Exquisite specificity and peptide epitope recognition promiscuity, properties shared by antibodies from sharks to humans

This review considers definitions of the specificity of antibodies including the development of recent concepts of recognition polyspecificity and epitope promiscuity. Using sets of homologous and unrelated peptides derived from the sequences of immunoglobulin and T cell receptor chains we offer operational definitions of cross-reactivity by investigating correlations of either identities in amino acid sequence, or in hydrophobicity/hydrophilicity profiles with degree of binding in enzyme-linked immunosorbent assays. Polyreactivity, or polyspecificity, are terms used to denote binding of a monoclonal antibody or purified antibody preparation to large complex molecules that are structurally unrelated, such as thyroglobulin and DNA. As a first approximation, there is a linear correlation between degree of sequence identity or hydrophobicity/hydrophilicity and antigenic cross-binding. However, catastrophic interchanges of amino acids can occur where changing of one amino acid out of 16 in a synthetic peptide essentially eliminates binding to certain antibodies. An operational definition of epitope promiscuity for peptides is the case where two peptides show little or no identity in amino acid sequence but bind strongly to the same antibody as shown by either direct binding or competitive inhibition. Analysis of antibodies of humans and sharks, the two most divergent species in evolution to express antibodies and the combinatorial immune response, indicates that the capacity for both exquisite specificity and epitope recognition promiscuity are essential conserved features of individual vertebrate antibodies.     

Antibodies against a peptide of cholera toxin differing in cross-reactivity with the toxin differ in their specific interactions with the peptide as observed by 1H NMR spectroscopy.

The interactions between the aromatic residues of the monoclonal antibody TE34, and its peptide antigen CTP3, have been studied by 2D TRNOE difference spectroscopy. The sequence of CTP3 corresponds to residues 50-64 of the B subunit of cholera toxin (VEVPGSQHIDSQKKA). Unlike two previously studied anti-CTP3 antibodies (TE32 and TE33), the TE34 antibody does not bind the toxin. The off-rate of CTP3 from TE34 was found to be too slow to measure strong TRNOE cross-peaks between the antibody and the peptide. Much faster off-rates, resulting in a strong TRNOE, were obtained for two peptide analogues: (a) CTP3 with an amide in the C-terminus (VEVPGSQHIDSQKKA-NH2) and (b) a truncated version of the peptide (N-acetyl-IDSQKKA). These modifications do not interfere significantly either with the interactions of the unmodified part of the peptide with the antibody or with intramolecular interactions occurring in the epitope recognized by the antibody. The combined use of these peptides allows us to study the interactions between the antibody and the whole peptide. Two tyrosine residues and one or more tryptophan and phenylalanine residues have been found to interact with histidine-8, isoleucine-9, aspartate-10, lysine-13 and/or lysine-14, and alanine-15 of the peptide. In the bound peptide, we observe interactions of a lysine residue with aspartate-10 beta protons. While the peptide epitope recognized by TE34 is between histidine-8 and the negatively charged C-terminus, that recognized by TE32 and TE33 is between residues 3 and 10 of the peptide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Soft docking an L and a D peptide to an anticholera toxin antibody using internal coordinate mechanics.

BACKGROUND: The tremendous increase in sequential and structural information is a challenge for computer-assisted modelling to predict the binding modes of interacting biomolecules. One important area is the structural understanding of protein-peptide interactions, information that is increasingly important for the design of biologically active compounds. RESULTS: We predicted the three-dimensional structure of a complex between the monoclonal antibody TE33 and its cholera-toxin-derived peptide epitope VPGSQHID. Using the internal coordinate mechanics (ICM) method of flexible docking, the bound conformation of the initially extended peptide epitope to the antibody crystal or modelled structure reproduced the known binding conformation to a root mean square deviation of between 1.9 A and 3.1 A. The predicted complexes are in good agreement with binding data obtained from substitutional analyses in which each epitope residue is replaced by all other amino acids. Furthermore, a de novo prediction of the recently discovered TE33-binding D peptide dwGsqhydp (single-letter amino acid code where D amino acids are represented by lower-case letters) explains results obtained from binding studies with 172 peptide analogues. CONCLUSIONS: Despite the difficulties arising from the huge conformational space of a peptide, this approach allowed the prediction of the correct binding orientation and the majority of essential binding features of a peptide-antibody complex.     

