NMR mapping and secondary structure determination of the major acetylcholine receptor alpha-subunit determinant interacting with alpha-bungarotoxin.

The alpha-subunit of the nicotinic acetylcholine receptor (alphaAChR) contains a binding site for alpha-bungarotoxin (alpha-BTX), a snake-venom-derived alpha-neurotoxin. Previous studies have established that the segment comprising residues 173-204 of alphaAChR contains the major determinant interacting with the toxin, but the precise boundaries of this determinant have not been clearly defined to date. In this study, we applied NMR dynamic filtering to determine the exact sequence constituting the major alphaAChR determinant interacting with alpha-BTX. Two overlapping synthetic peptides corresponding to segments 179-200 and 182-202 of the alphaAChR were complexed with alpha-BTX. HOHAHA and ROESY spectra of these complexes acquired with long mixing times highlight the residues of the peptide that do not interact with the toxin and retain considerable mobility upon binding to alpha-BTX. These results, together with changes in the chemical shifts of the peptide protons upon complex formation, suggest that residues 184-200 form the contact region. At pH 4, the molecular mass of the complex determined by dynamic light scattering (DLS) was found to be 11.2 kDa, in excellent agreement with the expected molecular mass of a 1:1 complex, while at pH >5 the DLS measurement of 20 kDa molecular mass indicated dimerization of the complex. These results were supported by T(2) measurements. Complete resonance assignment of the 11.2 kDa complex of alpha-BTX bound to the alphaAChR peptide comprising residues 182-202 was obtained at pH 4 using homonuclear 2D NMR spectra measured at 800 MHz. The secondary structures of both alpha-BTX and the bound alphaAChR peptide were determined using 2D (1)H NMR experiments. The peptide folds into a beta-hairpin conformation, in which residues (R)H186-(R)V188 and (R)Y198-(R)D200 form the two beta-strands. Residues (R)Y189-(R)T191 form an intermolecular beta-sheet with residues (B)K38-(B)V40 of the second finger of alpha-BTX. These results accurately pinpoint the alpha-BTX-binding site on the alphaAChR and pave the way to structure determination of this important alphaAChR determinant involved in binding acetylcholine and cholinergic agonists and antagonists.

Probing hydrogen bonds in the antibody-bound HIV-1 gp120 V3 loop by solid state NMR REDOR measurements.

We describe solid state NMR measurements on frozen solutions of the complex of the 24-residue HIV-1 gp120 V3 loop peptide RP135 with the Fab fragment of the anti-gp120 antibody 0.5beta, using rotational echo double resonance (REDOR). In order to probe possible hydrogen bonding between arginine side chains and glycine backbone carbonyls in the region of the conserved Gly-Pro-Gly-Arg (GPGR) motif of the V3 loop, RP135 samples were prepared with 15N labels at the eta nitrogen positions of arginine side chains and 13C labels at glycine carbonyl positions and 13C-detected 13C-15N REDOR measurements were performed on peptide/antibody complexes of these labeled samples. Such hydrogen bonding was previously observed in a crystal structure of the V3 loop peptide/antibody complex RP142/59.1 [Ghiara et al. (1994) Science, 264, 82-85], but is shown by the REDOR measurements to be absent in the RP135/0.5beta complex. These results confirm the antibody-dependent conformational differences in the GPGR motif suggested by previously reported solid state NMR measurements of phi and psi backbone dihedral angles in the RP135/0.53 complex. In addition, we describe REDOR measurements on the helical synthetic peptide MB(i+4)EK in frozen solution that establish our ability to detect 13C-15N dipole-dipole couplings in the distance range appropriate to these hydrogen bonding studies. We also report the results of molecular modeling calculations on the central portion RP135, using a combination of the solid state NMR restraints of Weliky et al. [Nat. Struct. Biol., 6, 141-145, 1999] and the liquid state NMR restraints of Tugarinov et al. (Nat. Struct. Biol., 6, 331-335, 1999]. The dynamics calculations demonstrate the mutual compatibility of the two sets of experimental structural restraints and reduce ambiguities in the solid state NMR restraints that result from symmetry and signal-to-noise considerations.

