Opsin stability and folding: the role of Cys185 and abnormal disulfide bond formation in the intradiscal domain

The structure in the extracellular, intradiscal domain of rhodopsin surrounding the Cys110–Cys187 disulfide bond has been shown to be important for correct folding of this receptor in vivo. Retinitis pigmentosa misfolding mutants of the apoprotein opsin (such as P23H) misfold, as defined by a deficiency in ability to bind 11-cis retinal and form rhodopsin. These mutants also possess an abnormal Cys185–Cys187 disulfide bond in the intradiscal domain. Here, by mutating Cys185 to alanine, we eliminate the possibility of forming this abnormal disulfide bond and investigate the effect of combining the C185A mutation with the retinitis pigmentosa mutation P23H. Both the P23H and P23H/C185A double mutant suffer from low expression and poor 11-cis retinal binding. Our data suggest that misfolding events occur that do not have an absolute requirement for abnormal Cys185–Cys187 disulfide bond formation. In the detergent-solubilised, purified state, the C185A mutation allows formation of rhodopsin at wild-type (WT) levels, but has interesting effects on protein stability. C185A rhodopsin is less thermally stable than WT, whereas C185A opsin shows the same ability to regenerate rhodopsin in detergent as WT. Purified C185A and WT opsins, however, have contrasting 11-cis retinal binding kinetics. A high proportion of C185A opsin binds 11-cis retinal with a slow rate that reflects a denatured state of opsin reverting to a fast-binding, open-pocket conformation. This slower rate is not observed in a stabilising lipid/detergent system, 1,2-dimyristoyl-sn-glycero-3-phosphocholine/Chaps, in which C185A exhibits WT (fast) retinal binding. We propose that the C185A mutation destabilises the open-pocket conformation of opsin in detergent resulting in an equilibrium between correctly folded and denatured states of the protein. This equilibrium can be driven towards the correctly folded rhodopsin state by the binding of 11-cis retinal.     

Endothelial CD146 is required for in vitro tumor-induced angiogenesis: The role of a disulfide bond in signaling and dimerization

Tumor angiogenesis, induced by tumor-secreted pro-angiogenic factors, is an essential process for cancer development and metastasis. CD146 is identified as an endothelial cell adhesion molecule and implicated in blood vessel formation, however, its exact role in angiogenesis, particularly tumor angiogenesis, and its potential function of mediating downstream signaling are still unclear. In present study, we evidenced that silencing endogenous endothelial CD146 by RNAi significantly impaired hepatocarcinoma cell secretions-promoted tubular morphogenesis and -enhanced motility of endothelial cells. Biochemical studies revealed that CD146 was required for the activation of p38/IKK/NFκB signaling cascade and up-regulation of NFκB downstream pro-angiogenic genes, notably IL-8, ICAM-1 and MMP9, in response to tumor secretions. Interestingly, specific anti-CD146 mAb AA98, which bound a conformational epitope depending on C452–C499 disulfide bond, could abrogate NFκB activation and tumor angiogenesis, whereas another anti-CD146 mAb AA1 recognizing a linear epitope containing aa50–54 did not have such effects. Further structure–function analysis identified that C452–C499 disulfide bond within the fifth extracellular Ig domain was indispensible for CD146-mediated signaling and tube formation. Moreover, dimerization of CD146, which was enhanced by tumor secretions and suppressed by AA98 but not AA1, also relied on C452 and C499. Together, this study for the first time uncovered the pro-angiogenic role of CD146 and also pinpointed the key structural basis responsible for its signaling function and dimerization. These findings also suggested that CD146 might serve as not just a cell adhesion molecule but also a membrane signal receptor in tumor-induced angiogenesis.     

Characterization of the Ligand Binding Functionality of the Extracellular Domain of Activin Receptor Type IIB