Linear epitope mapping by native mass spectrometry

The identification of antigenic epitopes is important for the optimization of monoclonal antibodies (mAbs) intended as therapeutic agents. MS has proven to be a powerful tool for the study of noncovalent molecular interactions such as those involved in antibody-antigen (Ab-Ag) binding. In this work, we described a novel methodology for mapping a linear epitope based on direct mass spectrometric measurement of Ab-Ag complexes. To demonstrate the utility of our methodology, we employed two approaches, epitope excision and epitope extraction, to study a model system consisting of a Fab antibody fragment with specificity toward the peptide aβ(1-40). In epitope excision, the Fab and aβ(1-40) complex was treated with proteolytic enzymes and the digested complexes were directly monitored by MS under native conditions. Mass differences between the Fab-aβ complex and the Fab control revealed the size of epitope peptides that were bound to the Fab. Using the epitope extraction approach, aβ(1-40) was first digested by Lys-C, and the fragment containing the epitope was selected by Fab binding. Data analysis allowed mapping of the epitope to aβ(16-27) which is in good agreement with previously unpublished data. The utility of the methodology was demonstrated by elucidating the binding epitopes for two full-length anti-aβ(1-40) mAbs.     

NMR study of the complexes between a synthetic peptide derived from the B subunit of cholera toxin and three monoclonal antibodies against it

The contact interactions between a synthetic peptide and three different anti-peptide monoclonal antibodies have been studied by nuclear magnetic resonance (NMR). The synthetic peptide is CTP3 (residues 50-64 of the B subunit of cholera toxin) suggested as a possible epitope for synthetic vaccine against cholera. The hybridoma cell lines TE33 and TE32 derived after immunization with CTP3 produce antibodies cross-reactive with the native toxin. The cell line TE34 produces anti-CTP3 antibodies that do not bind the toxin. Selective deuteriation of the antibodies has been used to simplify the proton NMR spectra and to assign resonances to specific types of amino acids. The difference spectra between the proton NMR spectrum of the peptide-Fab complex and that of Fab indicate that the combining site structures of TE32 and TE33 are very similar but differ considerably from the combining site structure of TE34. By magnetization transfer experiments with selectively deuteriated Fab fragment of the antibody, we have found that in TE32 and TE33 the histidine residue of the peptide is buried in a hydrophobic pocket of the antibody combining site, formed by a tryptophan and two tyrosine residues. The hydrophobic nature of the pocket is further demonstrated by the lack of any pH titration effect on the chemical shift of the C4H of the bound peptide histidine. In contrast, for TE34 we have found only one tyrosine residue in contact with the histidine of the peptide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induced peptide conformations in different antibody complexes: molecular modeling of the three-dimensional structure of peptide-antibody complexes using NMR-derived distance restraints.

