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PubMed2002

Cloning and molecular characterization of a SDS-activated tyrosinase from Marinomonas mediterranea.

The sequence of the tyrosinase gene cloned from Marinomonas mediterranea is reported. It is the second tyrosinase cloned from a Gram negative bacterium. Its size is higher than that of Gram positive tyrosinases from Streptomyces, and more similar to the eukaryotic enzymes. Its sequence shares the features of copper-binding sites found in all tyrosinases. Based in the comparison of tyrosinases from all types of organisms, an extension of the characteristic signatures existing at Prosite is proposed. This tyrosinase shares with some plant and amphibian tyrosinases a strong specific activation by submicellar concentrations of SDS. Intrinsic fluorescence and kinetic properties indicate that the activation is caused by an SDS-dependent conformational change that facilitates the substrate accessibility to the dicopper active site.

Amino Acid Sequence↗
PubMed1999

Substrate and inhibitor-induced conformational changes in the structurally related enzymes UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) and 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS).

UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) and 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) have both a unique three-dimensional topology and overall reaction mechanism in common. In the case of MurA, the substrate-free, unliganded protein exhibits an "open" conformation. Upon binding of substrates, the protein forms a much more tightly packed so-called "closed" form following an induced fit mechanism. In this closed form, the substrates are properly positioned for catalysis. On the basis of the structural and mechanistic similarities of MurA and EPSPS, a similar conformational change is likely to occur in EPSPS to generate a catalytically competent active site. However, there is currently little experimental evidence available to support the occurrence of such a conformational change in EPSPS. Using limited tryptic digestion of MurA,(1) it could be shown that formation of the "closed" conformation of MurA is accompanied by a marked increase of stability toward proteolytic degradation. Formation of the closed conformation was achieved by addition of either an excess of both substrates or the sugar nucleotide substrate in conjunction with the antibiotic fosfomycin. Analysis of the MurA tryptic fragments by MALDI-TOF mass spectrometry demonstrates that the protection of the protein in either case is caused by (1) a specific shielding of regions thereby becoming less accessible as a result of the conformational change, and (2) an unspecific overall protection of the whole protein due to an apparently reduced flexibility of the peptide backbone in the binary and ternary complexes. The establishment of methods to describe the effects of tryptic digestion on MurA under various conditions was then extended to EPSPS. Although EPSPS was found to be much more stable toward proteolysis than MurA, the presence of shikimate 3-phosphate (S3P) and the inhibitor glyphosate led to a pronounced suppression of proteolytic degradation. When unliganded EPSPS was treated with trypsin, three of the peptide fragments obtained could be identified by mass spectrometry. Two of these are located in a region corresponding to the "catalytic" loop in MurA which participates in the conformational change. This indicates a conformational change in EPSPS, similar to the one observed in MurA, leading to the protection mentioned above. Corroborating evidence was obtained using a conformational sensitive monoclonal antibody against EPSPS which showed a 20-fold reduced affinity toward the protein complexed with S3P and glyphosate as compared to the unliganded enzyme.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1999

Conformational changes in the NS3 protease from hepatitis C virus strain Bk monitored by limited proteolysis and mass spectrometry.

Conformational changes occurring within the NS3 protease domain from the hepatitis C virus Bk strain (NS3(1-180)) under different physico-chemical conditions either in the absence or in the presence of its cofactor Pep4A were investigated by limited proteolysis experiments. Because the surface accessibility of the protein is affected by conformational changes, when comparative experiments were carried out on NS3(1-180) either at different glycerol concentrations or in the presence of Pep4A, differential peptide maps were obtained from which protein regions involved in the structural changes could be inferred. The surface topology of isolated NS3(1-180) in solution was essentially consistent with the crystal structure of the protein with the N-terminal segment showing a high conformational flexibility. At higher glycerol concentration, the protease assumed a more compact structure showing a decrease in the accessibility of the N-terminal segment that either was forced to interact with the protein or originate intermolecular interactions with neighboring molecules. Binding of the cofactor Pep4A caused the displacement of the N-terminal arm from the protein moiety, leading this segment to again adopt an open and flexible conformation, thus suggesting that the N-terminus of the protease contributes only marginally to the stability of the complex. The observed conformational changes might be directly correlated with the activation mechanism of the protease by either the cosolvent or the cofactor peptide because they lead to tighter packing of the substrate binding site.

