizing salt bridges: R59-D67 and D62-R131. Charge-inverting and neutralizing mutations at these residues had drastic effects on opening and closing rates, inactivation, and especially recovery from inactivation. Disulfide pairs introduced at these salt bridge positions support their proximity as predicted by the model. A cryo-EM characterization of the disulfide cross-linked channel is in progress.

Genome-wide investigation of defensin genes in peanut (Arachis hypogaea L.) reveals AhDef2.2 conferring resistance to bacterial wilt

Peanut (Arachis hypogea L.) bacterial wilt (BW) is a devastating disease caused by Ralstonia solanacearum that results in severe yield and quality losses. Plant defensins are short cysteine-rich peptides with antimicrobial activity. The role of defensin genes (AhDef) in peanut is unclear. A genome-wide investigation of AhDef genes was undertaken, and 12 identified AhDef genes were classified into two groups containing the gamma-thionin domain formed by four disulfide pairs: Cys1-Cys8, Cys2-Cys5, Cys3-Cys6, and Cys4-Cys7. In silico analysis revealed that AhDef genes showed highly conserved architectural features and contained cis-elements associated with phytohormone signaling and defense responses. A highly resistant cultivar, H108 (R) and a susceptible accession, H107 (S) were tested by R. solanacearum inoculation. H108 (R) showed fewer symptoms than H107 (S) owing to inhibition of bacterial reproduction and spread in the vascular bundles of roots and stems. In a transcriptomic expression profile, AhDef genes, particularly AhDef1.6 and AhDef2.2, were up-regulated in H108 (R) compared with H107 (S) under R. solanacearum infection and phytohormone treatment. Subcellular localization showed that the AhDef1.6 and AhDef2.2 proteins were both expressed specifically on the plasma membrane. Overexpression of protein fusion AhDef2.2-YFP in Nicotiana benthamiana and peanut leaves increased resistance to R. solanacearum, suggesting its role in response to BW infection. AhDef2.2 may be valuable for peanut resistance breeding.

InVivo Reoxidation Kinetics of Free Thiols in Multiple Domains of IgG1 Antibodies in Rats.

While free thiols in monoclonal antibodies (mAbs) have been extensively characterized by invitro studies to probe its effect on antibody function and stability, their invivo biotransformation has not been comprehensively studied. In this study, a panel of five recombinant IgG1 mAbs with elevated free thiols in the VH, VL, and CH2 domains were intravenously administered into Wistar rats. Invivo biotransformation of thirty-five free thiol sites in total (7 disulfide pairs in VL, CL, VH, CH1, HH, CH2, CH3 domains across the 5 mAbs) were monitored using a denaturing differential isotopic tagging procedure on immunopurified timepoints followed by LC-MS of tryptic digests. The free thiol levels in two VH domain and one CH2 domain disulfide sites decreased invivo following first order kinetics. Free thiol levels of the remaining 32 sites were remarkably stable invivo. Further analytical characterization highlighted a positive association between a free thiol's solvent accessibility and a free thiol's reoxidation propensity. The data and discussion presented here shed valuable insights into the invivo fate of free thiols in several recombinant IgG1s and its implications for free thiols as a product quality attribute in therapeutic mAb products.

Oxysulfide Ba5(VO2S2)2(S2)2 Combining Disulfide Channels and Mixed-Anion Tetrahedra and Its Third-Harmonic-Generation Properties.

Mixed-anion compounds are among the most promising systems to design functional materials with enhanced properties. In particular, heteroleptic environments around transition metals allow tuning of the polarity or band-gap engineering for instance. We present the original oxysulfide Ba5(VO2S2)2(S2)2, the fifth member in the quaternary system Ba-V-S-O. It exhibits the mixed-anion building units V5+O2S2 and isolated disulfide pairs (S2)2-. The structure is solved by combining single-crystal and powder X-ray diffraction and transmission electron microscopy. First-principles calculations were combined in order to highlight the anion roles. In particular, our density functional theory study shows that the 3p states of the disulfide pairs dictate the band gap. In this study, we point out anionic tools for band-gap engineering that can be useful for the design of phases for numerous applications. Finally, third harmonic generation (THG) was measured and compared to the large THG observed for Cu2O, which reveals the potential for nonlinear-optical properties that should be further investigated.

Identifying Reducing and Capping Sites of Protein-Encapsulated Gold Nanoclusters.