Intramolecular interactions in bound cholera toxin peptide (CTP3) in three antibody complexes were studied by two-dimensional transferred NOE spectroscopy. These measurements together with previously recorded spectra that show intermolecular interactions in these complexes were used to obtain restraints on interproton distances in two of these complexes (TE32 and TE33). The NMR-derived distance restraints were used to dock the peptide into calculated models for the three-dimensional structure of the antibody combining site. It was found that TE32 and TE33 recognize a loop comprising the sequence VPGSQHID and a beta-turn formed by the sequence VPGS. The third antibody, TE34, recognizes a different epitope within the same peptide and a beta-turn formed by the sequence IDSQ. Neither of these two turns was observed in the free peptide. The formation of a beta-turn in the bound peptide gives a compact conformation that maximizes the contact with the antibody and that has greater conformational freedom than alpha-helix or beta-sheet secondary structure. A total of 15 antibody residues are involved in peptide contacts in the TE33 complex, and 73% of the contact area in the antibody combining site consists of the side chains of aromatic amino acids. A comparison of the NMR-derived models for CTP3 interacting with TE32 and TE33 with the previously derived model for TE34 reveals a relationship between amino acid sequence and combining site structure and function. (a) The three aromatic residues that interact with the peptide in TE32 and TE33 complexes, Tyr 32L, Tyr 32H, and Trp 50H, are invariant in all light chains sharing at least 65% identity with TE33 and TE32 and in all heavy chains sharing at least 75% identity with TE33. Although TE34 differs from TE32 and TE33 in its fine specificity, these aromatic residues are conserved in TE34 and interact with its antigen. Therefore, we conclude that the role of these three aromatic residues is to participate in nonspecific hydrophobic interactions with the antigen. (b) Residues 31, 31c, and 31e of CDR1 of the light chain interact with the antigen in all three antibodies that we have studied. The amino acids in these positions in TE34 differ from those in TE32 and TE33, and they are involved in specific polar interactions with the antigen. (c) CDR3 of the heavy chain varies considerably both in length and in sequence between TE34 and the two other anti-CTP3 antibodies. These changes modify the shape of the combining site and the hydrophobic and polar interactions of CDR3 with the peptide antigen.     

Structure of an anti-cholera toxin antibody Fab in complex with an epitope-derived D-peptide: a case of polyspecific recognition

The structure of a complex of the anti-cholera toxin antibody TE33 Fab (fragment antibody) with the D-peptide vpGsqhyds was solved to 1.78 A resolution. The D-peptide was derived from the linear L-peptide epitope VPGSQHIDS by a stepwise transformation. Despite the very similar amino acid sequence-the only difference is a tyrosine residue in position 7-there are marked differences in the individual positions with respect to their contribution to the peptide overall affinity as ascertained by a complete substitutional analysis. This is reflected by the X-ray structure of the TE33 Fab/D-peptide complex where there is an inverted orientation of the D-peptide as compared with the known structure of a corresponding complex containing the epitope L-peptide, with the side chains establishing different contacts within the binding site of TE33. The D- and L-peptide affinities are comparable and the surface areas buried by complex formation are almost the same. Thus the antibody TE33 provides a typical example for polyspecific binding behavior of IgG family antibodies.     

Functional fine-mapping and molecular modeling of a conserved loop epitope of the measles virus hemagglutinin protein.

Neutralizing and protective monoclonal antibodies (mAbs) were used to fine-map the highly conserved hemagglutinin noose epitope (H379-410, HNE) of the measles virus. Short peptides mimicking this epitope were previously shown to induce virus-neutralizing antibodies [El Kasmi et al. (2000) J. Gen. Virol.81, 729-735]. The epitope contains three cysteine residues, two of which (Cys386 and Cys394) form a disulfide bridge critical for antibody binding. Substitution and truncation analogues revealed four residues critical for binding (Lys387, Gly388, Gln391 and Glu395) and suggested the binding motif X7C[KR]GX[AINQ]QX2CEX5 for three distinct protective mAbs. This motif was found in more than 90% of the wild-type viruses. An independent molecular model of the core epitope predicted an amphiphilic loop displaying a remarkably stable and rigid loop conformation. The three hydrophilic contact residues Lys387, Gln391 and Glu395 pointed on the virus towards the solvent-exposed side of the planar loop and the permissive hydrophobic residues Ile390, Ala392 and Leu393 towards the solvent-hidden side of the loop, precluding antibody binding. The high affinity (Kd = 7.60 nm) of the mAb BH216 for the peptide suggests a high structural resemblance of the peptide with the natural epitope and indicates that most interactions with the protein are also contributed by the peptide. Improved peptides designed on the basis of these findings induced sera that crossreacted with the native measles virus hemagglutinin protein, providing important information about a lead structure for the design of more stable antigens of a synthetic or recombinant subunit vaccine.     

