Publications

The Nterminal segment of the chemokine receptor CCR5, NtCCR5, contains four tyrosine residues, Y3, Y10, Y14 and Y15. Sulfation of at least two of these tyrosine residues was found to be essential for high affinity binding of CCR5 to its chemokine ligands. Here we show that among the monosulfated NtCCR5(820) peptide surrogates (sNtCCR5) those sulfated at Y15 and Y14 have the highest affinity for the CCL5 chemokine in comparison with monosulfation at position Y10. Sulfation at Y3 was not investigated. A peptide sulfated at both Y14 and Y15 has the highest affinity for CCL5 by up to a factor of 3, in comparison with the other disulfated (sNtCCR5) peptides. Chemical shift perturbation analysis and TRNOE measurements indicate that the sulfated tyrosine residues interact with the same CCL5 binding pocket and that each of the sulfated tyrosines at positions 10, 14 and 15 can occupy individually the binding site on CCL5 in a similar manner, although with somewhat different affinity, suggesting the possibility of allovalency in sulfated NtCCR5 peptides. The affinity of the disulfated peptides to CCL5 could be increased by this allovalency and by stronger electrostatic interactions.

Infection by HIV-1 requires protein-protein interactions involving gp120, CD4 and CCR5. We have previously demonstrated that the transferred nuclear Overhauser effect (TRNOE), in combination with asymmetric deuteration of a protein and a peptide ligand can be used to detect intermolecular interactions in large protein complexes with molecular weights up to similar to 100 kDa. Here, using this approach, we reveal interactions between tyrosine residues of a 27-residue peptide corresponding to the N-terminal segment of the CCR5 chemokine receptor, and a dimeric extended core (YU2) gp120 envelope protein of HIV-1 complexed with a CD4-mimic miniprotein. The TRNOE crosspeaks in the ternary complex were assigned to the specific Tyr protons in the CCR5 peptide and to methyl protons of isoleucine, leucine and/or valine residues of gp120. Site directed mutagenesis combined with selective deuteration and TRNOE resulted in the first discernment by a biophysical method of specific pairwise interactions between gp120 residues in the bridging sheet of gp120 and the N-terminus of CCR5.

NMR is a powerful tool for studying structural details of protein/peptide complexes exhibiting weak to medium binding (K_D > 10 μm). However, it has been assumed that intermolecular nuclear Overhauser effect (NOE) interactions are difficult to observe in such complexes. We demonstrate that intermolecular NOEs can be revealed by combining the ¹³C-edited/¹³C-filtered experiment with the transferred NOE effect (TRNOE). Due to the TRNOE phenomenon, intermolecular NOE cross peaks are characterized by both the chemical shifts (CSs) of the protein protons and the average CSs of the peptide protons, which are dominated by the CSs of the protons of the free peptide. Previously, the TRNOE phenomenon was used almost exclusively to investigate the conformation of small ligands bound to large biomolecules. Here, we demonstrate that TRNOE can be extended to enable the study of intermolecular interactions in small- and medium-sized protein complexes. We used the ¹³C-edited/¹³C-filtered TRNOE experiment to study the interactions of the chemokine regulated upon activation, normal T cell, expressed and secreted (RANTES) with a 27-residue peptide, containing two sulfotyrosine residues, representing the N-terminal segment of the CCR5 receptor ((Nt-CCR5(127). The TRNOE phenomenon led to more than doubling of the signal-to-noise ratios (SNRs) for the intermolecular NOEs observed in the ¹³C-edited/¹³C-filtered experiment for the 11.5-kDa monomeric RANTES/Nt-CCR5(127) complex. An even better improvement in the SNR was achieved with dimeric Nt-CCR5(127)/RANTES (23 kDa), especially in comparison with the spectra measured with a 1 : 1 protein to peptide ratio. In principle, the isotope-edited/isotope-filtered TRNOE spectrum can discern all intermolecular interactions involving nonexchangeable protons in the complex.