The single transmembrane domain serine/threonine kinase activin receptor type IIB (ActRIIB) has been proposed to bind key regulators of skeletal muscle mass development, including the ligands GDF-8 (myostatin) and GDF-11 (BMP-11). Here we provide a detailed kinetic characterization of ActRIIB binding to several low and high affinity ligands using a soluble activin receptor type IIB-Fc chimera (ActRIIB.Fc). We show that both GDF-8 and GDF-11 bind the extracellular domain of ActRIIB with affinities comparable with those of activin A, a known high affinity ActRIIB ligand, whereas BMP-2 and BMP-7 affinities for ActRIIB are at least 100-fold lower. Using site-directed mutagenesis, we demonstrate that ActRIIB binds GDF-11 and activin A in different ways such as, for example, substitutions in ActRIIB Leu⁷⁹ effectively abolish ActRIIB binding to activin A yet not to GDF-11. Native ActRIIB has four isoforms that differ in the length of the C-terminal portion of their extracellular domains. We demonstrate that the C terminus of the ActRIIB extracellular domain is crucial for maintaining biological activity of the ActRIIB.Fc receptor chimera. In addition, we show that glycosylation of ActRIIB is not required for binding to activin A or GDF-11. Together, our findings reveal binding specificity and activity determinants of the ActRIIB receptor that combine to effect specificity in the activation of distinct signaling pathways.     

Expression cloning of an activin receptor, a predicted transmembrane serine kinase.

Activins are involved in the regulation of multiple biological events, ranging from early development to pituitary function. To characterize the cellular mechanisms involved in these processes, cDNAs coding for an activin receptor were cloned from AtT20 mouse corticotropic cells by screening COS cell transfectants for binding of 125I-activin A. The cDNAs code for a protein of 494 amino acids comprising a ligand-binding extracellular domain, a single membrane-spanning domain, and an intracellular kinase domain with predicted serine/threonine specificity. 125I-activin A binds to transfected COS cells with an affinity of 180 pM and can be competed by activin A, activin B, and inhibin A, but not by transforming growth factor beta 1. The kinase domain, but not the extracellular sequence, of the activin receptor is most closely related to the C. elegans daf-1 gene product, a putative transmembrane serine/threonine-specific protein kinase for which the ligand is not known.     

A flexible activin explains the membrane-dependent cooperative assembly of TGF-beta family receptors

A new crystal structure of activin in complex with the extracellular domain of its type II receptor (ActRIIb-ECD) shows that the ligand exhibits an unexpected flexibility. The motion in the activin dimer disrupts its type I receptor interface, which may account for the disparity in its affinity for type I versus type II receptors. We have measured the affinities of activin and its antagonist inhibin for ActRIIb-ECD and found that the affinity of the 2-fold symmetric homodimer activin for ActRIIb-ECD depends on the availability of two spatially coupled ActRIIb-ECD molecules, whereas the affinity of the heterodimer inhibin does not. Our results indicate that activin's affinity for its two receptor types is greatly influenced by their membrane-restricted setting. We propose that activin affinity is modulated by the ligand flexibility and that cooperativity is achieved by binding to two ActRII chains that immobilize activin in a type I binding-competent orientation.     

Molecular cloning and binding properties of the human type II activin receptor.

A full-length cDNA for the type II human activin receptor was cloned by hybridization from a human testis cDNA library. The sequence encodes a 513 amino acid protein that is 99% identical, at the amino acid level, with the mouse type II activin receptor. The type II human activin receptor consists of an extracellular domain that specifically binds activin A with a Kd of 360 pM, a single-membrane spanning domain, and an intracellular kinase domain with predicted serine/threonine specificity.     

Agonist-regulated Cleavage of the Extracellular Domain of Parathyroid Hormone Receptor Type 1

The receptor for parathyroid hormone (PTHR) is a main regulator of calcium homeostasis and bone maintenance. As a member of class B of G protein-coupled receptors, it harbors a large extracellular domain, which is required for ligand binding. Here, we demonstrate that the PTHR extracellular domain is cleaved by a protease belonging to the family of extracellular metalloproteinases. We show that the cleavage takes place in a region of the extracellular domain that belongs to an unstructured loop connecting the ligand-binding parts and that the N-terminal 10-kDa fragment is connected to the receptor core by a disulfide bond. Cleaved receptor revealed reduced protein stability compared with noncleaved receptor, suggesting degradation of the whole receptor. In the presence of the agonistic peptides PTH(1–34), PTH(1–14), or PTH(1–31), the processing of the PTHR extracellular domain was inhibited, and receptor protein levels were stabilized. A processed form of the PTHR was also detected in human kidney. These findings suggest a new model of PTHR processing and regulation of its stability.     