Molecular characterization of the β‐amyloid(4‐10) epitope of plaque specific Aβ antibodies by affinity‐mass spectrometry using alanine site mutation

Alzheimer disease is a neurodegenerative disease affecting an increasing number of patients worldwide. Current therapeutic strategies are directed to molecules capable to block the aggregation of the β‐amyloid(1‐42) (Aβ) peptide and its shorter naturally occurring peptide fragments into toxic oligomers and amyloid fibrils. Aβ‐specific antibodies have been recently developed as powerful antiaggregation tools. The identification and functional characterization of the epitope structures of Aβ antibodies contributes to the elucidation of their mechanism of action in the human organism. In previous studies, the Aβ(4‐10) peptide has been identified as an epitope for the polyclonal anti‐Aβ(1‐42) antibody that has been shown capable to reduce amyloid deposition in a transgenic Alzheimer disease mouse model. To determine the functional significance of the amino acid residues involved in binding to the antibody, we report here the effects of alanine single‐site mutations within the Aβ‐epitope sequence on the antigen‐antibody interaction. Specific identification of the essential affinity preserving mutant peptides was obtained by exposing a Sepharose‐immobilized antibody column to an equimolar mixture of mutant peptides, followed by analysis of bound peptides using high‐resolution MALDI‐Fourier transform‐Ion Cyclotron Resonance mass spectrometry. For the polyclonal antibody, affinity was preserved in the H6A, D7A, S8A, and G9A mutants but was lost in the F4, R5, and Y10 mutants, indicating these residues as essential amino acids for binding. Enzyme‐linked immunosorbent assays confirmed the binding differences of the mutant peptides to the polyclonal antibody. In contrast, the mass spectrometric analysis of the mutant Aβ(4‐10) peptides upon affinity binding to a monoclonal anti‐Aβ(1‐17) antibody showed complete loss of binding by Ala‐site mutation of any residue of the Aβ(4‐10) epitope. Surface plasmon resonance affinity determination of wild‐type Aβ(1‐17) to the monoclonal Aβ antibody provided a binding constant K_D in the low nanomolar range. These results provide valuable information in the elucidation of the binding mechanism and the development of Aβ‐specific antibodies with improved therapeutic efficacy.     

Epitope‐specificity of recombinant antibodies reveals promiscuous peptide‐binding properties

Protein–peptide interactions are a common occurrence and essential for numerous cellular processes, and frequently explored in broad applications within biology, medicine, and proteomics. Therefore, understanding the molecular mechanism(s) of protein–peptide recognition, specificity, and binding interactions will be essential. In this study, we report the first detailed analysis of antibody–peptide interaction characteristics, by combining large‐scale experimental peptide binding data with the structural analysis of eight human recombinant antibodies and numerous peptides, targeting tryptic mammalian and eukaryote proteomes. The results consistently revealed that promiscuous peptide‐binding interactions, that is, both specific and degenerate binding, were exhibited by all antibodies, and the discovery was corroborated by orthogonal data, indicating that this might be a general phenomenon for low‐affinity antibody–peptide interactions. The molecular mechanism for the degenerate peptide‐binding specificity appeared to be executed through the use of 2–3 semi‐conserved anchor residues in the C‐terminal part of the peptides, in analogue to the mechanism utilized by the major histocompatibility complex–peptide complexes. In the long‐term, this knowledge will be instrumental for advancing our fundamental understanding of protein–peptide interactions, as well as for designing, generating, and applying peptide specific antibodies, or peptide‐binding proteins in general, in various biotechnical and medical applications.     

