The Role of the Disulfide Bridge in the Stability and Structural Integrity of Ovalbumin Evaluated by Site-Directed Mutagenesis

To provide a molecular explanation of the role of the disulfide (SS) bridge in the thermostability and structural integrity of ovalbumin (OVA), we prepared SS-mutated OVAs in which SS-forming residues were replaced by Ala or Ser (C73A, C73S, C120A, and C73/120A), and compared the conformation, thermostability, susceptibility to elastase, and formation of heat-stable OVA (S-OVA) with those of the wild-type. The circular dichroism (CD) and tryptophan fluorescence spectra revealed that the SS-mutated OVAs assumed a native-like conformation similar to the wild-type. The thermal denaturation temperature for the SS-mutated OVAs was significantly lower than that for the wild-type. C73S, C120A, and C73/120A mutants converted to S-OVA on alkaline treatment. Analyses for elastase digestion fragments showed that a non-native SS bridge was generated in all SS-mutated OVAs, but non-native SS-pairing did not contribute to thermostability. Hence, we concluded that the presence of the original SS bridge in OVA contributes to conformational stability but is not directly related to the conversion to S-OVA.     

Improvement of oxidative and thermostability of N-carbamyl-d-amino Acid amidohydrolase by directed evolution

N-Carbamyl-D-amino acid amidohydrolase (N-carbamoylase), which is currently employed in the industrial production of unnatural D-amino acid in conjunction with D-hydantoinase, has low oxidative and thermostability. We attempted the simultaneous improvement of the oxidative and thermostability of N-carbamoylase from Agrobacterium tumefaciens NRRL B11291 by directed evolution using DNA shuffling. In a second generation of evolution, the best mutant 2S3 with improved oxidative and thermostability was selected, purified and characterized. The temperature at which 50% of the initial activity remains after incubation for 30 min was 73 degrees C for 2S3, whereas it was 61 degrees C for wild-type enzyme. Treatment of wild-type enzyme with 0.2 mM hydrogen peroxide for 30 min at 25 degrees C resulted in a complete loss of activity, but 2S3 retained about 79% of the initial activity under the same conditions. The K(m) value of 2S3 was estimated to be similar to that of wild-type enzyme; however k(cat) was decreased, leading to a slightly reduced value of k(cat)/K(m), compared with wild-type enzyme. DNA sequence analysis revealed that six amino acid residues were changed in 2S3 and substitutions included Q23L, V40A, H58Y, G75S, M184L and T262A. The stabilizing effects of each amino acid residue were investigated by incorporating mutations individually into wild-type enzyme. Q23L, H58Y, M184L and T262A were found to enhance both oxidative and thermostability of the enzyme and of them, T262A showed the most significant effect. V40A and G75S gave rise to an increase only in oxidative stability. The positions of the mutated amino acid residues were identified in the structure of N-carbamoylase from Agrobacterium sp. KNK 712 and structural analysis of the stabilizing effects of each amino acid substitution was also carried out.     

Refined crystal structures of human Ca(2+)/Zn(2+)-binding S100A3 protein characterized by two disulfide bridges

S100A3, a member of the EF-hand-type Ca²⁺-binding S100 protein family, is unique in its exceptionally high cysteine content and Zn²⁺ affinity. We produced human S100A3 protein and its mutants in insect cells using a baculovirus expression system. The purified wild-type S100A3 and the pseudo-citrullinated form (R51A) were crystallized with ammonium sulfate in N,N-bis(2-hydroxyethyl)glycine buffer and, specifically for postrefolding treatment, with Ca²⁺/Zn²⁺ supplementation. We identified two previously undocumented disulfide bridges in the crystal structure of properly folded S100A3: one disulfide bridge is between Cys30 in the N-terminal pseudo-EF-hand and Cys68 in the C-terminal EF-hand (SS1), and another disulfide bridge attaches Cys99 in the C-terminal coil structure to Cys81 in helix IV (SS2). Mutational disruption of SS1 (C30A + C68A) abolished the Ca²⁺ binding property of S100A3 and retarded the citrullination of Arg51 by peptidylarginine deiminase type III (PAD3), while SS2 disruption inversely increased both Ca²⁺ affinity and PAD3 reactivity in vitro. Similar backbone structures of wild type, R51A, and C30A + C68A indicated that neither Arg51 conversion by PAD3 nor SS1 alters the overall dimer conformation. Comparative inspection of atomic coordinates refined to 2.15−1.40 Å resolution shows that SS1 renders the C-terminal classical Ca²⁺-binding loop flexible, which are essential for its Ca²⁺ binding properties, whereas SS2 structurally shelters Arg51 in the metal-free form. We propose a model of the tetrahedral coordination of a Zn²⁺ by (Cys)₃His residues that is compatible with SS2 formation in S100A3.     