The reducing and capping sites along with their local structure impact photo properties of the red bovine serum albumin-capped Au nanocluster (BSA-AuNC), however, they are hard to identify. We developped a workflow and relevant techniques using mass spectrometry (MS) to identify the reducing and capping sites of BSA-AuNCs involved in their formation and fluorescence. Digestion without disulfide cleavages yielded an Au core fraction exhibiting red fluorescence and [AunSm] ion signals and a non-core fraction exhibiting neither of them. The core fraction was identified to mainly be comprised of peptides containing cysteine residues. The fluorescence and [AunSm] signals were quenched by tris(2-carboxyethyl)phosphine, confirming that disulfide groups were required for nanocluster stabilization and fluorescence. By MS sequencing, the disulfide pairs, C75–C91/C90–C101 in domain IA, C315–C360/C359–C368 in domain IIB, and C513–C558/C557–C566 in domain IIIB, were identified to be main capping sites of red AuNCs. Peptides containing oxidized cysteines (sulfinic or cysteic acid) were identified as reducing sites mainly in the non-core fraction, suggesting that disulfide cleavages by oxidization and conformational changes contributed to the subsequent growth of nanoclusters at nearby intact disulfide pairs. This is the first report on precise identification of the reducing and capping sites of BSA-AuNCs.

Simplified identification of disulfide, trisulfide, and thioether pairs with 213 nm UVPD.

Disulfide heterogeneity and other non-native crosslinks introduced during therapeutic antibody production and storage could have considerable negative effects on clinical efficacy, but tracking these modifications remains challenging. Analysis must also be carried out cautiously to avoid introduction of disulfide scrambling or reduction, necessitating the use of low pH digestion with less specific proteases. Herein we demonstrate that 213 nm ultraviolet photodissociation streamlines disulfide elucidation through bond-selective dissociation of sulfur-sulfur and carbon-sulfur bonds in combination with less specific backbone dissociation. Importantly, both types of fragmentation can be initiated in a single MS/MS activation stage. In addition to disulfide mapping, it is also shown that thioethers and trisulfides can be identified by characteristic fragmentation patterns. The photochemistry resulting from 213 nm excitation facilitates a simplified, two-tiered data processing approach that allows observation of all native disulfide bonds, scrambled disulfide bonds, and non-native sulfur-based linkages in a pepsin digest of Rituximab. Native disulfides represented the majority of bonds according to ion count, but the highly solvent-exposed heavy/light interchain disulfides were found to be most prone to modification. Production and storage methods that facilitate non-native links are discussed. Due to the importance of heavy and light chain connectivity for antibody structure and function, this region likely requires particular attention in terms of its influence on maintaining structural fidelity.

In vitro Reconstitution to Understand the Mechanism of Bam

The outer membrane of Gram-negative bacteria is an essential asymmetric lipid bilayer that serves as a protective barrier and performs required cellular functions, such as nutrient uptake and export. Within the outer membrane are trans-membrane proteins that adopt unique beta-barrel structures. These outer membrane proteins (Omps) are folded and inserted by a multi-protein complex embedded in the membrane called the Bam complex, which performs this function without the use of chemical energy (i.e. ATP or proton motive force). The Bam complex is made of five proteins, BamA-E. BamA is a beta-barrel itself and is considered the central component of the complex. Crystal structures and molecular dynamics simulations suggests that BamA has a destabilized barrel seam that can open and close to potentially nucleate new beta-barrels. Locking the barrel seam in vivo with engineered disulfides is lethal, suggesting that this lateral gate is essential for BamA function. We tested the lateral gate hypothesis in a minimal in vitro system with BamA and a substrate Omp, OmpX in synthetic liposomes. In this system, locking the barrel has no affect on the activity of BamA – locked barrels had the same activity as unlocked barrels. This suggests that either the lateral gate is not important for function or we are not capturing the full activity of BamA. Interestingly, disulfide cross-linking experiments suggest that the barrel seam is highly dynamic, since disulfide pairs separated by up to 4 residues are able to form. To further probe the lateral gate hypothesis, we have moved into a fully reconstituted system with the Bam complex in native E. coli lipids. This system will help us differentiate between these two possibilities. Understanding how the Bam complex assists in protein folding will help answer fundamental questions about membrane protein folding.