Intermolecular NOE interactions are invaluable for structure determination of biomolecular complexes by NMR and they represent the \u201cgold-standard\u201d amongst NMR measurements for characterizing interfaces. These NOEs constitute only a small fraction of the observed NOEs in a complex and are usually weaker than many of the intramolecular NOEs. A number of methods have been developed to remove the intramolecular NOEs that interfere with the identification of intermolecular NOEs. NMR experiments used to observe intermolecular NOE interactions in large protein complexes must cope with the short T2 relaxation time of the protons and heteronuclei in these complexes because they result in severe losses in sensitivity. The isotope-edited/isotope-filtered experiment is a powerful method for extraction of intermolecular NOEs in biomolecular complexes. Its application to large protein complexes is limited because of severe losses in signal-to-noise ratio caused by delays in the pulse sequence necessary for the multiple magnetization transfer steps between protons and heteronuclei. Isotope-edited/isotope-edited experiments, in which one protein is usually labeled with ¹³C and the other is labeled with ¹⁵N, reduce possible artifacts in the filtering experiments and improve somewhat the sensitivity of these experiments. Sensitivity can also be improved by deuteration of the components of the complex in order to replace either or both of the filtering or editing steps. Asymmetric deuteration, where aromatic residues in one protein and non-aromatic amino acids in the other are reverse protonated, can eliminate the editing and the filtering steps altogether, thus maintaining high sensitivity even for large proteins complexes. Difference spectroscopy and the use of 2D NOESY experiments without using editing or filtering steps can significantly increase the signal-to-noise ratio in experiments aimed at observing intermolecular NOEs. The measurement of NOESY spectra of three different preparations of a heterodimeric complex under investigation in which one or neither of the components is uniformly deuterated, and calculation of a double difference spectrum provides information on all intermolecular NOEs of non-exchangeable protons. Recent studies indicate that many protein-protein interactions are actually between a protein and a linear peptide recognition motif of the second protein, and determinants represented by linear peptides contribute significantly to the binding energy. NMR is a very versatile method to study peptide-protein interactions over a wide range of binding affinities and binding kinetics. Protein-peptide interactions in complexes exhibiting tight binding can be studied using single and/or multiple deuteration of the peptide residues and measuring a difference NOESY spectrum. This difference spectrum will show exclusively intra- and intermolecular interactions of the peptide protons that were deuterated. Transferred nuclear Overhauser spectroscopy (TRNOE) extends NMR to determine interactions within and between a weakly-bound rapidly-exchanging peptide and its protein target. TRNOE, together with asymmetric deuteration, is applicable to complexes up to ∼100 KDa and is highly sensitive, taking advantage of the long average T2 of the peptide protons. Among the methods described in this review, TRNOE has the best potential to determine intermolecular NOEs for the upper molecular weight limit of proteins that can be studied in detail by NMR.

The peptide T20, which corresponds to a sequence in the C-terminal segment of the HIV-1 transmembrane glycoprotein gp41, is a strong entry inhibitor of HIV-1. It has been assumed that T20 inhibits HIV-1 infection by binding to the trimer formed by the N-terminal helical region (HR1) of gp41, preventing the formation of a six helix bundle by the N- and C-terminal helical regions of gp41. In addition to binding to gp41, T20 was found to bind to gp120 of X4 viruses and this binding was suggested to be responsible for an alternative mechanism of HIV-1 inhibition by this peptide. In the present study, T20 also was found to bind R5 gp120. Using NMR spectroscopy, the segments of T20 that interact with both gp120 and a gp120/CD4M33 complex were mapped. A peptide corresponding to the fourth constant region of gp120, sC4, was found to partially recapitulate gp120 binding to T20 and the segment of this peptide interacting with T20 was mapped. The present study concludes that an amphiphilic helix on the T20 C-terminus binds through mostly hydrophobic interactions to a nonpolar gp120 surface formed primarily by the C4 region. The ten- to thousand-fold difference between the EC₅₀ of T20 against viral fusion and the affinity of T20 to gp120 implies that binding to gp120 is not a major factor in T20 inhibition of HIV-1 fusion. Nevertheless, this hydrophobic gp120 surface could be a target for anti-HIV therapeutics. Enfuvirtide (T20) is a peptide of the HIV-1 transmembrane glycoprotein gp41 that inhibits HIV-1 entry by interfering with the formation of the six-helix bundle by gp41. In addition, T20 was shown to bind gp120 of X4 viruses. Anglister and colleagues now report that T20 also binds to the gp120 protein of R5 viruses that cause the majority of HIV-1 infections. This binding was mapped to specific residues in T20 and the C4 region of gp120 using solution NMR techniques. The identified gp120 binding surface could be a target for anti-HIV therapeutics. This article is accompanied by a podcast, listen now. Or listen in iTunes.