In Silico Rational Design and Systems Engineering of Disulfide Bridges in the Catalytic Domain of an Alkaline α-Amylase from Alkalimonas amylolytica To Improve Thermostability

High thermostability is required for alkaline α-amylases to maintain high catalytic activity under the harsh conditions used in textile production. In this study, we attempted to improve the thermostability of an alkaline α-amylase from Alkalimonas amylolytica through in silico rational design and systems engineering of disulfide bridges in the catalytic domain. Specifically, 7 residue pairs (P35-G426, Q107-G167, G116-Q120, A147-W160, G233-V265, A332-G370, and R436-M480) were chosen as engineering targets for disulfide bridge formation, and the respective residues were replaced with cysteines. Three single disulfide bridge mutants—P35C-G426C, G116C-Q120C, and R436C-M480C—of the 7 showed significantly enhanced thermostability. Combinational mutations were subsequently assessed, and the triple mutant P35C-G426C/G116C-Q120C/R436C-M480C showed a 6-fold increase in half-life at 60°C and a 5.2°C increase in melting temperature compared with the wild-type enzyme. Interestingly, other biochemical properties of this mutant also improved: the optimum temperature increased from 50°C to 55°C, the optimum pH shifted from 9.5 to 10.0, the stable pH range extended from 7.0 to 11.0 to 6.0 to 12.0, and the catalytic efficiency (k_cat/K_m) increased from 1.8 × 10⁴ to 2.4 × 10⁴ liters/g · min. The possible mechanism responsible for these improvements was explored through comparative analysis of the model structures of wild-type and mutant enzymes. The disulfide bridge engineering strategy used in this work may be applied to improve the thermostability of other industrial enzymes.     

Structural basis for the thermostability of ferredoxin from the cyanobacterium Mastigocladus laminosus

Plant-type ferredoxins (Fds) carry a single [2Fe-2S] cluster and serve as electron acceptors of photosystem I (PSI). The ferredoxin from the thermophilic cyanobacterium Mastigocladus laminosus displays optimal activity at 65 degrees C. In order to reveal the molecular factors that confer thermostability, the crystal structure of M.laminosus Fd (mFd) was determined to 1.25 A resolution and subsequently analyzed in comparison with four similar plant-type mesophilic ferredoxins. The topologies of the plant-type ferredoxins are similar, yet two structural determinants were identified that may account for differences in thermostability, a salt bridge network in the C-terminal region, and the flexible L1,2 loop that increases hydrophobic accessible surface area. These conclusions were verified by three mutations, i.e. substitution of L1,2 into a rigid beta-turn ((Delta)L1,2) and two point mutations (E90S and E96S) that disrupt the salt bridge network at the C-terminal region. All three mutants have shown reduced electron transfer (ET) capabilities and [2Fe-2S] stability at high temperatures in comparison to the wild-type mFd. The results have also provided new insights into the involvement of the L1,2 loop in the Fd interactions with its electron donor, the PSI complex.     

Increasing the thermal stability of cellulase C using rules learned from thermophilic proteins: a pilot study

Some structural features underlying the increased thermostability of enzymes from thermophilic organisms relative to their homologues from mesophiles are known from earlier studies. We used cellulase C from Clostridium thermocellum to test whether thermostability can be increased by mutations designed using rules learned from thermophilic proteins. Cellulase C has a TIM barrel fold with an additional helical subdomain. We designed and produced a number of mutants with the aim to increase its thermostability. Five mutants were designed to create new electrostatic interactions. They all retained catalytic activity but exhibited decreased thermostability relative to the wild-type enzyme. Here, the stabilizing contributions are obviously smaller than the destabilization caused by the introduction of the new side chains. In another mutant, the small helical subdomain was deleted. This mutant lost activity but its melting point was only 3 degrees C lower than that of the wild-type enzyme, which suggests that the subdomain is an independent folding unit and is important for catalytic function. A double mutant was designed to introduce a new disulfide bridge into the enzyme. This mutant is active and has an increased stability (deltaT(m)=3 degrees C, delta(deltaG(u))=1.73 kcal/mol) relative to the wild-type enzyme. Reduction of the disulfide bridge results in destabilization and an altered thermal denaturation behavior. We conclude that rules learned from thermophilic proteins cannot be used in a straightforward way to increase the thermostability of a protein. Creating a crosslink such as a disulfide bond is a relatively sure-fire method but the stabilization may be smaller than calculated due to coupled destabilizing effects.     