Structural basis for polyspecificity in the POT family of proton‐coupled oligopeptide transporters

An enigma in the field of peptide transport is the structural basis for ligand promiscuity, as exemplified by PepT1, the mammalian plasma membrane peptide transporter. Here, we present crystal structures of di‐ and tripeptide‐bound complexes of a bacterial homologue of PepT1, which reveal at least two mechanisms for peptide recognition that operate within a single, centrally located binding site. The dipeptide was orientated laterally in the binding site, whereas the tripeptide revealed an alternative vertical binding mode. The co‐crystal structures combined with functional studies reveal that biochemically distinct peptide‐binding sites likely operate within the POT/PTR family of proton‐coupled symporters and suggest that transport promiscuity has arisen in part through the ability of the binding site to accommodate peptides in multiple orientations for transport.     

Delineation of a conserved B cell epitope on bonnet monkey (Macaca radiata) and human zona pellucida glycoprotein-B by monoclonal antibodies demonstrating inhibition of sperm-egg binding

To circumvent autoimmune oophoritis after immunization with zona pellucida (ZP) glycoproteins, synthetic peptides encompassing B cell epitope(s) and devoid of oophoritogenic T cell epitopes as immunogens have been proposed. In this study, bonnet monkey (Macaca radiata) ZP glycoprotein-B (bmZPB) was expressed as polyhistidine fusion protein in Escherichia coli. Rabbit polyclonal antibodies against recombinant bmZPB (r-bmZPB) significantly inhibited human sperm-oocyte binding. To map B cell epitopes on ZPB, a panel of 7 murine monoclonal antibodies (mAbs) was generated against r-bmZPB. All 7 mAbs, when tested in an indirect immunofluorescence assay, reacted with bonnet monkey ZP, and only 6 recognized human zonae. Monoclonal antibodies MA-809, -811, -813, and -825 showed significant inhibition in the binding of human spermatozoa to human ZP in a hemizona assay. Epitope-mapping studies using multipin peptide synthesis strategy revealed that these 4 mAbs recognized a common epitope corresponding to amino acids (aa) 136-147 (DAPDTDWCDSIP). Competitive binding studies revealed that the synthetic peptide corresponding to the identified epitope (aa 136-147) inhibited the binding of MA-809, -811, -813, and -825 to r-bmZPB in an ELISA and to bonnet monkey ZP in an indirect immunofluorescence assay. The epitopic domain corresponding to aa 136-147 of bmZPB was completely conserved in human ZPB. These studies will further help in designing ZP-based synthetic peptide immunogens incorporating relevant B cell epitope for fertility regulation in humans.     

Functional reconstruction and synthetic mimicry of a conformational epitope using CLIPS technology

This paper describes immunization studies with CLIPS-constrained peptides covering only the major part (beta3-loop) of a structurally complex antigenic site on human Follicle Stimulating Hormone beta-subunit (FSH-beta). In cases where linear and SS-constrained peptides fail, the CLIPS-constrained peptides generate polyclonal antibodies with high neutralizing activity for hFSH. The sera were shown to be specific for hFSH over human Luteinizing Hormone (hLH) and human Chorionic Gonadotropin (hCG). ELISA-competition studies and circular dichroism (CD)-measurements illustrate clearly that activity of the peptides in antibody binding and generation relates directly to precise and appropriate fixation of the peptide conformation. Design of the CLIPS-peptides was entirely based on epitope mapping studies with two neutralizing anti-hFSH mAbs. Both mAbs were shown to bind to a conformational epitope located at the top of the beta1-beta3-loop covering the amino acid sequences Y58-P77 (beta3-loop). The results described in this paper show that CLIPS-constrained peptides covering the Y58-P77 sequence provide the minimally required structural entity necessary to generate reproducibly sera with high hFSH-neutralizing activity.     

