Calculations of the CD spectrum of bovine pancreatic ribonuclease

CD spectra of bovine pancreatic ribonuclease A (RNase A) and its subtilisin-modified form (RNase S) have been calculated, based upon high-resolution structures from x-ray diffraction. All known transitions in the peptide and side-chain groups, especially the aromatic and disulfide groups, have been included. Calculations have been performed with both the matrix method and with first-order perturbation theory. A newly developed method for treating the electrostatic interactions among transition charge densities and between static charge distributions and transition charge densities is used. The effects of local electrostatic fields upon the group transition energies are included for all transitions. Rotational strengths generated by the matrix method were combined with Gaussian band shapes to generate theoretical CD spectra. The calculated spectra reproduce the signs and approximate magnitudes of the near-uv CD bands of both RNase A and S. Agreement is most satisfactory for the negative 275 nm band, dominated by tyrosine contributions. In agreement with two previous studies by other workers, coupling between Tyr 73 and Tyr 115 is the single most important factor in this band. The positive band observed near 240 nm is dominated by disulfide contributions, according to our results. The far-uv CD spectrum is poorly reproduced by the calculations. The observed 208 nm band, characteristic of α-helices, is absent from the calculated spectrum, probably because the helices in RNase are short. A strong positive couplet centered near 190 nm is predicted but not observed. Possible reasons for these incorrect predictions of the current theoretical model in the far-uv are discussed. © 1997 John Wiley & Sons, Inc.     

Complexes of DNA with histones f2a2 and f3. Circular dichroism studies.

Two histones from calf thymus, the slightly lysine-rich histone f2a2 and the arginine-rich f3, were combined separately, with homologous DNA. The complexes were reconstituted by means of guanidine hydrochloride gradient dialysis, and their circular dichroic (CD) spectra were examined in 0.14 M NaCl. The CD spectra of f2a2-DNA complexes are characterized by a positive band at 272 nm which is blue-shifted and greatly enhanced relative to the corresponding band for native DNA. This type of CD change was noted previously with f2a1-DNA and f2b-DNA complexes. In contrast, f3 histone causes only minor distortions in the DNA CD spectrum, and their character depends upon the state of the two sulfhydryl groups in f3. When the cysteines are reduced, f3-DNA complexes have a slightly increased positive band with a small blue shift; when oxidized disulfide is the predominant form, this CD band becomes slightly smaller than native DNA value. This laboratory has now examined complexes reconstituted from DNA and all five histones of calf thymus. The sum of the CD spectra of these complexes, although very similar to the CD curve for reconstituted complexes containing whole histone, does not approximate that of chromatin; the consequence of this observation is discussed.     

Lima bean proteinase inhibitor. Origins of circular dichroism bands and modification by Br2- and (CNS)2-.

Origins of CD bands in lima bean proteinase inhibitor were deduced from an acetylation-deacetylation study of the sole tyrosyl residue in the protein (Tyr 69), and by analogy with Bowman-Birk soybean proteinase inhibitor, a homologous protein with similar spectral properties. Tyr 69 is relatively inaccessible to N-acetylimidazole; 100-fold molar excess of the reagent in the presence of 6 M guanidine hydrochloride elicited about 70 to 80% O-acetylation. A broad negative CD band centered around 280 nm arises mainly from the longest wavelength transition of cystinyl side chains (epsilon L--epsilon R approximately equal to -0.8 M-1 cm-1 per disulfide). The second cystinyl transition gives rise to a positive CD band of a comparable intensity at 247 nm. The Lb vibronic transition of Tyr 69 has negative CD around 280 nm, contributing approximately 10% of the total CD intensity at 278 nm (epsilon L--epsilon R approximately equal to -0.5 M-1 cm-1). The 232 nm positive shoulder is from the La vibronic transition of Tyr 69. Radical anions, Br2- and (CNS)2-, generated by the irradiation of N2O-saturated inhibitor solutions containing KBr or KCNS, reduced tyrosyl CD without affecting disulfide CD bands, indicating that the radical anions damaged Tyr 69 without altering protein conformation. The inhibitor modified at Tyr 69 by Br2- and (CNS)2- retained full activity toward trypsin and chymotrypsin. The irradiation of the inhibitor in the air-saturated solution led to loss in tyrosyl as well as cystinyl CD bands and decline in both antiproteinase activities.     