Determination of disulfide bond arrangement in bombyxin-IV, an insulin superfamily peptide from the silkworm,Bombyx mori, by combination of thermolysin digestion of natural peptide and selective synthesis of disulfide bond isomers

The mode of disulfide linkages in bombyxin-IV, an insulin superfamily peptide consisting of A- and B-chains, was determined as A6–A11, A7–B10, and A20–B22. An intermolecular bond of A20–B22 was identified by sequencing and mass spectrometric analysis of the fragments generated by thermolysin digestion of natural bombyxin-IV. The mode of the remaining two bridges was determined by chemical and selective synthesis of three possible disulfide bond isomers of bombyxin-IV. A- and B-chains were synthesized by solid-phase method, and three disulfide bonds were bridged stepwise and in a fully controlled manner. Retention time on reversed-phase high-performance liquid chromatography (HPLC), thermolysin digests, and biological activity of the synthetic [A6–A11, A7–B10, A20–B22-cystine]-bombyxin-IV revealed that it was identical with the natural bombyxin-IV. Two other isomers with respect to disulfide bond arrangement, [A6–A7, A11–B10, A20–B22-cystine]- and [A6–B10, A7–A11, A20–B22-cystine]-bombyxin-IVs, were distinguishable from the natural one by use of HPLC, thermolysin digestion, and bioassay.     

Extracellular intersubunit interactions modulate epithelial Na+ channel gating

Epithelial Na⁺ channels (ENaCs) and related channels have large extracellular domains where specific factors interact and induce conformational changes, leading to altered channel activity. However, extracellular structural transitions associated with changes in ENaC activity are not well defined. Using crosslinking and two-electrode voltage clamp in Xenopus oocytes, we identified several pairs of functional intersubunit contacts where mouse ENaC activity was modulated by inducing or breaking a disulfide bond between introduced Cys residues. Specifically, crosslinking E499C in the β-subunit palm domain and N510C in the α-subunit palm domain activated ENaC, whereas crosslinking βE499C with αQ441C in the α-subunit thumb domain inhibited ENaC. We determined that bridging βE499C to αN510C or αQ441C altered the Na⁺ self-inhibition response via distinct mechanisms. Similar to bridging βE499C and αQ441C, we found that crosslinking palm domain αE557C with thumb domain γQ398C strongly inhibited ENaC activity. In conclusion, we propose that certain residues at specific subunit interfaces form microswitches that convey a conformational wave during ENaC gating and its regulation.     

Mobility of Lower MA-Helices for Ion Conduction through Lateral Portals in 5-HT3A Receptors

The intracellular domain of the serotonin type 3A receptor, a pentameric ligand-gated ion channel, is crucial for regulating conductance. Ion permeation through the extracellular vestibule and the transmembrane channel is well understood, whereas the specific ion conduction pathway through the intracellular domain is less clear. The intracellular domain starts with a short loop after the third transmembrane segment, followed by a short α-helical segment, a large unstructured loop, and finally, the membrane-associated MA-helix that continues into the last transmembrane segment. The MA-helices from all five subunits form the extension of the transmembrane ion channel and shape what has been described as a “closed vestibule,” with their lateral portals obstructed by loops and their cytosolic ends forming a tight hydrophobic constriction. The question remains whether the lateral portals or cytosolic constriction conduct ions upon channel opening. In our study, we used disulfide bond formation between pairs of engineered cysteines to probe the proximity and mobility of segments of the MA-helices most distal to the membrane bilayer. Our results indicate that the proximity and orientation for cysteine pairs at I409C/R410C, in close proximity to the lateral windows, and L402C/L403C, at the cytosolic ends of the MA-helices, are conducive for disulfide bond formation. Although conformational changes associated with gating promote cross-linking for I409C/R410C, which in turn decreases channel currents, cross-linking of L402C/L403C is functionally silent in macroscopic currents. These results support the hypothesis that concerted conformational changes open the lateral portals for ion conduction, rendering ion conduction through the vertical portal unlikely.     

Human CD300C Delivers an Fc Receptor-γ-dependent Activating Signal in Mast Cells and Monocytes and Differs from CD300A in Ligand Recognition