Thermostabilization of ovalbumin by alkaline treatment: Examination of the possible roles of d-serine residues

It was revealed from the crystal structure analysis of S-ovalbumin (S-OVA) formed by alkaline treatment that Ser164, Ser236, and Ser320 take the d-amino acid residue configuration (Yamasaki et al., J Biol Chem 2003; 278:35524–35530). To address the implications of a d-configuration for these Ser residues in S-OVA formation, three mutant OVAs (S164A, S236A, and S320A) were generated to compare their thermostabilities before and after alkaline treatment. Following alkaline treatment, S236A showed a marked increase in melting temperature similar to the wild type (ΔT_m, +9°C) which corresponded to the formation of S-OVA, whereas the increment in T_m for both S164A and S320A was only 4.5°C. Furthermore, the T_m value of the double mutant S164/320A remained unchanged after alkaline treatment, supporting the relevance of Ser164 and Ser320 for thermostabilization of OVA. As Arg142 was predicted to interact with D-Ser164 upon S-OVA formation, it was substituted to Ala to generate R142A. The resulting increment in T_m of mutant R142A after alkaline treatment was 5.8°C. The double mutant R142/S320A was therefore prepared to eliminate the participation of Ser320 in thermostabilization, and its T_m value was compared before and after alkaline treatment. As expected, the increase in T_m for the double mutant was only 1.2°C. Taken together, the data suggest that d-configuration of Ser164 caused by alkaline treatment favors interaction with Arg142 through conformational changes of the side chain. These results strongly supported the participation of the configurational inversion of both Ser164 and Ser320 residues in the formation of S-OVA.     

Introduction of a non-native disulfide bridge to human lysozyme by cysteine scanning mutagenesis

To examine whether the disulfide bridge between residues 65 and 81 can be replaced by a non-native disulfide bridge in the mutant h-lysozyme C77/95A and whether the formation of such a new disulfide bridge affects the folding of the protein, cysteine scanning mutagenesis has been performed within two discontinuous segments (residues 61-67 for the mutant C65/77/95A, and 74-84 for the mutant C77/81/95A). The position of the Cys residue at 65 or 81 was continuously shifted by site-directed mutagenesis. Of the mutants, only substitution of Cys for Trp64 allowed the secretion of mutant h-lysozyme(W64C) into the medium in a sufficient amount for analysis. After the purification, the mutant enzyme was obtained as two components (W64C-A and W64C-B). The only difference between A and B was that A had a peptide bond cleaved between Ala77 and His78. A non-native disulfide bridge between residues 64-81 was found in both components. Little difference was observed in CD spectra among wild-type and mutant enzymes. It is likely that the tertiary structure of the W64C mutant might be distorted at the location, because the directions of amino acid side chains at positions of 64 and 81 are shown to be opposite to each other in wild-type h-lysozyme by X-ray crystallographic analysis.     

Improving the thermostability and activity of Melanocarpus albomyces cellobiohydrolase Cel7B

Two different types of approach were taken to improve the hydrolytic activity towards crystalline cellulose at elevated temperatures of Melanocarpus albomyces Cel7B (Ma Cel7B), a single-module GH-7 family cellobiohydrolase. Structure-guided protein engineering was used to introduce an additional tenth disulphide bridge to the Ma Cel7B catalytic module. In addition, a fusion protein was constructed by linking a cellulose-binding module (CBM) and a linker from the Trichoderma reesei Cel7A to the C terminus of Ma Cel7B. Both approaches proved successful. The disulphide bridge mutation G4C/M70C located near the N terminus, close to the entrance of the active site tunnel of Ma Cel7B, led to improved thermostability (ΔT _m = 2.5°C). By adding the earlier found thermostability-increasing mutation S290T (ΔT _m = 1.5°C) together with the disulphide bridge mutation, the unfolding temperature was increased by 4°C (mutant G4C/M70C/S290T) compared to that of the wild-type enzyme, thus showing an additive effect on thermostability. Both disulphide mutants had increased activity towards microcrystalline cellulose (Avicel) at 75°C, apparently solely because of their improved thermostability. The addition of a CBM also improved the thermostability (ΔT _m = 2.5°C) and caused a clear (sevenfold) increase in the hydrolysis activity of Ma Cel7B towards Avicel at 70°C.     