Domain swapping of CD4 upon dimerization

It has recently been shown that disulfide bond Cys130-Cys159 in domain 2 of monomeric CD4 is involved in the formation of CD4 disulfide-bonded dimers on cell surfaces and that it can influence the permissiveness of cells to HIV infection. Because this disulfide bond is buried in the monomer, a large conformational change must take place in order to allow for such disulfide exchange. Using standard optimization techniques, whose efficiency was first checked in the well-documented CD2 case, we have shown that 3D domain swapping is a likely candidate for the conformational change, the hinge loop, or linker, being loop E-F. Indeed, as a consequence of domain swapping, because Cys130 and Cys159 belong to beta-strands C and F, respectively, two disulfide bonds become established between Cys130 in one monomer and Cys159 in the other one. Such a disulfide exchange has already been observed when the nuclear magnetic resonance (NMR) structure of the prion protein was compared to the crystallographic, dimeric one. In both cases, domain swapping implies disulfide exchange because the linker is located in the sequence between two disulfide-bonded cysteines. As in the CD2 case, the proposed configuration of the CD4 dimer is found as a pair of neighboring monomers in the crystallographic unit cell. Moreover, because in this configuration the epitope of monoclonal antibody MT151, which does not compete with Gp120 for CD4 binding, is in the cleft between the pair of CD4 monomers, it is suggested that MT151 achieves its HIV-blocking activity by interfering with the formation of CD4 domain-swapped dimers on cell surface.     

Conformation of malformin A: a proton magnetic resonance and a circular dichroism study

Malformin A is a cyclic pentapeptide with an intramolecular disulfide bridge. The conformation in solution of this molecule has been studied by NMR and CD. The 270 MHz Proton spectrum in dimethyl sulfoxide is well resolved and the peaks corresponding to the five residues have been assigned. From the temperature dependence of chemical shifts of the peptide protons and from the exchange rate of these protons, it is concluded that the NH proton of one Cys is shielded from the solvent. This observation and H? N? _αC? H angles, estimated from the corresponding coupling constants, a proposed conformation of the peptide backbone. From the H? _βC? _αC? H coupling constants, a P chirality for the disulfide bridge is proposed. Such a conformation is confirmed by the circular dichroism spectrum which shows a negative band at λ > 250 nm. It is concluded that the conformation of malformin A is rigid and that the disulfide bridge is exposed to interact with biological receptors.     

The alpha-chain of murine CD8 lacks an invariant Ig-like disulfide bond but contains a unique intrachain loop instead

The CD8 Ag is a cell surface heterodimer which demarcates predominantly cytotoxic T cells which are restricted by class I MHC Ag. The disulfide bonds within the murine structure were assigned in this study and the alpha-beta-interchain bond involves one or more cysteine residues located in each chain proximal to the plasma membrane or included within it. The location of the intrachain disulfide loop within the CD8 beta-chain confirms its proposed structural homology to an IgV domain but no corresponding disulfide loop is present within the alpha-chain. The invariant IgV disulfide loop has been replaced by a unique, short loop involving an unusual cysteine which is conserved in the CD8 alpha-chains of man, mouse, and rat. Despite its lack of precedent in other Ig-related structures, this unusual disulfide loop can be parsimoniously accommodated into a modified domain which has retained the major features of the Ig structural motif.     