CD300C is highly homologous with an inhibitory receptor CD300A in an immunoglobulin-like domain among the human CD300 family of paired immune receptors. To clarify the precise expression and function of CD300C, we generated antibodies discriminating between CD300A and CD300C, which recognized a unique epitope involving amino acid residues CD300A(F56-L57) and CD300C(L63-R64). Notably, CD300C was highly expressed in human monocytes and mast cells. Cross-linking of CD300C by its specific antibody caused cytokine/chemokine production of human monocytes and mast cells. Fc receptor γ was indispensable for both efficient surface expression and activating functions of CD300C. To identify a ligand for CD300A or CD300C, we used reporter cell lines expressing a chimera receptor harboring extracellular CD300A or CD300C and intracellular CD3ζ, in which its unknown ligand induced GFP expression. Our results indicated that phosphatidylethanolamine (PE) among the lipids tested and apoptotic cells were possible ligands for both CD300C and CD300A. PE and apoptotic cells more strongly induced GFP expression in the reporter cells through binding to extracellular CD300A as compared with CD300C. Differential recognition of PE by extracellular CD300A and CD300C depended on different amino acid residues CD300A(F56-L57) and CD300C(L63-R64). Interestingly, GFP expression induced by extracellular CD300C-PE binding in the reporter cells was dampened by co-expression of full-length CD300A, indicating the predominance of CD300A over CD300C in PE recognition/signaling. PE consistently failed to stimulate cytokine production in monocytes expressing CD300C with CD300A. In conclusion, specific engagement of CD300C led to Fc receptor γ-dependent activation of mast cells and monocytes.     

Molecular cloning and characterization of a novel rat activin receptor.

We report the isolation of a full-length rat cDNA for a new activin receptor. The deduced amino acid sequence of this receptor shows 67 percent overall identity with that of a previously identified mouse activin receptor. As predicted for the mouse activin receptor, the amino acid sequence of the rat receptor is consistent with a polypeptide containing an extracellular ligand binding domain, a hydrophobic transmembrane domain, and a serine/threonine kinase intracellular domain. In an expression assay, this new receptor was found to bind I125 radiolabeled activin.     

Structure and Activity of the Photosystem II Manganese-Stabilizing Protein: Role of the Conserved Disulfide Bond

The 33-kDa manganese-stabilizing protein (MSP) of Photosystem II (PS II) maintains the functional stability of the Mn cluster in the enzyme’s active site. This protein has been shown to possess characteristics similar to those of the intrinsically disordered, or natively unfolded proteins [Lydakis-Simantiris et al. (1999b) Biochemistry 38: 404–414]. Alternately it was proposed that MSP should be classified as a molten globule, based in part on the hypothesis that its lone disulfide bridge is necessary for structural stability and function in solution [Shutova et al. (2000) FEBS Lett. 467: 137–140]. A site-directed mutant MSP (C28A,C51A) that eliminates the disulfide bond reconstitutes O₂ evolution activity and binds to MSP-free PS II preparations at wild-type levels [Betts et al. (1996) Biochim. Biophys. Acta 1274: 135–142]. This mutant was further characterized by incubation at 90 °C to determine the effect of loss of the disulfide bridge on MSP thermostability and solution structure. After heating at 90 °C for 20 min, C28A,C51A MSP was still able to bind to PS II preparations at molar stoichiometries similar to those of WT MSP and reconstitute O₂ evolution activity. A fraction of the protein aggregates upon heating, but after resolubilization, it regains the ability to bind to PS II and reconstitute O₂ evolution activity. Characterization of the solution structure of C28A,C51A MSP, using CD spectroscopy, UV absorption spectroscopy, and gel filtration chromatography, revealed that the mutant has a more disordered solution structure than WT MSP. The disulfide bond is therefore unnecessary for MSP function and the intrinsically disordered characteristics of MSP are not dependent on its presence. However, the disulfide bond does play a role in the solution structure of MSP in vivo, as evidenced by the lability of a C20S MSP mutation in Synechocystis 6803 [Burnap et al. (1994) Biochemistry 33: 13712–13718].     

The Full-Length, Cytoplasmic C-Terminus of the β2-Adrenergic Receptor Expressed in E. coli Acts as a Substrate for Phosphorylation by Protein Kinase A, Insulin Receptor Tyrosine Kinase, GRK2, but Not Protein Kinase C and Suppresses Desensitization when Expressed in Vivo