NMR structure of an anti-gp120 antibody complex with a V3 peptide reveals a surface important for co-receptor binding.

BACKGROUND: The protein 0.5beta is a potent strain-specific human immunodeficiency virus type 1 (HIV-1) neutralizing antibody raised against the entire envelope glycoprotein (gp120) of the HIV-1(IIIB) strain. The epitope recognized by 0.5beta is located within the third hypervariable region (V3) of gp120. Recently, several HIV-1 V3 residues involved in co-receptor utilization and selection were identified. RESULTS: Virtually complete sidechain assignment of the variable fragment (Fv) of 0.5beta in complex with the V3(IIIB) peptide P1053 (RKSIRIQRGPGRAFVTIG, in single-letter amino acid code) was accomplished and the combining site structure of 0.5beta Fv complexed with P1053 was solved using multidimensional nuclear magnetic resonance (NMR). Five of the six complementarity determining regions (CDRs) of the antibody adopt standard canonical conformations, whereas CDR3 of the heavy chain assumes an unexpected fold. The epitope recognized by 0.5beta encompasses 14 of the 18 P1053 residues. The bound peptide assumes a beta-hairpin conformation with a QRGPGR loop located at the very center of the binding pocket. The Fv and peptide surface areas buried upon binding are 601 A and 743 A(2), respectively, in the 0.5beta Fv-P1053 mean structure. The surface of P1053 interacting with the antibody is more extensive and the V3 peptide orientation in the binding site is significantly different compared with those derived from the crystal structures of a V3 peptide of the HIV-1 MN strain (V3(MN)) complexed to three different anti-peptide antibodies. CONCLUSIONS: The surface of P1053 that is in contact with the anti-protein antibody 0.5beta is likely to correspond to a solvent-exposed region in the native gp120 molecule. Some residues of this region of gp120 are involved in co-receptor binding, and in discrimination between different chemokine receptors utilized by the protein. Several highly variable residues in the V3 loop limit the specificity of the 0.5beta antibody, helping the virus to escape from the immune system. The highly conserved GPG sequence might have a role in maintaining the beta-hairpin conformation of the V3 loop despite insertions, deletions and mutations in the flanking regions.

A model of a gp120 V3 peptide in complex with an HIV-neutralizing antibody based on NMR and mutant cycle-derived constraints.

The 0.5beta monoclonal antibody is a very potent strain-specific HIV-neutralizing antibody raised against gp120, the envelope glycoprotein of HIV-1. This antibody recognizes the V3 loop of gp120, which is a major neutralizing determinant of the virus. The antibody-peptide interactions, involving aromatic and negatively charged residues of the antibody 0.5beta, were studied by NMR and double-mutant cycles. A deuterated V3 peptide and a Fab containing deuterated aromatic amino acids were used to assign these interactions to specific V3 residues and to the amino acid type and specific chain of the antibody by NOE difference spectroscopy. Electrostatic interactions between negatively charged residues of the antibody Fv and peptide residues were studied by mutagenesis of both antibody and peptide residues and double-mutant cycles. Several interactions could be assigned unambiguously: F96(L) of the antibody interacts with Pro13 of the peptide, H52(H) interacts with Ile7, Ile9 and Gln10 and D56(H) interacts with Arg11. The interactions of the light-chain tyrosines with Pro13 and Gly14 could be assigned to either Y30a(L) and Y32(L), respectively, or Y32(L) and Y49(L), respectively. Three heavy-chain tyrosines interact with Ile7, Ile20 and Phe17. Several combinations of assignments involving Y32(H), Y53(H), Y96(H) and Y100a(H) may satisfy the NMR and mutagenesis constraints, and therefore at this stage the interactions of the heavy-chain tyrosines were not taken into account. The unambiguous assignments [F96(L), H52(H) and D56(H)] and the two possible assignments of the light-chain tyrosines were used to dock the peptide into the antibody-combining site. The peptide converges to a unique position within the binding site, with the RGPG loop pointing into the center of the groove formed by the antibody complementary determining regions while retaining the beta-hairpin conformation and the type-VI RGPG turn [Tugarinov, V., Zvi, A., Levy, R. & Anglister, J. (1999) Nat. Struct. Biol. 6, 331-335].