Inhibitors of protein-disulfide isomerase prevent cleavage of disulfide bonds in receptor-bound glycoprotein 120 and prevent HIV-1 entry

We previously reported that monoclonal antibodies to protein-disulfide isomerase (PDI) and other membrane-impermeant PDI inhibitors prevented HIV-1 infection. PDI is present at the surface of HIV-1 target cells and reduces disulfide bonds in a model peptide attached to the cell membrane. Here we show that soluble PDI cleaves disulfide bonds in recombinant envelope glycoprotein gp120 and that gp120 bound to the surface receptor CD4 undergoes a disulfide reduction that is prevented by PDI inhibitors. Concentrations of inhibitors that prevent this reduction and inhibit the cleavage of surface-bound disulfide conjugate prevent infection at the level of HIV-1 entry. The entry of HIV-1 strains differing in their coreceptor specificities is similarly inhibited, and so is the reduction of gp120 bound to CD4 of coreceptor-negative cells. PDI inhibitors also prevent HIV envelope-mediated cell-cell fusion but have no effect on the entry of HIV-1 pseudo-typed with murine leukemia virus envelope. Importantly, PDI coprecipitates with both soluble and cellular CD4. We propose that a PDI.CD4 association at the cell surface enables PDI to reach CD4-bound virus and to reduce disulfide bonds present in the domain of gp120 that binds to CD4. Conformational changes resulting from the opening of gp120-disulfide loops may drive the processes of virus-cell and cell-cell fusion. The biochemical events described identify new potential targets for anti-HIV agents.     

The CD of two-chain coiled coils: experiments on tropomyosin and tropomyosin segments in the tyrosine/disulfide spectral region

CD experiments are reported for several coiled-coil species in the tyrosine/disulfide (approximately 250-350-nm) region. Intact noncross-linked tropomyosin (approximately 3 degrees C) shows a negative nonsymmetric band maximal at 280 nm. This spectrum is the sum over six tyrosines/chain, and has conformational significance, since it disappears on denaturation. Experiments on an excised coiled-coil segment, each of whose chains comprise residues 11-127 of the tropomyosin sequence and only one tyrosine (Y60), reveal that not all tyrosines are alike. The spectrum at 3 degrees C shows a small negative maximum at approximately 285 nm and a substantial, hitherto unknown, positive band at approximately 270 nm, the latter masked in the parent protein by the negative contribution from the other tyrosines. A noncross-linked coiled-coil segment comprising residues 142-281, in which Y60 is absent, shows no such positive band. This peculiarity of Y60 is confirmed by absorbance spectra, with the extinction coefficient of Y60 larger in benign media than the average of the other tyrosines. Intact (3 degrees C) C190 cross-linked tropomyosin is known to yield, besides tyrosine contributions, a positive maximum at approximately 300 nm. Subtracting the corresponding data for noncross-linked tropomyosin shows that the disulfide spectrum itself actually has two equal, partly resolved bands at, respectively, 250 and 280 nm. The existence of a chiral disulfide argues for a relatively rigid, perhaps strained, local coiled coil. A C190 cross-linked segment comprising residues 142-281 shows a chiral disulfide spectrum like tropomyosin's, but another segment, comprising residues 168-284, shows none; thus removal of residues 142-167 causes loss of chirality at C190, over 20 residues away. These spectra thus contain important information on the subtle local differences in coiled-coil structures.     

Spectroscopic analysis of thermal denaturation of Cajanus cajan proteinase inhibitor at neutral and acidic pH by circular dichroism

The conformational changes accompanying thermal denaturation under neutral, acidic and reducing conditions of Cajanus cajan proteinase inhibitor were investigated using near- and far-ultraviolet circular dichroism (CD) spectroscopy. The protein inhibitor shows a reversible N<-->D transition at neutral pH with a Tm approximately equal to 63 degrees C. The negative CD band intensities at 200 nm (far-UV) and near about 280 nm (near-UV) decrease as a result of thermal stress. The effect is more pronounced at low pH and in the presence of dithiothreitol. Only partial reversibility is observed under acidic conditions. Significant changes in the near- as well as far-ultraviolet CD spectrum are observed in the presence of dithiothreitol suggestive of the importance of disulfide linkages in maintaining the structure of C. cajan proteinase inhibitor.     

Normal ligand binding and signaling by CD47 (integrin-associated protein) requires a long range disulfide bond between the extracellular and membrane-spanning domains.