Introduction of a disulfide bridge enhances the thermostability of a <Emphasis Type="Italic">Streptomyces olivaceoviridis</Emphasis> xylanase mutant

Substitution of the N-terminus of Streptomyces olivaceoviridis xylanase XYNB to generate mutant TB has been previously shown to increase the thermostability of the enzyme. To further improve the stability of this mutant, we introduced a disulfide bridge (C109–C153) into the TB mutant, generating TS. To assess the effect of the disulfide bridge in the wild-type enzyme, the S109C-N153C mutation was also introduced into XYNB, resulting in XS. The mutants were expressed in Pichia pastoris, the recombinant enzymes were purified, and the effect of temperature and pH on enzymatic activity was characterized. Introduction of the disulfide bridge (C109–C153) into XYNB (XS variant) and TB (TS variant) increased the thermostability up to 2.8-fold and 12.4-fold, respectively, relative to XYNB, after incubation at 70°C, pH 6.0, for 20 min. In addition, a synergistic effect of the disulfide bridge and the N-terminus replacement was observed, which extended the half-life of XYNB from 3 to 150 min. Moreover, XS and TS displayed better resistance to acidic conditions compared with the respective enzymes that did not contain a disulfide bridge.     

Enhancement of the thermostability of thermophilic bacterium PS-3 PPase on substitution of Ser-89 with carboxylic amino acids

Serine 89 of the inorganic pyrophosphatase (PPase) subunit from thermophilic bacterium PS-3 (PS-3) was replaced with glycine, alanine, threonine, glutamic acid, or aspartic acid by the PCR-mutagenesis method with Mut-1 in order to determine the contribution of this serine residue to the thermostability and structural integrity of the enzyme molecule. S89G, S89A, and S89T showed reduced catalytic activity, whereas S89D and S89E showed increased enzyme activity. S89G, S89A, and S89T as well as the wild-type PPase were stable in the presence of 5 mM MgCl(2) at 70 degrees C for 1 h, but were inactivated rapidly with time at 80 degrees C. On the contrary, S89D and S89E were stable at 80 degrees C, showing more than 95% of the original activity after 1 h incubation. The wild-type PPase, S89D and S89E were each a hexamer before and after incubation at 80 degrees C for 1 h, while S89G and S89A comprised a mixture of a hexamer and a trimer both before and after incubation at 80 degrees C for 1 h. On the other hand, S89T was a mixture of a hexamer, a trimer and a monomer, and it was partially precipitated during heat treatment at 80 degrees C. The CD spectra of the recombinant enzymes in the far-ultraviolet region were the same as that of the wild-type PPase, whereas those of S89G, S89A, and S89T as well as the wild-type PPase were markedly different after heat treatment, although those of S89D and S89E did not change. The present study suggested that local small change(s) in the network of interactions among amino acid residues on replacement at position 89 led to the PS-3 PPase molecule being unable to form a hexamer from trimers or to dissociate into monomers in some cases without a significant change in the backbone conformation. It was also suggested that the partial disordering of the conformation of PS-3 PPase caused by heat depended on the degree of hydrophilicity in the vicinity of position 89.     

Oral delivery of antigens in liposomes with some lipid compositions modulates oral tolerance to the antigens

Several liposomes containing ovalbumin (OVA), a model antigen, with different lipid compositions were prepared in order to evaluate their ability to induce oral tolerance. Oral administration of these liposomal OVAs induced suppression of the proliferative responses of popliteal lymph node cells from the treated mice to OVA, suggesting that these treated mice were tolerized. The efficiency of the induction of oral tolerance was affected by the liposome composition. OVA entrapment in these liposomes could modulate the tolerizing dose of OVA itself. These results suggest that some liposomes can be suitable antigen-delivery systems for modulated and/or effective induction of oral tolerance.     