Crystal structure and thermodynamic analysis of diagnostic mAb 106.3 complexed with BNP 5‐13 (C10A)

B‐type natriuretic peptide (BNP) is a naturally secreted regulatory hormone that influences blood pressure and vascular water retention in human physiology. The plasma BNP concentration is a clinically recognized biomarker for various cardiovascular diseases. Quantitative detection of BNP can be achieved in immunoassays using the high‐affinity monoclonal IgG1 antibody 106.3, which binds an epitope spanning residues 5‐13 of the mature bioactive peptide. To understand the structural basis of this molecular recognition, we crystallized the Fab fragment complexed with the peptide epitope and determined the three‐dimensional structure by X‐ray diffraction to 2.1 Å resolution. The structure reveals the detailed interactions that five of the complementarity‐determining regions make with the partially folded peptide. Thermodynamic measurements using fluorescence spectroscopy suggest that the interaction is enthalpy driven, with an overall change in free energy of binding, ΔG = ?54 kJ/mol, at room temperature. The parameters are interpreted on the basis of the structural information. The kinetics of binding suggest a diffusion‐limited mechanism, whereby the peptide easily adopts a bound conformation upon interaction with the antibody. Moreover, comparative analysis with alanine‐scanning results of the epitope explains the basis of selectivity for BNP over other related natriuretic peptides. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Epitope analysis using kinetic measurements of antibody binding to synthetic peptides presenting single amino acid substitutions

The fine modulation of peptide–antibody interactions was investigated with anti-peptide monoclonal antibodies recognizing peptide 125–136 of the coat protein of tobacco mosaic virus. Nine synthetic peptides presenting single amino acid substitutions were selected for detailed analysis on the basis of their reactivity in ELISA. Kinetic measurements of the binding of four antibodies to these peptides performed with a biosensor instrument (BIAcore^TM, Pharmacia) were used to quantify the contribution of individual residues to antibody binding. The results showed that even conservative exchanges of some residues in the epitope results in a small but significant decrease of the equilibrium affinity constant due mostly to a higher dissociation rate constant of the monoclonal antibodies. Two amino acid residues directly adjacent to the epitope, which appeared to play no role when tested by ELISA, were shown to influence the kinetics of binding. These data should be useful for computer modelling of the peptide–antibody interactions.     

Epitope recognition by diverse antibodies suggests conformational convergence in an antibody response

Crystal structures of distinct mAbs that recognize a common epitope of a peptide Ag have been determined and analyzed in the unbound and bound forms. These Abs display dissimilar binding site structures in the absence of the Ag. The dissimilarity is primarily expressed in the conformations of complementarity-determining region H3, which is responsible for defining the epitope specificity. Interestingly, however, the three Abs exhibit similar complementarity-determining region conformations in the Ag binding site while recognizing the common epitope, indicating that different pathways of binding are used for Ag recognition. The epitope also exhibits conformational similarity when bound to each of these Abs, although the peptide Ag was otherwise flexible. The observed conformational convergence in the epitope and the Ag binding site was facilitated by the plasticity in the nature of interactions.     

Insights into the substrate specificity of plant peptide deformylase, an essential enzyme with potential for the development of novel biotechnology applications in agriculture

CD151, a member of the tetraspanin family of proteins, forms a stable complex with integrin alpha 3 beta 1 and regulates integrin-mediated cell-substrate adhesion. However, the molecular basis of the stable association of CD151 with integrin alpha 3 beta 1 remains poorly understood. In the present study, we show that a panel of anti-human CD151 mAbs (monoclonal antibodies) could be divided into three groups on the basis of their abilities to co-immunoprecipitate integrin alpha 3: Group-1 mAbs were devoid of sufficient activities to co-precipitate integrin alpha 3 under both low- and high-stringency detergent conditions; Group-2 mAbs co-precipitated integrin alpha 3 under low-stringency conditions; and Group-3 mAbs exhibited strong co-precipitating activities under both conditions. Group-1 mAbs in particular exhibited increased reactivity toward integrin alpha 3 beta 1-unbound CD151, indicating that the binding sites for Group-1 mAbs are partly blocked by bound integrin alpha 3 beta 1. Epitope mapping using a series of CD151 mutants with substitutions at amino acid residues that are not conserved between human and mouse CD151 revealed that Gly(176)/Gly(177), Leu(191) and Gln(194) comprise epitopes characteristic of Group-1 mAbs. Replacement of short peptide segments, each containing one of these epitopes, with those of other tetraspanins lacking stable interactions with integrin alpha 3 beta 1 demonstrated that the segment from Cys(185) to Cys(192), including Leu(191), was involved in the stable association of CD151 with integrin alpha 3 beta 1, as was the Gln(194)-containing QRD peptide. Taken together these results indicate that two consecutive segments including two Group-1 epitopes, Leu(191) and Gln(194), comprise an interface between CD151 and integrin alpha 3 beta 1, and, along with the epitope including Gly(176)/Gly(177), are concealed by bound integrin.     