The effect of disulfide bond introduction and related Cys/Ser mutations on the stability of a cyclohexanone monooxygenase

Baeyer–Villiger monooxygenases (BVMO) belong to the class B of flavin-dependent monooxygenases (type I BVMOs) and catalyze the oxidation of (cyclic) ketones into esters and lactones. The prototype BVMO is the cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. NCIMB 9871. This enzyme shows an impressive substrate scope with a high chemo-, regio- and/or enantioselectivity. BVMO reactions are often difficult, if not impossible to achieve by chemical approaches and this makes these enzymes thus highly desired candidates for industrial applications. Unfortunately, the industrial use is hampered by several factors related to the lack of stability of these biocatalysts. Thus, the aim of this study was to improve the CHMO’s long-term stability, one of the most relevant parameter for biocatalytic processes, and additionally its stability against oxidation. We used an easy computational method for the prediction of stabilizing disulfide bonds in the CHMO-scaffold. The three most promising predicted disulfide pairs were created and biochemically characterized. The most oxidatively stable variant (Y411C-A463C) retained nearly 60% activity after incubation with 25mM H2O2 whereas the wild type retained only 16%. In addition, one extra disulfide pair (T415C-A463C) was created and tested for increased stability. The melting temperature (Tm) of this variant was increased by 5°C with simultaneous improved long-term stability.After verification by ABD-F labeling that this mutant does not form a disulfide bond, single and double Cys/Ser mutants were prepared and investigated. Subsequent analysis revealed that the T415C single point variant is the most stable variant with a 30-fold increased long-term stability (33% residual activity after 24h incubation at 25°C) showcasing a great achievement for practical applications.

The MIA pathway: a key regulator of mitochondrial oxidative protein folding and biogenesis.

ConspectusMitochondria are fundamental intracellular organelles with key roles in important cellular processes like energy production, Fe/S cluster biogenesis, and homeostasis of lipids and inorganic ions. Mitochondrial dysfunction is consequently linked to many human pathologies (cancer, diabetes, neurodegeneration, stroke) and apoptosis. Mitochondrial biogenesis relies on protein import as most mitochondrial proteins (about 10–15% of the human proteome) are imported after their synthesis in the cytosol. Over the last several years many mitochondrial translocation pathways have been discovered. Among them, the import pathway that targets proteins to the intermembrane space (IMS) stands out as it is the only one that couples import to folding and oxidation and results in the covalent modification of the incoming precursor that adopt internal disulfide bonds in the process (the MIA pathway). The discovery of this pathway represented a significant paradigm shift as it challenged the prevailing dogma that the endoplasmic reticulum is the only compartment of eukaryotic cells where oxidative folding can occur.The concept of the oxidative folding pathway was first proposed on the basis of folding and import data for the small Tim proteins that have conserved cysteine motifs and must adopt intramolecular disulfides after import so that they are retained in the organelle. The introduction of disulfides in the IMS is catalyzed by Mia40 that functions as a chaperone inducing their folding. The sulfhydryl oxidase Erv1 generates the disulfide pairs de novo using either molecular oxygen or, cytochrome c and other proteins as terminal electron acceptors that eventually link this folding process to respiration. The solution NMR structure of Mia40 (and supporting biochemical experiments) showed that Mia40 is a novel type of disulfide donor whose recognition capacity for its substrates relies on a hydrophobic binding cleft found adjacent to a thiol active CPC motif. Targeting of the substrates to this pathway is guided by a novel type of IMS targeting signal called ITS or MISS. This consists of only 9 amino acids, found upstream or downstream of a unique Cys that is primed for docking to Mia40 when the substrate is accommodated in the Mia40 binding cleft. Different routes exist to complete the folding of the substrates and their final maturation in the IMS. Identification of new Mia40 substrates (some even without the requirement of their cysteines) reveals an expanded chaperone-like activity of this protein in the IMS. New evidence on the targeting of redox active proteins like thioredoxin, glutaredoxin, and peroxiredoxin into the IMS suggests the presence of redox-dependent regulatory mechanisms of the protein folding and import process in mitochondria. Maintenance of redox balance in mitochondria is crucial for normal cell physiology and depends on the cross-talk between the various redox signaling processes and the mitochondrial oxidative folding pathway.

Conformational landscape and pathway of disulfide bond reduction of human alpha defensin.