Amino Acid Sequence↗
PubMed2005

Kinetic characterization of tetrapropylammonium inhibition reveals how ATP and Pi alter access to the Na+-K+-ATPase transport site.

Current models of the Na(+)-K(+)-ATPase reaction cycle have ATP binding with low affinity to the K(+)-occluded form and accelerating K(+) deocclusion, presumably by opening the inside gate. Implicit in this situation is that ATP binds after closing the extracellular gate and thus predicts that ATP binding and extracellular cation binding to be mutually exclusive. We tested this hypothesis. Accordingly, we needed a cation that binds outside and not inside, and we determined that tetrapropylammonium (TPA) behaves as such. TPA competed with K(+) (and not Na(+)) for ATPase, TPA was unable to prevent phosphoenzyme (EP) formation even at low Na(+), and TPA decreased the rate of EP hydrolysis in a K(+)-competitive manner. Having established that TPA binding is a measurement of extracellular access, we next determined that TPA and inorganic phosphate (P(i)) were not mutually exclusive inhibitors of para-nitrophenylphosphatase (pNPPase) activity, implying that when P(i) is bound, the transport site has extracellular access. Surprisingly, we found that ATP and TPA also were not mutually exclusive inhibitors of pNPPase activity, implying that when the cation transport site has extracellular access, ATP can still bind. This is consistent with a model in which ATP speeds up the conformational changes that lead to intracellular or extracellular access, but that ATP binding is not, by itself, the trigger that causes opening of the cation site to the cytoplasm.

4-Nitrophenylphosphatase↗
PubMed2006

Fibrillogenic and non-fibrillogenic ensembles of SDS-bound human alpha-synuclein.

Fibril formation of alpha-synuclein is associated with several neurodegenerative diseases, including Parkinson's disease in humans. The anionic detergent sodium dodecyl sulfate (SDS) can accelerate the fibril formation in vitro. However, the molecular basis of this acceleration is not clear. Our study shows that native alpha-synuclein exhibits relatively less fibril growth despite providing fibril seeds for nucleation. The presence of SDS promotes the seeded fibril growth in a concentration-dependent manner, with an optimal concentration of 0.5-0.75 mM. We used isothermal calorimetry, hydrophobic dye binding and circular dichroism spectroscopy to characterize the protein-detergent interactions as a function of the concentration of SDS. Interaction of SDS with alpha-synuclein when studied by isothermal titration calorimetry and hydrophobic dye-binding reveals a similar characteristic optimal behavior between 0.5 mM and 0.75 mM SDS. The study shows two types of ensembles of alpha-synuclein and SDS: the fibrillogenic ensembles formed with optimal concentration of SDS around 0.5-0.75 mM are characterized by enhanced accessible hydrophobic surfaces and extended to partially helical conformation, while the less or non-fibrillogenic ensembles formed above 2 mM SDS are characterized by less accessible hydrophobic surfaces and maximal helical content. Little or no fibrillogenicity of the ensembles observed above 2 mM SDS could be partly because of the observed intrinsic instability of the fibrils under the condition.

Circular Dichroism↗
PubMed1998

Structural determinants of metal-induced conformational changes in HIV-1 integrase.

Human immunodeficiency virus type 1 (HIV-1) integrase (IN) undergoes a reversible metal-induced conformational change that activates the enzyme (Asante-Appiah, E., and Skalka, A. M. (1997) J. Biol. Chem. 272, 16196-16205). In this report, key structural features that mediate this conformational change have been identified by site-directed mutagenesis, limited proteolysis, and mass spectrometry studies. The results reveal two separable metal-induced effects. One depends on residues in the N-terminal domain (amino acids 1-50) and a C-terminal tail (amino acids 274-288) and is detected by increased resistance of the full-length protein to proteolytic digestion. This effect appears to depend on metal binding at an undefined location distinct from the known sites in the N-terminal and catalytic core domains. The second conformational change depends on metal binding at the active site in the catalytic core domain. Substitution of acidic residues Asp64 or Glu152 in the catalytic core D,D(35)E motif or truncation of the Src homology 3 (SH3)-like domain in the C-terminal region of the enzyme abolishes this metal-induced change. Comparison of tryptic digests of an HIV-1 IN derivative competent for metal-induced conformational change and a conformation-defective D64N derivative identified specific regions in HIV-1 IN that are affected by this second change. A region in the N terminus that spans Lys14, an extended loop and the adjacent region in the core domain (including lysines 136, 156, and 160 and Arg173), and residues at the C terminus beyond the SH3-like domain all become less accessible to proteolysis in the conformation-competent protein. In contrast, a region that encompasses Lys258 in the putative DNA binding groove of the SH3-like domain becomes more sensitive to proteolysis in the presence of Mn2+. The results are consistent with a model in which the binding of the metal ion by residues of the D,D(35)E motif elicits specific changes in all three domains of HIV-1 IN, inducing the restructuring of the enzyme for catalytic competence.