CD47 is a unique member of the Ig superfamily with a single extracellular Ig domain followed by a multiply membrane-spanning (MMS) domain with five transmembrane segments, implicated in both integrin-dependent and -independent signaling cascades. Essentially all functions of CD47 require both the Ig and MMS domains, raising the possibility that interaction between the two domains is required for normal function. Conservation of Cys residues among CD47 homologues suggested the existence of a disulfide bond between the Ig and MMS domains that was confirmed by chemical digestion and mapped to Cys(33) and Cys(263). Subtle changes in CD47 conformation in the absence of the disulfide were suggested by decreased binding of two anti-Ig domain monoclonal antibodies, decreased SIRPalpha1 binding, and reduced CD47/SIRPalpha1-mediated cell adhesion. Mutagenesis to prevent formation of this disulfide completely disrupted CD47 signaling independent of effects on ligand binding, as assessed by T cell interleukin-2 secretion and Ca(2+) responses. Loss of the disulfide did not affect membrane raft localization of CD47 or its association with alpha(v)beta(3) integrin. Thus, a disulfide bond between the Ig and MMS domains of CD47 is required for normal ligand binding and signal transduction.     

A comparison of spectral and physicochemical properties of yeast iso-1 cytochrome c and Cys 102-modified derivatives of the protein

Derivatives of yeast iso-1 cytochrome c, chemically modified at Cys-102 (Cys-102 acetamide-derivatized monomer, Cys-102 thionitrobenzoate-derivatized monomer, Cys-102 S-methylated monomer, and the disulfide dimer), exhibit different spectral and physicochemical properties relative to the native, unmodified protein, depending on the nature of the modifying group. The results of proton NMR studies on the Cys-102 acetamidederivatized monomer of iso-1 ferricytochrome c indicate that the conformational characteristics of the heme environment in this protein derivative are intermediate between those of the unmodified monomer and disulfide dimer forms of the protein. Measurements of the pK_a of the alkaline transitions of the five forms of iso-1 ferricytochrome c provided values of 8.89, 8.82, 8.67, 8.47, and 8.50 for the unmodified monomer, S-methylated monomer, acetamide-derivatized monomer, thionitrobenzoate-derivatized monomer, and disulfide dimer, respectively. The results of proton NMR studies of the reduced form of these proteins suggest that the heme environments of the unmodified monomer and disulfide dimer derivatives of iso-1 ferrocytochrome c are similar and indicate that treatment of the thionitrobenzoate-derivatized and disulfide dimer forms of the protein with sodium dithionite results in cleavage of the disulfide bonds at position 102. Circular dichroism studies reveal that only the disulfide dimer form of iso-1 ferricytochrome c exhibits a Soret CD spectrum which differs from the native, unmodified monomer in that the intensity of the negative band at approximately 420 nm is diminished in the spectrum of the dimer relative to the spectrum of the monomer. Soret CD spectra of the ascorbate-reduced form of all protein derivatives are similar. The process of autoreduction of yeast iso-1 ferricytochrome c is shown to occur in the absence of a free sulfhydryl group at position 102 and is exacerbated under moderately high pH conditions. These results are suggestive of the presence of a redox-active amino acid, perhaps a tyrosine, in yeast iso-1 cytochrome c.     

CD44 Binding to Hyaluronic Acid Is Redox Regulated by a Labile Disulfide Bond in the Hyaluronic Acid Binding Site

CD44 is the primary leukocyte cell surface receptor for hyaluronic acid (HA), a component of the extracellular matrix. Enzymatic post translational cleavage of labile disulfide bonds is a mechanism by which proteins are structurally regulated by imparting an allosteric change and altering activity. We have identified one such disulfide bond in CD44 formed by Cys77 and Cys97 that stabilises the HA binding groove. This bond is labile on the surface of leukocytes treated with chemical and enzymatic reducing agents. Analysis of CD44 crystal structures reveal the disulfide bond to be solvent accessible and in the–LH hook configuration characteristic of labile disulfide bonds. Kinetic trapping and binding experiments on CD44-Fc chimeric proteins show the bond is preferentially reduced over the other disulfide bonds in CD44 and reduction inhibits the CD44-HA interaction. Furthermore cells transfected with CD44 no longer adhere to HA coated surfaces after pre-treatment with reducing agents. The implications of CD44 redox regulation are discussed in the context of immune function, disease and therapeutic strategies.     