Regulation of yeast actin behavior by interaction of charged residues across the interdomain cleft

His(73) participates in the regulation of the nucleotide binding cleft conformation in yeast actin. Earlier molecular dynamics studies suggested that Asp(184) interacts with His(73) thereby stabilizing a "closed-cleft" G-actin. However, beta-actin in the open-cleft state shows a closer interaction of His(73) with Asp(179) than with Asp(184). We have thus assessed the relative importance of Asp(184) and Asp(179) on yeast actin stability and function. Neutral substitutions at 184 or 179 alone had little adverse effect on the monomer and polymerization behavior of actin. Arg or His at 184 in H73E actin partially rescued the monomeric properties of H73E actin, as demonstrated by near-normal thermostability and wild-type (WT)-like protease digestion patterns. ATP exchange was still considerably faster than with WT-actin although slower than that of H73E alone. However, polymerization of H73E/D184R and H73E/D184H is worse than with H73E alone. Conversely, D179R rescued all monomeric properties of H73E to near WT values and largely restored polymerization rate and filament thermostability. These results and new simulations of G-actin in the "open" state underscore the importance of the His(73)-Asp(179) interaction and suggest that the open and not the closed state of yeast actin may be favored in the absence of the methyl group of His(73).     

通过定点诱变法研究耐热碱性磷酸酯酶的活性位点、耐热性和亲热性

通过对耐热碱性磷酸酯酶ＴＡＰＮＤ２７活性位点Ｓ（６９）两侧的Ｅ（６８）和Ｓ（７０）的定点突变,得到了３个突变子Ｅ６８Ｓ、Ｓ７０Ａ和Ｅ６８ＳＳ７０Ａ。在蛋白纯化的基础上测定了３个变体的一些酶学性质,与ＴＡＰＮＤ２７相比,Ｅ６８Ｓ的比活力上升８倍,Ｔｍ下降了３℃,最适反应温度上升了２０℃;Ｓ７０Ａ的比活力上升了１部,Ｔｍ下降了２℃,最适反应温度上升了５℃;Ｅ６８ＳＳ７０Ａ的比活力下降了５０％,Ｔｍ下降     

Thermostability of protein studied by molecular dynamics simulation

The thermostability of protein thermostable cathechol 2,3-dixoygenase (TC23O) has been studied by the parallel molecular dynamics simulations. By analysis of the exponent beta, which is related to the scattering spectrum and constant-pressure heat capacity Cp, we reveal the respective contribution of a specific residue 228 proline; a specific salt bridge, Lys188N-Glu291OE1; four ions; and a different water environment to the thermostability of TC23O. The dynamic transition temperature of the mutants, Pro228Ser and Glu291Gly of the TC23O, was decreased about 10 degrees C and 19 degrees C respectively. The displacement of the four ions had no significant effect on the thermostability of TC23O. Water affects the thermostability by influencing the changes of accessible conformation to a certain extent. All these results agree with the known experimental results.     

Structural characteristics of hen egg ovalbumin expressed in yeast Pichia pastoris

The recombinant ovalbumin (OVA) produced in yeast Pichia pastoris was purified from the culture medium by anion exchange chromatography, and its structural characteristics were compared with those of hen egg OVA, mainly from the point of view of posttranslational modification. The expressed OVA consisted of two molecular species immmunoreactive with antibody for hen egg OVA. The two molecular species, 45 and 47 kDa in molecular size, were thought to correspond to mono-glycosylated form and di-glycosylated form respectively. The non-glycosylated form was not produced in the system. The other posttranslational modifications (N-terminal acetylation and phosphorylation) observed in hen egg OVA were not detected in either of the molecular species. The two recombinant proteins displayed almost exactly the same circular dichroism and intrinsic tryptophan fluorescence spectra as hen egg OVA. The melting temperature, Tm, which was determined from the thermal unfolding curve, was almost identical in the two recombinant proteins, despite the difference in glycosylation levels, while it decreased by about 2.5 degrees C as compared with that of hen egg OVA (77.3 degrees C). These data indicate that the additional glycosylation to Asn-311 in the recombinant protein does not affect protein conformation or thermostability.     