Human alpha defensins are a class of antimicrobial peptides with additional antiviral activity. Such antimicrobial peptides constitute a major part of mammalian innate immunity. Alpha defensins contain six cysteines, which form three well defined disulfide bridges under oxidizing conditions. Residues C3-C31, C5-C20, and C10-C30 form disulfide pairs in the native structure of the peptide. The major tissue in which HD5 is expressed is the crypt of the small intestine, an anaerobic niche that should allow for substantial pools of both oxidized and (partly) reduced HD5. We used ion mobility coupled to mass spectrometry to track the structural changes in HD5 upon disulfide bond reduction. We found evidence of stepwise unfolding of HD5 with sequential reduction of the three disulfide bonds. Alkylation of free cysteines followed by tandem mass spectrometry of the corresponding partially reduced states revealed a dominant pathway of reductive unfolding. The majority of HD5 unfolds by initial reduction of C5-C20, followed by C10-C30 and C3-C31. We find additional evidence for a minor pathway that starts with reduction of C3-C31, followed by C5-C20 and C10-C30. Our results provide insight into the pathway and conformational landscape of disulfide bond reduction in HD5.

Dicarba analogues of α-conotoxin RgIA. Structure, stability, and activity at potential pain targets.

α-Conotoxin RgIA is both an antagonist of the α9α10 nicotinic acetylcholine receptor (nAChR) subtype and an inhibitor of high-voltage-activated N-type calcium channel currents. RgIA has therapeutic potential for the treatment of pain, but reduction of the disulfide bond framework under physiological conditions represents a potential liability for clinical applications. We synthesized four RgIA analogues that replaced native disulfide pairs with nonreducible dicarba bridges. Solution structures were determined by NMR, activity assessed against biological targets, and stability evaluated in human serum. [3,12]-Dicarba analogues retained inhibition of ACh-evoked currents at α9α10 nAChRs but not N-type calcium channel currents, whereas [2,8]-dicarba analogues displayed the opposite pattern of selectivity. The [2,8]-dicarba RgIA analogues were effective in HEK293 cells stably expressing human Cav2.2 channels and transfected with human GABAB receptors. The analogues also exhibited improved serum stability over the native peptide. These selectively acting dicarba analogues may represent mechanistic probes to explore analgesia-related biological receptors.

Serine‐Selective Aerobic Cleavage of Peptides and a Protein Using a Water‐Soluble Copper–Organoradical Conjugate

AbstractThe site‐specific cleavage of peptide bonds is an important chemical modification of biologically relevant macromolecules. The reaction is not only used for routine structural determination of peptides, but is also a potential artificial modulator of protein function. Realizing the substrate scope beyond the conventional chemical or enzymatic cleavage of peptide bonds is, however, a formidable challenge. Here we report a serine‐selective peptide‐cleavage protocol that proceeds at room temperature and near neutral pH value, through mild aerobic oxidation promoted by a water‐soluble copper–organoradical conjugate. The method is applicable to the site‐selective cleavage of polypeptides that possess various functional groups. Peptides comprising D‐amino acids or sensitive disulfide pairs are competent substrates. The system is extendable to the site‐selective cleavage of a native protein, ubiquitin, which comprises more than 70 amino acid residues.

Serine-selective aerobic cleavage of peptides and a protein using a water-soluble copper-organoradical conjugate.

The site-specific cleavage of peptide bonds is an important chemical modification of biologically relevant macromolecules. The reaction is not only used for routine structural determination of peptides, but is also a potential artificial modulator of protein function. Realizing the substrate scope beyond the conventional chemical or enzymatic cleavage of peptide bonds is, however, a formidable challenge. Here we report a serine-selective peptide-cleavage protocol that proceeds at room temperature and near neutral pH value, through mild aerobic oxidation promoted by a water-soluble copper-organoradical conjugate. The method is applicable to the site-selective cleavage of polypeptides that possess various functional groups. Peptides comprising D-amino acids or sensitive disulfide pairs are competent substrates. The system is extendable to the site-selective cleavage of a native protein, ubiquitin, which comprises more than 70 amino acid residues.

Structure, Function, and Chemical Synthesis of Vaejovis mexicanus Peptide 24: A Novel Potent Blocker of Kv1.3 Potassium Channels of Human T Lymphocytes

Animal venoms are rich sources of ligands for studying ion channels and other pharmacological targets. Proteomic analyses of the soluble venom from the Mexican scorpion Vaejovis mexicanus smithi showed that it contains more than 200 different components. Among them, a 36-residue peptide with a molecular mass of 3864 Da (named Vm24) was shown to be a potent blocker of Kv1.3 of human lymphocytes (K(d) ∼ 3 pM). The three-dimensional solution structure of Vm24 was determined by nuclear magnetic resonance, showing the peptide folds into a distorted cystine-stabilized α/β motif consisting of a single-turn α-helix and a three-stranded antiparallel β-sheet, stabilized by four disulfide bridges. The disulfide pairs are formed between Cys6 and Cys26, Cys12 and Cys31, Cys16 and Cys33, and Cys21 and Cys36. Sequence analyses identified Vm24 as the first example of a new subfamily of α-type K(+) channel blockers (systematic number α-KTx 23.1). Comparison with other Kv1.3 blockers isolated from scorpions suggests a number of structural features that could explain the remarkable affinity and specificity of Vm24 toward Kv1.3 channels of lymphocytes.