岩豆凝集素分子修饰与圆二色性的关系研究

天然态岩豆凝集素（ＭＤＬ）远紫外圆二色性（ＣＤ）谱显示２１６ｎｍ处单一负峰,是一种高β－折叠构象凝集素;近紫外ＣＤ谱呈现２８２ｎｍ处负峰和２６０～２７５ｎｍ及２９５ｎｍ处的负肩,经Ｎ－乙基顺丁烯二酰亚胺（ＮＥＭＩ）和对氯汞苯甲酸（ＰＣＭＢ）修饰ＭＤＬ的巯基,其近紫外ＣＤ谱未发生变化,远紫外ＣＤ谱仅发生细微变化,ＭＤＬ凝集活性保持不变;ＰＣＭＢ过量时,ＣＤ谱呈现典型的无规卷曲谱形,ＭＤＬ完全丧失凝集活性,去除ＰＣＭＢ后,活性又全部恢复．二硫苏糖醇（ＤＤＴ）修饰ＭＤＬ的二硫键并用碘乙酸（ＩＣＨ２ＣＯＯＨ）保护巯基,ＭＤＬ远紫外ＣＤ谱２１６ｎｍ处的负峰红移至２２５ｎｍ,且显著减小;同时,近紫外ＣＤ谱２８２ｎｍ处负峰几乎消失,两负肩分别保持完整,分子中α－螺旋降低,无规卷曲增加较多,ＭＤＬ凝集活性未发生变化．用Ｎ－溴代丁二酰亚胺（ＮＢＳ）修饰ＭＤＬ分子中的色氨酸,导致２１６ｎｍ负峰蓝移至２０８ｎｍ且变小,分子中无规卷曲和α－螺旋增加,β折叠减少,近紫外ＣＤ谱２９５ｎｍ负肩消失,２８２ｎｍ负峰红移至２８７ｎｍ,ＭＤＬ凝集活性完全丧失．     

Interactions in nonnative and truncated forms of staphylococcal nuclease as indicated by mutational free energy changes.

Several mixed disulfide variants of staphylococcal nuclease have been produced by disulfide bond formation between nuclease V23C and methane, ethane, 1-propane, 1-n-butane, and 1-n-pentane thiols. Although CD spectroscopy shows that the native state is largely unperturbed, the stability toward urea-induced unfolding is highly dependent on the nature of the group at this position, with the methyl disulfide protein being the most stable. The variant produced by modification with iodoacetic acid, however, gives a CD spectrum indicative of an unfolded polypeptide. Thiol-disulfide exchange equilibrium constants between nuclease V23C and 2-hydroxyethyl disulfide have been measured as a function of urea concentration. Because thiol-disulfide exchange and unfolding are thermodynamically linked, the effects of a mutation (disulfide exchange) can be partitioned between various conformational states. In the case of unmodified V23C and the 2-hydroxyethyl protein mixed disulfide, significant effects in the nonnative states of nuclease are observed. Truncated forms of staphylococcal nuclease are thought to be partially folded and may be good models for early folding intermediates. We have characterized a truncated form of nuclease comprised of residues 1-135 with a V23C mutation after chemical modification of the cysteine residue. High-resolution size-exclusion chromatography indicates that modification brings about significant changes in the Stokes radius of the protein, and CD spectroscopy indicates considerable differences in the amount of secondary structure present. Measurement of the disulfide exchange equilibrium constant between this truncated protein and 2-hydroxyethyl disulfide indicate significant interactions between position 23 and the rest of the protein when the urea concentration is lower than 1.5 M.(ABSTRACT TRUNCATED AT 250 WORDS)     

Near-UV circular dichroism of trypsin inhibitor of adzuki beans attributable to disulfide groups.