Solution Structure of an Antibody-Bound HIV-1(IIIB) V3 Peptide: A Cis Proline Turn Linking Two ß-hairpin Strands.

Abstract The refined solution structure of a peptide representing the full epitope of the HIV-1(IIIB) V3 loop in complex with the anti-gp120 antibody Fv fragment was determined using isotope-filtered and isotope-edited NMR. Both the (15)N-labeled peptide in complex with the unlabeled Fv and the unlabeled peptide complexed with the uniformly (15)N,(13)C-labeled Fv were investigated. The backbone of the bound peptide adopts a well defined ß-hairpin conformation with two twisted anti-parallel ß-strands linked by a type VI tight turn comprising residues RGPG. The central glycine and proline residues of the turn are linked by a cis peptide bond. (15)N{(1)H} NOE measurements demonstrated that the backbone of the bound peptide including the central QRGPGR loop is well ordered in the bound state. The V3 loop peptide solution structure is significantly different from the peptide conformation in the X-ray structures of three anti-peptide antibody/V3(MN) peptide complexes. These differences seem to be dictated by the antibody dependence and HIV strain-specificity of the V3 peptide fold.

2D-NMR and ATR-FTIR study of the structure of a cell-selective diastereomer of melittin and its orientation in phospholipids.

Melittin, a 26 residue, non-cell-selective cytolytic peptide, is the major component of the venom of the honey bee Apis mellifera. In a previous study, a diastereomer ([D]-V(5,8),I(17),K(21)-melittin, D-amino acids at positions V(5,8),I(17),K(21)) of melittin was synthesized and its function was investigated [Oren, Z., and Shai, Y. (1997) Biochemistry 36, 1826-1835]. [D]-V(5,8),I(17),K(21)-melittin lost its cytotoxic effects on mammalian cells; however, it retained antibacterial activity. Furthermore, [D]-V(5,8),I(17),K(21)-melittin binds strongly and destabilizes only negatively charged phospholipid vesicles, in contrast to native melittin, which binds strongly also zwitterionic phospholipids. To understand the differences in the properties of melittin and its diastereomer, 2D-NMR experiments were carried out with [D]-V(5,8),I(17),K(21)-melittin, and polarized attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy experiments were done with both melittin and [D]-V(5,8), I(17),K(21)-melittin. The structure of the diastereomer was characterized by NMR in water, as well as in three different membrane-mimicking environment, 40% 2,2,2-trifluoroethanol (TFE)/water, methanol, and dodecylphosphocholine/phosphatidylglycerol (DPC/DMPG) micelles. The NMR data revealed an amphipathic alpha-helix only in the C-terminal region of the diastereomer in TFE/water and methanol solutions and in DPC/DMPG micelles. ATR-FTIR experiments revealed that melittin and [D]-V(5,8),I(17),K(21)-melittin are oriented parallel to the membrane surface. This study indicates the role of secondary structure formation in selective cytolytic activity of [D]-V(5,8), I(17),K(21)-melittin. While the N-terminal helical structure is not required for the cytolytic activity toward negatively charged membranes and bacterial cells, it appears to be a crucial structural element for binding and insertion into zwitterionic membranes and for hemolytic activity.

A cis proline turn linking two beta-hairpin strands in the solution structure of an antibody-bound HIV-1IIIB V3 peptide.