Role of conserved cysteine residues in hepatitis C virus glycoprotein e2 folding and function.

Hepatitis C virus glycoprotein E2 contains 18 conserved cysteines predicted to form nine disulfide pairs. In this study, a comprehensive cysteine-alanine mutagenesis scan of all 18 cysteine residues was performed in E1E2-pseudotyped retroviruses (HCVpp) and recombinant E2 receptor-binding domain (E2 residues 384 to 661 [E2(661)]). All 18 cysteine residues were absolutely required for HCVpp entry competence. The phenotypes of individual cysteines and pairwise mutation of disulfides were largely the same for retrovirion-incorporated E2 and E2(661), suggesting their disulfide arrangements are similar. However, the contributions of each cysteine residue and the nine disulfides to E2 structure and function varied. Individual Cys-to-Ala mutations revealed discordant effects, where removal of one Cys within a pair had minimal effect on H53 recognition and CD81 binding (C486 and C569) while mutation of its partner abolished these functions (C494 and C564). Removal of disulfides at C581-C585 and C452-C459 significantly reduced the amount of E1 coprecipitated with E2, while all other disulfides were absolutely required for E1E2 heterodimerization. Remarkably, E2(661) tolerates the presence of four free cysteines, as simultaneous mutation of C452A, C486A, C569A, C581A, C585A, C597A, and C652A (M+C597A) retained wild-type CD81 binding. Thus, only one disulfide from each of the three predicted domains, C429-C552 (DI), C503-C508 (DII), and C607-C644 (DIII), is essential for the assembly of the E2(661) CD81-binding site. Furthermore, the yield of total monomeric E2 increased to 70% in M+C597A. These studies reveal the contribution of each cysteine residue and the nine disulfide pairs to E2 structure and function.

Crystal Structure of the Ectodomain Complex of the CGRP Receptor, a Class-B GPCR, Reveals the Site of Drug Antagonism

Dysregulation of the calcitonin gene-related peptide (CGRP), a potent vasodilator, is directly implicated in the pathogenesis of migraine. CGRP binds to and signals through the CGRP receptor (CGRP-R), a heterodimer containing the calcitonin receptor-like receptor (CLR), a class B GPCR, and RAMP1, a receptor activity-modifying protein. We have solved the crystal structure of the CLR/RAMP1 N-terminal ectodomain heterodimer, revealing how RAMPs bind to and potentially modulate the activities of the CLR GPCR subfamily. We also report the structures of CLR/RAMP1 in complex with the clinical receptor antagonists olcegepant (BIBN4096BS) and telcagepant (MK0974). Both drugs act by blocking access to the peptide-binding cleft at the interface of CLR and RAMP1. These structures illustrate, for the first time, how small molecules bind to and modulate the activity of a class B GPCR, and highlight the challenges of designing potent receptor antagonists for the treatment of migraine and other class B GPCR-related diseases.

Redox Regulation of Transglutaminase 2 Activity

Transglutaminase 2 (TG2) in the extracellular matrix is largely inactive but is transiently activated upon certain types of inflammation and cell injury. The enzymatic activity of extracellular TG2 thus appears to be tightly regulated. As TG2 is known to be sensitive to changes in the redox environment, inactivation through oxidation presents a plausible mechanism. Using mass spectrometry, we have identified a redox-sensitive cysteine triad consisting of Cys(230), Cys(370), and Cys(371) that is involved in oxidative inactivation of TG2. Within this triad, Cys(370) was found to participate in disulfide bonds with both Cys(230) and its neighbor, Cys(371). Notably, Ca(2+) was found to protect against formation of these disulfide bonds. To investigate the role of each cysteine residue, we created alanine mutants and found that Cys(230) appears to promote oxidation and inactivation of TG2 by facilitating formation of Cys(370)-Cys(371) through formation of the Cys(230)-Cys(370) disulfide bond. Although vicinal disulfide pairs are found in several transglutaminase isoforms, Cys(230) is unique for TG2, suggesting that this residue acts as an isoform-specific redox sensor. Our findings suggest that oxidation is likely to influence the amount of active TG2 present in the extracellular environment.