Characterization of Ser73 in Arabidopsis thaliana Glutathione S-transferase zeta class

Glutathione S-transferases (GSTs) are ubiquitous detoxifying superfamily enzymes. The zeta class GST from Arabidopsis thaliana (AtGSTZ) can efficiently degrade dichloroacetic acid (DCA), which is a common carcinogenic contaminant in drinking water. Ser73 in AtGSTZ is a conserved residue at Glutathione binding site (G-site). Compared with the equivalent residues in other GSTs, the catalytic and structural properties of Ser73 were poorly investigated. In this article, site-saturation mutagenesis was performed to characterize the detailed role of Ser73. The DCA de.chlorinating (DCA-DC) activity showed that most of the mutants had less than 3% of the wild-type activity, except S73T and $73A showing 43.48% and 21.62% of the wild-type activity, respectively, indicating that position 73 in AtGSTZ showed low mutational substitutability. Kinetic experiments revealed that mutants S73T, $73A, and S73G showed low binding affinity and catalytic efficiency toward DCA, 1.8-, 3.1-, and 10.7- fold increases in KmDcA values and 4.0-, 9.6-, and 34.1- fold decreases in KcatDCA/KmDCA values, respectively, compared to the wild type. Thermostability and refolding experiments showed that the wild type maintalned more thermostability and recovered activity. These results demonstrated the important role of Set73 in catalytic activity and structural stability of the enzyme. Such properties of Set73 could be particularly crucial to the molecular evolution of AtGSTZ and might,therefore, help explain why Ser73 is conserved in all GSTs. This conclusion might provide insights into the directed evolution of the DCA-DC activity of AtGSTZ.     

Thermostabilization of Pichia stipitis xylitol dehydrogenase by mutation of structural zinc-binding loop

Xylitol dehydrogenase from Pichia stipitis (PsXDH) is one of the key enzymes for the bio-ethanol fermentation system from xylose. Previously, we constructed the C4 mutant (S96C/S99C/Y102C) with enhanced thermostability by introduction of structural zinc. In this study, for further improvement of PsXDH thermostability, we constructed the appropriate structural zinc-binding loop by comparison with other polyol dehydrogenase family members. A high thermostability of PsXDH was obtained by subsequent site-directed mutagenesis of the structural zinc-binding loop. The best mutant in this study (C4/F98R/E101F) showed a 10.8 degrees C higher thermal transition temperature (T(CD)) and 20.8 degrees C higher half denaturation temperature (T(1/2)) compared with wild-type.     

Rescue of the neuroblastoma mutant of the human nucleoside diphosphate kinase A/nm23-H1 by the natural osmolyte trimethylamine-N-oxide

The point mutation S120G in human nucleoside diphosphate kinase A, identified in patients with neuroblastoma, causes a protein folding defect. The urea-unfolded protein cannot refold in vitro, and accumulates as a molten globule folding intermediate. We show here that the trimethylamine-N-oxide (TMAO) corrects the folding defect and stimulated subunit association. TMAO also substantially increased the stability to denaturation by urea of both wild-type and S120G mutant. A non-native folding intermediate accumulated in the presence of 4.5-7 M urea and of 2 M TMAO. It was inactive, monomeric, contained some secondary structure but no tertiary structure and displayed a remarkable stability to denaturation.     

Mutagenic stabilization and/or disruption of a CD4-bound state reveals distinct conformations of the human immunodeficiency virus type 1 gp120 envelope glycoprotein

The human immunodeficiency virus type 1 (HIV-1) gp120 exterior envelope glycoprotein is conformationally flexible. Upon binding to the host cell receptor CD4, gp120 assumes a conformation that is recognized by the second receptor, CCR5 and/or CXCR4, and by the CD4-induced (CD4i) antibodies. Guided by the X-ray crystal structure of a gp120-CD4-CD4i antibody complex, we introduced changes into gp120 that were designed to stabilize or disrupt this conformation. One mutant, 375 S/W, in which the tryptophan indole group is predicted to occupy the Phe 43 cavity in the gp120 interior, apparently favors a gp120 conformation closer to that of the CD4-bound state. The 375 S/W mutant was recognized as well as or better than wild-type gp120 by CD4 and CD4i antibodies, and the large decrease in entropy observed when wild-type gp120 bound CD4 was reduced for the 375 S/W mutant. The recognition of the 375 S/W mutant by CD4BS antibodies, which are directed against the CD4-binding region of gp120, was markedly reduced compared with that of the wild-type gp120. Compared with the wild-type virus, viruses with the 375 S/W envelope glycoproteins were resistant to neutralization by IgG1b12, a CD4BS antibody, were slightly more sensitive to soluble CD4 neutralization and were neutralized more efficiently by the 2G12 antibody. Another mutant, 423 I/P, in which the gp120 bridging sheet was disrupted, did not bind CD4, CCR5, or CD4i antibodies, even though recognition by CD4BS antibodies was efficient. These results indicate that CD4BS antibodies recognize conformations of gp120 different from that recognized by CD4 and CD4i antibodies.