The refined solution structure of an 18-residue HIV-1IIIB V3 peptide in complex with the Fv fragment of an anti-gp120 antibody reveals an unexpected type VI beta-turn comprising residues RGPG at the center of a beta-hairpin. The central glycine and proline of this turn are linked by a cis peptide bond. The residues of the turn interact extensively with the antibody Fv. 15N[1H] NOE measurements show that the backbone of the peptide, including the central QRGPGR loop, is well ordered in the complex. The solution structure is significantly different from the X-ray structures of HIV-1MN V3 peptides bound to anti-peptide antibodies. These differences could be due to a two-residue (QR) insertion preceding the GPGR sequence in the HIV-1IIIB strain, and the much longer peptide epitope immobilized by the anti-gp120 antibody.

Solid-state NMR evidence for an antibody-dependent conformation of the V3 loop of HIV-1 gp120.

Solid-state NMR measurements have been carried out on frozen solutions of the complex of a 24-residue peptide derived from the third variable (V3) loop of the HIV-1 envelope glycoprotein gp120 bound to the Fab fragment of an anti-gp120 antibody. The measurements place strong constraints on the conformation of the conserved central GPGR motif of the V3 loop in the antibody-bound state. In combination with earlier crystal structures of V3 peptide-antibody complexes and existing data on the cross-reactivity of the antibodies, the solid-state NMR measurements suggest that the Gly-Pro-Gly-Arg (GPGR) motif adopts an antibody-dependent conformation in the bound state and may be conformationally heterogeneous in unbound, full-length gp120. These measurements are the first application of solid-state NMR methods in a structural study of a peptide-protein complex.

Conformation of the principal neutralizing determinant of human immunodeficiency virus type 1 in complex with an anti-gp120 virus neutralizing antibody studied by two-dimensional nuclear magnetic resonance difference spectroscopy.

The principal neutralizing determinant (PND) of human immunodeficiency virus type 1 (HIV-1) is located in the third hypervariable region (V3) of the virus envelope glycoprotein gp120. The conformation of a V3 peptide of HIV-1IIIB bound to the Fab fragment of an anti-gp120 HIV neutralizing antibody, 0.5beta, was studied by 1H NMR spectroscopy. This 18-residue peptide represents the epitope recognized by 0.5beta and encompasses most of the PND. The slow off-rate of the peptide prevents the observation of peptide/Fab interactions as well as intramolecular interactions within the bound peptide by transferred nuclear Overhauser enhancement (TRNOE). To detect and assign interactions within the bound peptide in the 52 kDa complex, NOESY difference spectra were measured using three strategies: (a) deuteration of peptide residues, (b) Arg two head right arrow Lys replacements, and (c) truncation of the peptide antigen. Each difference spectrum was calculated between NOESY spectra measured for two Fab complexes in which the bound peptides differed in their deuteration or in their sequence. The difference spectra revealed numerous interactions between the N-terminus of the epitope (Arg-4, Lys-5, Ser-6, Ile-7, and Ile-9) and its C-terminus (Phe-17, Val-18, Thr-19, and Ile-20). The assigned NOE interactions within the bound peptide were translated into distance restraints that were used to calculate the conformation of the bound peptide by the hybrid distance geometry/simulated annealing method. A total of 39 long-range (residues i - j >> 4), 14 short-range, and 69 intraresidue NOE interactions within the bound peptide have been assigned. Twelve structures without NOE constraint violations were obtained, having a 1.6 A rms deviation for the backbone atoms. The peptide forms a 10-residue loop, while the two segments flanking this loop, KSI and VTI, interact extensively with each other and possibly form antiparallel beta-strands. This loop conformation could be observed due to the unusual large size (17 residues) of the antigenic determinant recognized by 0.5beta.

Three-dimensional solution structure of the complex of alpha-bungarotoxin with a library-derived peptide.