Amino Acid Sequence↗
PubMed1986

Human fibroblast chromatin states as effectors of the DNA-binding characteristics of benzo[a]pyrene anti-7,8-dihydrodiol 9,10-epoxide and two nonalkylating DNA-binding molecules.

Pure populations of mitotic or nonmitotic diploid human fibroblasts (greater than 98% pure) were exposed to [3H]benzo [a]pyrene (CAS: 50-32-8) anti-7,8-dihydrodiol 9,10-epoxide: r-7,t-8 dihydroxy-t-9, 10-oxy-7,8,9,10-tetrahydrobenzo [a]pyrene (or anti-diol-epoxide). In addition, metaphase chromosomes, interphase chromatin, or naked DNA was isolated from the pure cell populations and then titrated to saturation with anti-diol-epoxide, chromomycin A3, or 3,8-diamino-5-ethyl-6-phenylphenanthridinium bromide (ethidium bromide). At saturation, anti-diol-epoxide had covalently modified 1.5% of the total deoxyguanosine residues in naked DNA, and this was reduced to 29 and 15% of this level in saturating the available anti-diol-epoxide-binding sites in chromosomes or chromatin, respectively. A similar hierarchy of accessible binding sites (naked DNA greater than chromosomes greater than chromatin) was also observed for the noncovalent interaction of chromomycin A3 or ethidium bromide with the human cell DNA. Deproteinization of the chromosome or chromatin preparations returned the level of drug binding to that seen with naked DNA. The results clarify the association between proteins and DNA in human chromatin and suggest how cell-cycle-dependent changes in DNA-associated proteins or higher-order changes in protein-DNA conformation can act to alter the access of molecules to DNA-binding sites.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1990

A mutant T4 lysozyme displays five different crystal conformations.

Phage T4 lysozyme consists of two domains between which is formed the active-site cleft of the enzyme. The crystallographically determined thermal displacement parameters for the protein suggested that the amino terminal of the two domains undergoes 'hinge-bending' motion about an axis passing through the waist of the molecule. Such conformational mobility may be important in allowing access of substrates to the active site of the enzyme. We report here a crystallographic study of a mutant T4 lysozyme which demonstrates further the conformational flexibility of the protein. A mutant form of the enzyme with a methionine residue (Met 6) replaced by isoleucine crystallizes with four independent molecules in the crystal lattice. These four molecules have distinctly different conformations. The mutant protein can also crystallize in standard form with a structure very similar to the wild-type protein. Thus the mutant protein can adopt five different crystal conformations. The isoleucine for methionine substitution at the intersection of the two domains of T4 lysozyme apparently enhances the hinge-bending motion presumed to occur in the wild-type protein, without significantly affecting the catalytic activity or thermal stability of the protein.

Binding Sites↗
PubMed2002

Structural basis for CO2 fixation by a novel member of the disulfide oxidoreductase family of enzymes, 2-ketopropyl-coenzyme M oxidoreductase/carboxylase.