The ability of the cytoplasmic, full-length C-terminus of the β2-adrenergic receptor (BAC1) expressed in Escherichia coli to act as a functional domain and substrate for protein phosphorylation was tested. BAC1 was expressed at high-levels, purified, and examined in solution as a substrate for protein phosphorylation. The mobility of BAC1 on SDS–PAGE mimics that of the native receptor itself, displaying decreased mobility upon chemical reduction of disulfide bonds. Importantly, the C-terminal, cytoplasmic domain of the receptor expressed in E. coli was determined to be a substrate for phosphorylation by several candidate protein kinases known to regulate G-protein-linked receptors. Mapping was performed by proteolytic degradation and matrix-assisted laser desorption ionization, time-of-flight mass spectrometry. Purified BAC1 is phosphorylated readily by protein kinase A, the phosphorylation occurring within the predicted motif RRSSSK. The kinetic properties of the phosphorylation by protein kinase A displayed cooperative character. The activated insulin receptor tyrosine kinase, which phosphorylates the beta-adrenergic receptor in vivo, phosphorylates BAC1. The Y364 residue of BAC1 was predominantly phosphorylated by the insulin receptor kinase. GRK2 catalyzed modest phosphorylation of BAC1. Phosphorylation of the human analog of BAC1 in which Cys341 and Cys378 were mutated to minimize disulfide bonding constraints, displayed robust phosphorylation following thermal activation, suggesting under standard conditions that the population of BAC1 molecules capable of assuming the “activated” conformer required by GRKs is low. BAC1 was not a substrate for protein kinase C, suggesting that the canonical site in the second cytoplasmic loop of the intact receptor is preferred. The functional nature of BAC1 was tested additionally by expression of BAC1 protein in human epidermoid carcinoma A431 cells. BAC1 was found to act as a dominant-negative, blocking agonist-induced desensitization of the beta-adrenergic receptor when expressed in mammalian cells. Thus, the C-terminal, cytoplasmic tail of this G-protein-linked receptor expressed in E. coli acts as a functional domain, displaying fidelity with regard to protein kinase action in vivo and acting as a dominant-negative with respect to agonist-induced desensitization.     

The Role of Interchain Heterodisulfide Formation in Activation of the Human Common β and Mouse βIL-3 Receptors

The cytokines, interleukin-3 (IL-3), interleukin-5 (IL-5), and granulocyte-macrophage colony-stimulating factor (GM-CSF), exhibit overlapping activities in the regulation of hematopoietic cells. In humans, the common β (βc) receptor is shared by the three cytokines and functions together with cytokine-specific α subunits in signaling. A widely accepted hypothesis is that receptor activation requires heterodisulfide formation between the domain 1 D-E loop disulfide in human βc (hβc) and unidentified cysteine residues in the N-terminal domains of the α receptors. Since the development of this hypothesis, new data have been obtained showing that domain 1 of hβc is part of the cytokine binding epitope of this receptor and that an IL-3Rα isoform lacking the N-terminal Ig-like domain (the “SP2” isoform) is competent for signaling. We therefore investigated whether distortion of the domain 1-domain 4 ligand-binding epitope in hβc and the related mouse receptor, β_IL-3, could account for the loss of receptor signaling when the domain 1 D-E loop disulfide is disrupted. Indeed, mutation of the disulfide in hβc led to both a complete loss of high affinity binding with the human IL-3Rα SP2 isoform and of downstream signaling. Mutation of the orthologous residues in the mouse IL-3-specific receptor, β_IL-3, not only precluded direct binding of mouse IL-3 but also resulted in complete loss of high affinity binding and signaling with the mouse IL-3Rα SP2 isoform. Our data are most consistent with a role for the domain 1 D-E loop disulfide of hβc and β_IL-3 in maintaining the precise positions of ligand-binding residues necessary for normal high affinity binding and signaling.     

Mechanisms for Kinase-mediated Dimerization of the Epidermal Growth Factor Receptor

We study a mechanism by which dimerization of the EGF receptor (EGFR) cytoplasmic domain is transmitted to the ectodomain. Therapeutic and other small molecule antagonists to the kinase domain that stabilize its active conformation, but not those that stabilize an inactive conformation, stabilize ectodomain dimerization. Inhibitor-induced dimerization requires an asymmetric kinase domain interface associated with activation. EGF and kinase inhibitors stimulate formation of identical dimer interfaces in the EGFR transmembrane domain, as shown by disulfide cross-linking. Disulfide cross-linking at an interface in domain IV in the ectodomain was also stimulated similarly; however, EGF but not inhibitors stimulated cross-linking in domain II. Inhibitors similarly induced noncovalent dimerization in nearly full-length, detergent-solubilized EGFR as shown by gel filtration. EGFR ectodomain deletion resulted in spontaneous dimerization, whereas deletion of exons 2–7, in which extracellular domains III and IV are retained, did not. In EM, kinase inhibitor-induced dimers lacked any well defined orientation between the ectodomain monomers. Fab of the therapeutic antibody cetuximab to domain III confirmed a variable position and orientation of this domain in inhibitor-induced dimers but suggested that the C termini of domain IV of the two monomers were in close proximity, consistent with dimerization in the transmembrane domains. The results provide insights into the relative energetics of intracellular and extracellular dimerization in EGFR and have significance for physiologic dimerization through the asymmetric kinase interface, bidirectional signal transmission in EGFR, and mechanism of action of therapeutics.     