The solution structure of the complex between alpha-bungarotoxin (alpha-BTX) and a 13-residue library-derived peptide (MRYYESSLKSYPD) has been solved using two-dimensional proton-NMR spectroscopy. The bound peptide adopts an almost-globular conformation resulting from three turns that surround a hydrophobic core formed by Tyr-11 of the peptide. The peptide fills an alpha-BTX pocket made of residues located at fingers I and II, as well as at the C-terminal region. Of the peptide residues, the largest contact area is formed by Tyr-3 and Tyr-4. These findings are in accord with the previous data in which it had been shown that substitution of these aromatic residues by aliphatic amino acids leads to loss of binding of the modified peptide with alpha-BTX. Glu-5 and Leu-8, which also remarkably contribute to the contact area with the toxin, are present in all the library-derived peptides that bind strongly to alpha-BTX. The structure of the complex may explain the fact that the library-derived peptide binds alpha-BTX with a 15-fold higher affinity than that shown by the acetylcholine receptor peptide (alpha185-196). Although both peptides bind to similar sites on alpha-BTX, the latter adopts an extended conformation when bound to the toxin [Basus, V., Song, G. & Hawrot, E. (1993) Biochemistry 32, 12290-12298], whereas the library peptide is nearly globular and occupies a larger surface area of alpha-BTX binding site.

Solution structures of a highly insecticidal recombinant scorpion alpha-toxin and a mutant with increased activity.

The solution structure of a recombinant active alpha-neurotoxin from Leiurus quinquestriatus hebraeus, Lqh(alpha)IT, was determined by proton two-dimensional nuclear magnetic resonance spectroscopy (2D NMR). This toxin is the most insecticidal among scorpion alpha-neurotoxins and, therefore, serves as a model for clarifying the structural basis for their biological activity and selective toxicity. A set of 29 structures was generated without constraint violations exceeding 0.4 A. These structures had root mean square deviations of 0.49 and 1.00 A with respect to the average structure for backbone atoms and all heavy atoms, respectively. Similarly to other scorpion toxins, the structure of Lqh(alpha)IT consists of an alpha-helix, a three-strand antiparallel beta-sheet, three type I tight turns, a five-residue turn, and a hydrophobic patch that includes tyrosine and tryptophan rings in a "herringbone" arrangement. Positive phi angles were found for Ala50 and Asn11, suggesting their proximity to functionally important regions of the molecule. The sample exhibited conformational heterogeneity over a wide range of experimental conditions, and two conformations were observed for the majority of protein residues. The ratio between these conformations was temperature-dependent, and the rate of their interconversions was estimated to be on the order of 1-5 s(-1) at 308 K. The conformation of the polypeptide backbone of Lqh(alpha)IT is very similar to that of the most active antimammalian scorpion alpha-toxin, AaHII, from Androctonus australis Hector (60% amino acid sequence homology). Yet, several important differences were observed at the 5-residue turn comprising residues Lys8-Cys12, the C-terminal segment, and the mutual disposition of these two regions. 2D NMR studies of the R64H mutant, which is 3 times more toxic than the unmodified Lqh(alpha)IT, demonstrated the importance of the spatial orientation of the last residue side chain for toxicity of Lqh(alpha)IT.

Contribution of arginine residues in the RP135 peptide derived from the V3 loop of gp120 to its interaction with the Fv fragment of the 0.5beta HIV-1 neutralizing antibody.