The NADPH:2-ketopropyl-coenzyme M oxidoreductase/carboxylase (2-KPCC) is the terminal enzyme in a metabolic pathway that results in the conversion of propylene to the central metabolite acetoacetate in Xanthobacter autotrophicus Py2. This enzyme is an FAD-containing enzyme that is a member of the NADPH:disulfide oxidoreductase (DSOR) family of enzymes that include glutathione reductase, dihydrolipoamide dehydrogenase, trypanothione reductase, thioredoxin reductase, and mercuric reductase. In contrast to the prototypical reactions catalyzed by members of the DSOR family, the NADPH:2-ketopropyl-coenzyme M oxidoreductase/carboxylase catalyzes the reductive cleavage of the thioether linkage of 2-ketopropyl-coenzyme M, and the subsequent carboxylation of the ketopropyl cleavage product, yielding the products acetoacetate and free coenzyme M. The structure of 2-KPCC reveals a unique active site in comparison to those of other members of the DSOR family of enzymes and demonstrates how the enzyme architecture has been adapted for the more sophisticated biochemical reaction. In addition, comparison of the structures in the native state and in the presence of bound substrate indicates the binding of the substrate 2-ketopropyl-coenzyme M induces a conformational change resulting in the collapse of the substrate access channel. The encapsulation of the substrate in this manner is reminiscent of the conformational changes observed in the well-characterized CO2-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxidase (Rubisco).

Acetone↗
PubMed1996

Site-directed fluorescence labeling of P-glycoprotein on cysteine residues in the nucleotide binding domains.

P-Glycoprotein is a member of the ABC superfamily of membrane transporters, and functions as an ATP-driven active efflux pump for natural products and chemotherapeutic drugs. Overexpression of P-glycoprotein is a major cause of multidrug resistance in human cancers. Sulfhydryl modification agents are known to inactivate both P-glycoprotein ATPase activity and transport function. In the present study, P-glycoprotein purified from CHRB30 cells was covalently labeled at two conserved Cys residues, one within each of the nucleotide binding domains, using 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid (MIANS). MIANS modification inactivated P-glycoprotein ATPase function, in a concentration-dependent fashion. Increasing concentrations of ATP blocked MIANS labeling with an IC50 of 0.37 mM (similar to the KM for ATP hydrolysis), which suggests that the label is located close to the site of ATP binding within the nucleotide binding domain. A blue shift in the fluorescence spectrum of MIANS bound to P-glycoprotein indicated that the labeled Cys residues are situated in a nonpolar environment. MIANS-labeled P-glycoprotein was still able to bind ATP, as demonstrated by quenching of the fluorescence, with a Kd of 0.46 mM. Addition of a variety of drugs and chemosensitizers to MIANS-labeled P-glycoprotein led to substantial quenching of the probe fluorescence within the nucleotide binding domains. Dissociation constants for drug binding measured by fluorescence quenching were in the range of 0.77 microM for vinblastine to 158 microM for colchicine. Quenching by ATP and drugs was independent and additive, suggesting that each produces a defined change in the protein. The rate of MIANS labeling of Pgp was reduced in the presence of drugs and chemosensitizers, implying that a long-range conformational change arises from drug binding which alters the accessibility of the nucleotide binding domains to MIANS. These results suggest that there is conformational communication between the drug binding site(s) of P-glycoprotein and the ATPase catalytic sites within the nucleotide binding domains.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

Monoclonal antibodies as probes for the changes in antigenicity of bovine and porcine aspartyl proteases with pH.

Two monoclonal antibodies, 918(4) and 139(7), directed against either bovine or porcine pepsin, respectively, were retained among 365 positive hybridoma clones. These monoclonal antibodies were characterized by using both indirect and sandwich ELISA. Characterization of these monoclonal antibodies was further performed by the biospecific interaction analysis (BIA-core analysis). Then, they were used as antigenic probes to study the changes in antigenicity of both bovine and porcine pepsins induced by pH. The results demonstrated the importance of the conformational change in both catalytic activities and antigenic determinant accessibility of bovine and porcine pepsins. Furthermore, our results suggest that changes in the conformation due to pH can be detected by specific monoclonal antibodies.

Animals↗
PubMed2000

Stepwise assembly of a glucocorticoid receptor.hsp90 heterocomplex resolves two sequential ATP-dependent events involving first hsp70 and then hsp90 in opening of the steroid binding pocket.