The trypsin inhibitor of adzuki (Phaseolus angularis) beans shows a CD spectrum with a negative extremum at 280 nm and a positive shoulder around 245 nm. Since the inhibitor lacks tryptophan and tyrosine, the observed CD spectrum can be attributed to the six disulfide groups in the molecule. The CD features completely disappeared on reduction of the disulfide groups, and converged into a single negative extremum at 270 nm when the groups were modified to form mixed disulfides with glutathione. These observations of the CD properties of the inhibitor strongly suggest the presence of disulfide groups constrained with respect to their dihedral angles.     

Photoactivation of rhodopsin involves alterations in cysteine side chains: detection of an S-H band in the Meta I-->Meta II FTIR difference spectrum.

FTIR difference spectroscopy has been used to study the role of cysteine residues in the photoactivation of rhodopsin. A positive band near 2550 cm-1 with a low frequency shoulder is detected during rhodopsin photobleaching, which is assigned on the basis of its frequency and isotope shift to the S-H stretching mode of one or more cysteine residues. Time-resolved studies at low temperature show that the intensity of this band correlates with the formation and decay kinetics of the Meta II intermediate. Modification of rhodopsin with the reagent NEM, which selectively reacts with the SH groups of Cys-140 and Cys-316 on the cytoplasmic surface of rhodopsin, has no effect on the appearance of this band. Four other cysteine residues are also unlikely to contribute to this band because they are either thio-palmitylated (Cys-322 and Cys-323) or form a disulfide bond (Cys-110 and Cys-187). On this basis, it is likely that at least one of the four remaining cysteine residues in rhodopsin is structurally active during rhodopsin photoactivation. The possibility is also considered that this band arises from a transient cleavage of the disulfide bond between cysteine residues 110 and 187.     

Interleukin-2 signalling is modulated by a labile disulfide bond in the CD132 chain of its receptor

Certain disulfide bonds present in leucocyte membrane proteins are labile and can be reduced in inflammation. This can cause structural changes that result in downstream functional effects, for example, in integrin activation. Recent studies have shown that a wide range of membrane proteins have labile disulfide bonds including CD132, the common gamma chain of the receptors for several cytokines including interleukin-2 and interleukin-4 (IL-2 and IL-4). The Cys(183)-Cys(232) disulfide bond in mouse CD132 is susceptible to reduction by enzymes such as thioredoxin (TRX), gamma interferon-inducible lysosomal thiolreductase and protein disulfide isomerase, which are commonly secreted during immune activation. The Cys(183)-Cys(232) disulfide bond is also reduced in an in vivo lipopolysaccharide (LPS)-induced acute model of inflammation. Conditions that lead to the reduction of the Cys(183)-Cys(232) disulfide bond in CD132 inhibit proliferation of an IL-2-dependent T cell clone and concomitant inhibition of the STAT-5 signalling pathway. The same reducing conditions had no effect on the proliferation of an IL-2-independent T cell clone, nor did they reduce disulfide bonds in IL-2 itself. We postulate that reduction of the Cys(183)-Cys(232) disulfide in CD132 inhibits IL-2 binding to the receptor complex. Published data show that the Cys(183)-Cys(232) disulfide bond is exposed at the surface of CD132 and in close contact with IL-2 and IL-4 in their respective receptor complexes. In addition, mutants in these Cys residues in human CD132 lead to immunodeficiency and loss of IL-2 binding. These results have wider implications for the regulation of cytokine receptors in general, as their activity can be modulated by a 'redox regulator' mechanism caused by the changes in the redox environment that occur during inflammation and activation of the immune system.     