The construction, expression, and purification of an active Fv fragment of the 0.5beta monoclonal human immunodeficiency virus type 1 (HIV-1) neutralizing antibody is reported. The interaction between the Fv fragment and the RP135 peptide derived from the V3 loop of gp120 from HIV-1IIIB was studied by varying the salt concentration and by mutating arginine residues in the peptide. The mutations R4A, R8A and R11A (which correspond to residues 311, 315, and 318 in gp120 of HIV-1IIIB) reduce the binding free energy by 0.22 (+/- 0. 20), 4.32 (+/- 0.16), and 1.58 (+/- 0.17) kcal mol-1, respectively. The salt-dependent components of their contributions to binding are 0.02 (+/- 0.22), -0.55 (+/- 0.18), and -0.97 (+/- 0.19) kcal mol-1, respectively. The magnitudes of the mutational effects and the extent of shielding by 1 M NaCl suggest that Arg-8 is involved in a buried salt bridge in the peptide-Fv fragment complex, whereas Arg-11 is involved in a more solvent-exposed electrostatic interaction.

NMR identification of calcineurin B residues affected by binding of a calcineurin A peptide.

Triple resonance 3D NMR methods have been used to study the interaction between calcineurin B and a peptide fragment of calcineurin A for which it has high affinity (KD approximately 4 x 10(-7) M). Although calcineurin B aggregates at NMR concentrations of approximately 1 mM, in the presence of a target peptide fragment of calcineurin A it becomes monomeric and yields NMR spectra that are very similar to those reported previously for calcineurin B solubilized by the zwitterionic detergent CHAPS. Changes in chemical shifts between CHAPS- and peptide-solubilized calcineurin B are small which is indicative of no differences in secondary structure. Residues most affected by binding to target peptide are found primarily on the hydrophobic faces of the four helices, present in each of the two globular domains in calcineurin B, and in the loops connecting helices II and III, IV and V, and possibly in the C-terminal 12 residues, which also exhibit a change in mobility.

The principal neutralizing determinant of HIV-1 located in V3 of gp120 forms a 12-residue loop by internal hydrophobic interactions.

The interactions of the peptide RP135a (RKSI-RIQRGPGRAFVT), corresponding to residues 311-326 of gp120 of HIV-1IIIB, with the anti-gp120 HIV-1IIIB neutralizing antibody 0.5 beta were studied by NMR. The NOESY difference spectra measured using specifically deuterated derivatives of the peptide show exclusively the interactions of the deuterated residues both within the bound peptide and with the Fab fragment of the antibody. These measurements reveal hydrophobic interactions within the bound peptide between Ile-4, Ile-6 and Val-15 that create a 12-residue loop with these residues at the base and the conserved GPGR sequence at its top.

Structural diversity in a conserved cholera toxin epitope involved in ganglioside binding.

Cholera is a widespread disease for which there is no efficient vaccine. A better understanding of the conformational rearrangements at the epitope might be very helpful for the development of a good vaccine. Cholera toxin (CT) as well as the closely related heat-labile toxin from Escherichia coli (LT) are composed of two subunits, A and B, which form an oligomeric assembly AB5. Residues 50-64 on the surface of the B subunits comprise a conserved loop (CTP3), which is involved in saccharide binding to the receptor on epithelial cells. This loop exhibits remarkable conformational plasticity induced by environmental constraints. The crystal structure of this loop is compared in the free and receptor-bound toxins as well as in the crystal and solution structures of a complex with TE33, a monoclonal antibody elicited against CTP3. In the toxins this loop forms an irregular structure connecting a beta-strand to the central alpha-helix. Ser 55 and Gln 56 exhibit considerable conformational variability in the five subunits of the unliganded toxins. Saccharide binding induces a change primarily in Ser 55 and Gln 56 to a conformation identical in all five copies. Thus, saccharide binding confers rigidity upon the loop. The conformation of CTP3 in complex with TE33 is quite different. The amino-terminal part of CTP3 forms a beta-turn that fits snugly into a deep binding pocket on TE33, in both the crystal and NMR-derived solution structure. Only 8 and 12 residues out of 15 are seen in the NMR and crystal structures, respectively. Despite these conformational differences, TE33 is cross-reactive with intact CT, albeit with a thousandfold decrease in affinity. This suggests a different interaction of TE33 with intact CT.

NMR mapping of the antigenic determinant recognized by an anti-gp120, human immunodeficiency virus neutralizing antibody.