A system of five purified proteins that assembles stable glucocorticoid receptor (GR)-hsp90 heterocomplexes has been reconstituted from reticulocyte lysate. Two proteins, hsp90 and hsp70, are required for the activation of steroid binding activity that occurs with heterocomplex assembly, and three proteins, Hop, hsp40, p23, act as co-chaperones that enhance activation and assembly (Morishima, Y., Kanelakis, K. C., Silverstein, A.M., Dittmar, K. D., Estrada, L., and Pratt, W. B. (2000) J. Biol. Chem. 275, 6894-6900). Here we demonstrate that the first step in assembly is the ATP-dependent and hsp40 (YDJ-1)-dependent binding of hsp70 to the GR. After elimination of free hsp70, these preformed GR.hsp70 complexes can be activated to the steroid binding state by the hsp70 free assembly system in a second ATP-dependent step. hsp90 is required for opening of the steroid binding pocket and is converted to its ATP-dependent conformation during this second step. We predict that hsp70 in its ATP-dependent conformation binds initially to the folded receptor and is then converted to the ADP-dependent form with high affinity for hydrophobic substrate. This conversion initiates the opening of the hydrophobic steroid binding pocket such that it can now accept the hydrophobic binding form of hsp90, which in turn must be converted to its ATP-dependent conformation for the pocket to be accessible by steroid.

Adenosine Triphosphate↗
PubMed2004

Molecular basis for TPR domain-mediated regulation of protein phosphatase 5.

Protein phosphatase 5 (Ppp5) is a serine/threonine protein phosphatase comprising a regulatory tetratricopeptide repeat (TPR) domain N-terminal to its phosphatase domain. Ppp5 functions in signalling pathways that control cellular responses to stress, glucocorticoids and DNA damage. Its phosphatase activity is suppressed by an autoinhibited conformation maintained by the TPR domain and a C-terminal subdomain. By interacting with the TPR domain, heat shock protein 90 (Hsp90) and fatty acids including arachidonic acid stimulate phosphatase activity. Here, we describe the structure of the autoinhibited state of Ppp5, revealing mechanisms of TPR-mediated phosphatase inhibition and Hsp90- and arachidonic acid-induced stimulation of phosphatase activity. The TPR domain engages with the catalytic channel of the phosphatase domain, restricting access to the catalytic site. This autoinhibited conformation of Ppp5 is stabilised by the C-terminal alphaJ helix that contacts a region of the Hsp90-binding groove on the TPR domain. Hsp90 activates Ppp5 by disrupting TPR-phosphatase domain interactions, permitting substrate access to the constitutively active phosphatase domain, whereas arachidonic acid prompts an alternate conformation of the TPR domain, destabilising the TPR-phosphatase domain interface.

Acyl Coenzyme A↗
PubMed2000

[Effect of surface-active agents (SAA) on peroxidase activity. III. Effect of nonionic SAA].

The effect of a series of nonionic surfactants on the initial rate of the peroxide oxidation of 5-aminosalicylic acid in solution catalyzed by horseradish peroxidase was studied. As the surfactant concentration increases, the peroxidation rate first increases, then decreases, and the increase/decrease cycle is repeated. The primary increase may be induced by a change in properties of the medium under the action of surfactants, and the following decrease, by the enzyme inhibition. The secondary increase may be explained by a change in the enzyme conformation and an increase in the accessibility of its active site for the substrate due to the immobilization of the protein in the surfactant aggregates, whereas the secondary decrease, by a shielding of the protein with these aggregates.

Catalysis↗
PubMed2005

The role of the third extracellular loop of the Na+,K+-ATPase alpha subunit in a luminal gating mechanism.

Na+,K+-ATPase is responsible for maintaining the cross-membrane Na+ and K+ gradients of animal cells. This P-type ATPase works via a complex transport cycle, during which it binds and occludes three intracellular Na+ ions and then two extracellular K+ ions, which it then releases on the other side of the membrane. The cation pathway through the protein, and the structures responsible for occluding cations inside the protein, have not yet been definitely identified. We used cysteine mutagenesis to explore the accessibility and the role of five conserved residues in the short third extracellular loop, between the fifth and the sixth transmembrane helices. The P801C and L802C mutants were not affected by extracellular sulfhydryl reagents. The presence of cysteine residues at three positions (G803C, T804C and V805C) conferred sensitivity to omeprazole, a known inhibitor of the gastric proton pump, and to [2-(trimethylammonium)-ethyl]methanethiosulphonate bromide (MTSET). The effects of omeprazole and MTSET were modulated by the presence of extracellular K+, indicating that the accessibility of these positions depended on the conformational state of the protein. MTSET binding to cysteine at position 803 partially inhibited the Na+,K+-pump function by decreasing its affinity towards extracellular K+, suggesting a restriction of the access of extracellular K+ ions to their binding sites. In contrast, MTSET binding to cysteine at position 805 partially inhibited the Na+,K+-pump function by reducing its maximum turnover rate, probably by slowing a rate-limiting conformational change. These residues occupy positions that are critical for either the cation pathway or the conformational modifications.