Disulfide bond that constrains the HIV-1 gp120 V3 domain is cleaved by thioredoxin

A functional disulfide bond in both the HIV envelope glycoprotein, gp120, and its immune cell receptor, CD4, is involved in viral entry, and compounds that block cleavage of the disulfide bond in these proteins inhibit HIV entry and infection. The disulfide bonds in both proteins are cleaved at the cell surface by the small redox protein, thioredoxin. The target gp120 disulfide and its mechanism of cleavage were determined using a thioredoxin kinetic trapping mutant and mass spectrometry. A single disulfide bond was cleaved in isolated and cell surface gp120, but not the gp160 precursor, and the extent of the reaction was enhanced when gp120 was bound to CD4. The Cys(32) sulfur ion of thioredoxin attacks the Cys(296) sulfur ion of the gp120 V3 domain Cys(296)-Cys(331) disulfide bond, cleaving the bond. Considering that V3 sequences largely determine the chemokine receptor preference of HIV, we propose that cleavage of the V3 domain disulfide, which is facilitated by CD4 binding, regulates chemokine receptor binding. There are 20 possible disulfide bond configurations, and, notably, the V3 domain disulfide has the same unusual -RHStaple configuration as the functional disulfide bond cleaved in CD4.     

Reduced monomeric CD4 is the preferred receptor for HIV

CD4 is a co-receptor for binding of T cells to antigen-presenting cells and the primary receptor for the human immunodeficiency virus type 1 (HIV). CD4 exists in three different forms on the cell surface defined by the state of the domain 2 cysteine residues: an oxidized monomer, a reduced monomer, and a covalent dimer linked through the domain 2 cysteines. The disulfide-linked dimer is the preferred immune co-receptor. The form of CD4 that is preferred by HIV was examined in this study. HIV entry and envelope-mediated cell-cell fusion were tested using cells expressing comparable levels of wild-type or disulfide bond mutant CD4 in which the domain 2 cysteines were mutated to alanine. Eliminating the domain 2 disulfide bond increased entry of HIV reporter viruses and enhanced HIV envelope-mediated cell-cell fusion 2-4-fold. These observations suggest that HIV enters susceptible cells preferably through monomeric reduced CD4, whereas dimeric CD4 is the preferred receptor for binding to antigen-presenting cells. Cleavage of the domain 2 disulfide bond is possibly involved in the conformational change in CD4 associated with fusion of the HIV and cell membranes.     

Stabilization of HIV-1 gp120-CD4 Receptor Complex through Targeted Interchain Disulfide Exchange

HIV-1 enters cells via interaction between the trimeric envelope (Env) glycoprotein gp120/gp41 and the host cell surface receptor molecule CD4. The requirement of CD4 for viral entry has rationalized the development of recombinant CD4-based proteins as competitive viral attachment inhibitors and immunotherapeutic agents. In this study, we describe a novel recombinant CD4 protein designed to bind gp120 through a targeted disulfide-exchange mechanism. According to structural models of the gp120-CD4 receptor complex, substitution of Ser⁶⁰ on the CD4 domain 1 α-helix with Cys positions a thiol in proximity of the gp120 V1/V2 loop disulfide (Cys¹²⁶–Cys¹⁹⁶), satisfying the stereochemical and geometric conditions for redox exchange between CD4 Cys⁶⁰ and gp120 Cys¹²⁶, and the consequent formation of an interchain disulfide bond. In this study, we provide experimental evidence for this effect by describing the expression, purification, refolding, receptor binding and antiviral activity analysis of a recombinant two-domain CD4 variant containing the S60C mutation (2dCD4-S60C). We show that 2dCD4-S60C binds HIV-1 gp120 with a significantly higher affinity than wild-type protein under conditions that facilitate disulfide exchange and that this translates into a corresponding increase in the efficacy of CD4-mediated viral entry inhibition. We propose that targeted redox exchange between conserved gp120 disulfides and nucleophilic moieties positioned strategically on CD4 (or CD4-like scaffolds) conceptualizes a new strategy in the development of high affinity HIV-1 Env ligands, with important implications for therapy and vaccine development. More generally, this chalcogen substitution approach provides a general means of stabilizing receptor-ligand complexes where the structural and biophysical conditions for disulfide exchange are satisfied.