The 24-amino-acid peptide RP135 (NNTRKSIRIQRGPGRAFVTIGKIG) corresponds in its amino acid sequence to the principal neutralizing determinant of the human immunodeficiency virus type-1, IIIB isolate (HIV-1IIIB, residues 308-331 of the envelope glycoprotein gp120). In order to map the antigenic determinant recognized by 0.5 beta, the complex of RP135 with an anti-gp120 HIV neutralizing antibody, 0.5 beta, which cross reacts with the peptide, was studied by using two-dimensional NMR spectroscopy. A combination of homonuclear Hartmann Hahn two-dimensional experiment and roating-frame Overhauser enhancement spectroscopy of the Fab/peptide complex measured in H2O was used to eliminate the resonances of the Fab and the tightly bound peptide residues and to obtain sequential assignments for those parts of the peptide which retain considerable mobility upon binding. In this manner, a total of 14 residues (Ser6-Thr19) were shown to be part of the antigenic determinant recognized by the antibody 0.5 beta. Lys5 and Ile20 were found to retain considerable mobility in the bound peptide while their amide protons undergo significant change in chemical shift upon binding. This observation suggests that these two residues are at the boundaries of the determinant recognized by the antibody. Competitive binding experiments using truncated peptides strongly support the NMR observations.

NMR observation of interactions in the combining site region of an antibody using a spin-labeled peptide antigen and NOESY difference spectroscopy.

A spin-labeled peptide antigen (TEMPOVEVPGSQHIDSQ) was used to measure NOESY difference spectra that show interactions in the binding site region of the Fab fragment of the anti-cholera toxin peptide antibody TE33. In addition to identification of peptide-Fab interactions and interactions within the bound peptide, these difference spectra show well-resolved cross peaks due to interactions within the large Fab fragment (50 kDa). These difference spectra indicate that the conformational changes in the Fab upon peptide binding are confined to the combining site region of the antibody. The NOESY difference spectra of selectively deuterated Fab molecules were used in combination with HOHAHA measurements to assign the interactions to amino acid type and to identify the interactions within the Fab as either inter- or intraresidue interactions. The assignment of interactions within the Fab to corresponding aromatic residues in the Fab sequence was facilitated by an earlier NMR-derived model calculated on the basis of NOE restraints on Fab-peptide and intra-bound-peptide distances. The new restraints on distances within the Fab, combined with the previously obtained restraints, were used to generate a refined NMR-derived model for the TE33-peptide complex.

Two-dimensional NMR studies of the interactions between a peptide of cholera toxin and monoclonal antibodies.

To increase our understanding of the molecular basis for antibody specificity and for the cross-reactivity of antipeptide antibodies with native proteins, it is important to study the three-dimensional structure of antibody complexes with their peptide antigens. For this purpose it may not be necessary to solve the structure of the whole antibody complex but rather to concentrate on elucidating the combining site structure, the interactions of the antibody with its antigen, and the bound peptide conformation. To extract the information about antibody-peptide interactions and intramolecular interactions in the bound ligand from the complicated and unresolved spectrum of the Fab-peptide complex (Fab: antibody fragment made of Fv--the antibody fragment composed of the variable regions of the light and heavy chains forming a single combining site for the antigen--the light chain, and the first heavy chain constant regions), an nmr methodology based on measurements of two-dimensional transferred nuclear Overhauser effect (NOE) difference spectra was developed. Using this methodology the interactions of three monoclonal antibodies with a cholera toxin peptide were studied. The observed interactions were assigned to the antibody protons involved by specific deuteration of aromatic amino acids and specific chain labeling, and by using a predicted model for the structure of the antibody combining site. The assigned NOE interactions were translated to restraints on interproton distances in the complex that were used to dock the peptide into calculated models for the antibodies' combining sites.(ABSTRACT TRUNCATED AT 250 WORDS)