Animals↗
PubMed2000

Characteristics of the adeno-associated virus preintegration site in human chromosome 19: open chromatin conformation and transcription-competent environment.

Adeno-associated virus (AAV) establishes latency in infected cells by integrating into the cellular genome, with a high preference for a unique region, called AAVS1, of the human chromosome 19. The AAV proteins Rep78 and -68 are postulated to initiate the site-specific integration process by binding to a Rep binding site (RBS) in AAVS1. We provide further evidence to corroborate this model by demonstrating that the AAVS1 RBS in human cell lines is located near a DNase I hypersensitive "open" chromatin region and therefore is potentially easily accessible to Rep proteins. This open conformation is maintained in transgenic rats which carry an AAVS1 3. 5-kb DNA fragment and are proficient for Rep-mediated site-specific integration. Interestingly, the core of the DNAse I hypersensitive site in AAVS1 corresponds to a sequence displaying transcriptional enhancer-like properties, suggesting that AAVS1 constitutes a transcription-competent environment. The implications of our findings for AAV physiology and gene therapy are discussed.

Animals↗
PubMed1997

Conformational analysis of potent sweet taste ligands by nuclear magnetic resonance, computer simulations and X-ray diffraction studies.

Four potent sweet-tasting molecules, N-(3,3-dimethylbutyl)-L-aspartyl-L-phenylalanine methylester 1 (7000 times more potent than sucrose), N-(3,3-dimethylbutyl)-L-aspartyl-D-valine (S)-alpha-ethylbenzylamide 2 (3000 time more potent than sucrose), L-aspartyl-D-valine (R)-alpha-methoxymethylbenzylamide 3 (1350 times more potent than sucrose and L-aspartyl-(1R,2S,4S)-1-methyl-2-hydroxy-4-phenylhexylamide 4 (2500 times more potent than sucrose) were studied by 1H NMR and computer simulations. These flexible molecules adopt multiple conformations in solution. The "L-shaped" structure, which we believe to be responsible for sweet taste is accessible to all four compounds in solution. Extended conformations with the AH and B-containing moieties in the +y-axis and the hydrophobic group X pointing in the y-axis have also been observed for all four sweeteners. For compounds 1 and 3, the solid-state conformations were determined by X-ray diffraction studies. These results demonstrate that compounds 1 and 3 adopt an "L-shaped" structure even in the crystalline state. The extraordinary potency of the N-alkylated compound 1 compared with the unsubstituted Asp-Phe-OMe may be explained by the effect of a second hydrophobic binding domain in addition to interactions arising from the "L-shaped" structure.

Computer Simulation↗
PubMed1997

The V3-directed immune response in natural human immunodeficiency virus type 1 infection is predominantly directed against a variable, discontinuous epitope presented by the gp120 V3 domain.

The specific binding of antibodies to the V3 loop in sera from human immunodeficiency type 1 (HIV-1)-infected individuals was investigated. Different V3 structures were analyzed as full-length loops or by pepscan. Our data show that on full-length V3 loops, both variable regions on either side of the tip of the loop (GPGRAF) contribute to a common epitope for type-specific antibodies. Type-specific antibodies bound strongly and at high titers to native V3 loops but negligibly once the loop was denatured. In contrast to the type-specific, discontinuous epitope, the linear, conserved epitopes presented by the full-length V3 loop, the tip, the amino-terminal base, and the carboxy-terminal base were not accessible to serum antibody. When the V3 sequences were analyzed with linear peptides, antibodies bound preferentially to peptides containing the conserved GPGRAF sequence. Thus, two different specificities of V3-directed antibodies were detected in patient sera. Unlike group-specific antibodies directed against GPGRAF peptides, lack of type-specific antibodies directed against the discontinuous epitope was correlated with viral escape from autologous neutralization. Our data suggest that the full-length conformation of the V3 loop is accessible predominantly to highly type-specific antibodies present in sera from HIV-1-infected individuals. These antibodies are directed against discontinuous V3 epitopes, not against conserved linear V3 targets. The implications of these findings for viral escape and blockade of infection with V3-based vaccines are discussed.

Amino Acid Sequence↗