The role of antibody polyspecificity and lipid reactivity in binding of broadly neutralizing anti-HIV-1 envelope human monoclonal antibodies 2F5 and 4E10 to glycoprotein 41 membrane proximal envelope epitopes

Two neutralizing human mAbs, 2F5 and 4E10, that react with the HIV-1 envelope gp41 membrane proximal region are also polyspecific autoantibodies that bind to anionic phospholipids. To determine the autoantibody nature of these Abs, we have compared their reactivities with human anti-cardiolipin mAbs derived from a primary antiphospholipid syndrome patient. To define the role of lipid polyreactivity in binding of 2F5 and 4E10 mAbs to HIV-1 envelope membrane proximal epitopes, we determined the kinetics of binding of mAbs 2F5 and 4E10 to their nominal gp41 epitopes vs liposome-gp41 peptide conjugates. Both anti-HIV-1 mAbs 2F5 and 4E10 bound to cardiolipin with K(d) values similar to those of autoimmune anti-cardiolipin Abs, IS4 and IS6. Binding kinetics studies revealed that mAb 2F5 and 4E10 binding to their respective gp41 peptide-lipid conjugates could best be defined by a two-step (encounter-docking) conformational change model. In contrast, binding of 2F5 and 4E10 mAbs to linear peptide epitopes followed a simple Langmuir model. A mouse mAb, 13H11, that cross-blocks mAb 2F5 binding to the gp41 epitope did not cross-react with lipids nor did it neutralize HIV-1 viruses. Taken together, these data demonstrate the similarity of 2F5 and 4E10 mAbs to known anti-cardiolipin Abs and support the model that mAb 2F5 and 4E10 binding to HIV-1 involves both viral lipid membrane and gp41 membrane proximal epitopes.     

Probing antibody diversity by 2D NMR: comparison of amino acid sequences, predicted structures, and observed antibody-antigen interactions in complexes of two antipeptide antibodies

The interactions between the aromatic amino acids of two monoclonal antibodies (TE32 and TE33) with specific amino acid residues of a peptide of cholera toxin (CTP3) have been determined by two-dimensional (2D) transferred NOE difference spectroscopy. Aromatic amino acids are found to play an important role in peptide binding. In both antibodies two tryptophan and two tyrosine residues and one histidine residue interact with the peptide. In TE33 there is an additional phenylalanine residue that also interacts with the peptide. The residues of the CTP3 peptide that have been found to interact with the antibody are val 3, pro 4, gly 5, gln 7, his 8, and asp 10. We have determined the amino acid sequences of the two antibodies by direct mRNA sequencing. Computerized molecular modeling has been used to build detailed all-atom models of both antibodies from the known conformations of other antibodies. These models allow unambiguous assignment of most of the antibody residues that interact with the peptide. A comparison of the amino acid sequences of the two anti-CTP3 antibodies with other antibodies from the same gene family reveals that the majority of the aromatic residues involved in the binding of CTP3 are conserved although these antibodies have different specificities. This similarity suggests that these aromatic residues create a general hydrophobic pocket and that other residues in the complementarity-determining regions (CDRs) modulate the shape and the polarity of the combining site to fit the specific antigens.