Isolation and characterization of the circulating form of human endostatin

Recently, fragments of extracellular proteins, including endostatin, were defined as a novel group of angiogenesis inhibitors. In this study, human plasma equivalent hemofiltrate was used as a source for the purification of high molecular weight peptides (10–20 kDa), and the isolation and identification of circulating human endostatin are described. The purification of this C-terminal fragment of collagen α1(XVIII) was guided by MALDI-MS and the exact molecular mass determined by ESI-MS was found to be 18 494 Da. N-terminal sequencing revealed the identity of this putative angiogenesis inhibitor and its close relation to mouse endostatin. The cysteine residues 1–3 and 2–4 in the molecule are linked by disulfide bridges. In vitro biological characterization of the native protein demonstrated no anti-proliferative activity on different endothelial cell types. These data indicate that human endostatin, which is a putative angiogenesis inhibitor, is present in the circulation.     

The role of conserved extracellular cysteine residues in vasopressin V2 receptor function and properties of two naturally occurring mutant receptors with additional extracellular cysteine residues

The G protein-coupled vasopressin V2 receptor (V2 receptor) contains a pair of conserved cysteine residues (C112 and C192) which are thought to form a disulfide bond between the first and second extracellular loops. The conserved cysteine residues were found to be important for the correct formation of the ligand binding domain of some G protein-coupled receptors. Here we have assessed the properties of the V2 receptor after site-directed mutagenesis of its conserved cysteine residues in transiently transfected human embryonic kidney (HEK 293) cells. Mutant receptors (C112S, C112A and C192S, C192A) were non-functional and located mostly in the cell's interior. The conserved cysteine residues of the V2 receptor are thus not only important for the structure of the ligand binding domain but also for efficient intracellular receptor transport. In addition to the functional significance of the conserved cysteine residues, we have also analyzed the defects of two mutant V2 receptors which cause X-linked nephrogenic diabetes insipidus (NDI) by the introduction of additional cysteine residues into the second extracellular loop (mutants G185C, R202C). These mutations are assumed to impair normal disulfide bond formation. Mutant receptor G185C and R202C were efficiently transported to the plasma membrane but were defective in ligand binding. Only in the case of the mutant receptor R202C, the more sensitive adenylyl cyclase activity assay revealed vasopressin-stimulated cAMP formation with a 35-fold increased EC(50) value and with a reduced EC(max), indicating that ligand binding is not completely abolished. Taking the unaffected intracellular transport of both NDI-causing mutant receptors into account, our results indicate that the observed impairment of ligand binding by the additional cysteine residues is not due to the prevention of disulfide bond formation between the conserved cysteine residues.     

Disulfide Bonds Play a Critical Role in the Structure and Function of the Receptor-binding Domain of the SARS-CoV-2 Spike Antigen

The current coronavirus pandemic is exerting a tremendously detrimental impact on global health. The Spike proteins of coronaviruses, responsible for cell receptor binding and viral internalization, possess multiple and frequently conserved disulfide bonds raising the question about their role in these proteins. Here, we present a detailed structural and functional investigation of the disulfide bonds of the SARS-CoV-2 Spike receptor-binding domain (RBD). Molecular dynamics simulations of the RBD predict increased flexibility of the surface loops when the four disulfide bonds of the domain are reduced. This flexibility is particularly prominent for the disulfide bond-containing surface loop (residues 456–490) that participates in the formation of the interaction surface with the Spike cell receptor ACE2. In vitro, disulfide bond reducing agents affect the RBD secondary structure, lower its melting temperature from 52 °C to 36–39 °C and decrease its binding affinity to ACE2 by two orders of magnitude at 37 °C. Consistent with these in vitro findings, the reducing agents tris(2-carboxyethyl)phosphine (TCEP) and dithiothreitol (DTT) were able to inhibit viral replication at low millimolar levels in cell-based assays. Our research demonstrates the mechanism by which the disulfide bonds contribute to the molecular structure of the RBD of the Spike protein, allowing the RBD to execute its viral function.