Chemokines constitute a large family of small proteins that regulate leukocyte trafficking to the site of inflammation by binding to specific cell-surface receptors belonging to the G-protein-coupled receptor (GPCR) superfamily. The interactions between N-terminal (Nt-) peptides of these GPCRs and chemokines have been studied extensively using NMR spectroscopy. However, because of the lower affinities of peptides representing the three extracellular loops (ECLs) of chemokine receptors to their respective chemokine ligands, information concerning these interactions is scarce. To overcome the low affinity of ECL peptides to chemokines, we linked two or three CC chemokine receptor 5 (CCR5) extracellular domains using either biosynthesis in Escherichia coli or chemical synthesis. Using such chimeras, CCR5 binding to RANTES was followed using ¹H-¹⁵N-HSQC spectra to monitor titration of the chemokine with peptides corresponding to the extracellular surface of the receptor. Nt-CCR5 and ECL2 were found to be the major contributors to CCR5 binding to RANTES, creating an almost closed ring around this protein by interacting with opposing faces of the chemokine. A RANTES positively charged surface involved in Nt-CCR5 binding resembles the positively charged surface in HIV-1 gp120 formed by the C4 and the base of the third variable loop of gp120 (V3). The opposing surface on RANTES, composed primarily of β2-β3 hairpin residues, binds ECL2 and was found to be analogous to a surface in the crown of the gp120 V3. The chemical and biosynthetic approaches for linking GPCR surface regions discussed herein should be widely applicable to the investigation of interactions of extracellular segments of chemokine receptors with their respective ligands.

NMR detection of intermolecular interactions between protons in large protein complexes is very challenging because it is difficult to distinguish between weak NOEs from intermolecular interactions and the much larger number of strong intramolecular NOEs. This challenging task is exacerbated by the decrease in signal-to-noise ratio in the often used isotope-edited and isotope-filtered experiments as a result of enhanced T(2) relaxation. Here, we calculate a double difference spectrum that shows exclusively intermolecular NOEs and manifests the good signal-to-noise ratio in 2D homonuclear NOESY spectra even for large proteins. The method is straightforward and results in a complete picture of all intermolecular interactions involving non exchangeable protons. Ninety-seven such (1)H-(1)H NOEs were assigned for the 44 KDa interferon-alpha 2/IFNAR2 complex and used for docking these two proteins. The symmetry of the difference spectrum, its superb resolution, and unprecedented signal-to-noise ratio in this large protein/receptor complex suggest that this method is generally applicable to study large biopolymeric complexes.

Type I interferons (IFNs) make up a family of homologous helical cytokines initiating strong antiviral and antiproliferative activity. All type I IFNs bind to a common cell surface receptor consisting of two subunits, IFNAR1 and IFNAR2, associating upon binding of interferon. We studied intermolecular interactions between IFNAR2-EC and IFNα2 using asymmetric reverse-protonation of the different complex components and two-dimensional homonuclear NOESY. This new approach revealed with an excellent signal-to-noise ratio 24 new intermolecular NOEs between the two molecules despite the low concentration of the complex (0.25 mM) and its high molecular mass (44 kDa). Sequential and side chain assignment of IFNAR2-EC and IFNα2 in their binary complex helped assign the intermolecular NOEs to the corresponding protons. A docking model of the IFNAR2-EC-IFNα2 complex was calculated on the basis of the intermolecular interactions found in this study as well as four double mutant cycle constraints, previously observed NOEs between a single pair of residues and the NMR mapping of the binding sites on IFNAR2-EC and IFNα2. Our docking model doubles the buried surface area of the previous model and significantly increases the number of intermolecular hydrogen bonds, salt bridges, and van der Waals interactions. Furthermore, our model reveals the participation of several new regions in the binding site such as the N-terminus and A helix of IFNα2 and the C domain of IFNAR2-EC. As a result of these additions, the orientation of IFNAR2-EC relative to IFNα2 has changed by 30° in comparison with a previously calculated model that was based on NMR mapping of the binding sites and double mutant cycle constraints. In addition, the new model strongly supports the recently proposed allosteric changes in IFNα2 upon binding of IFNAR1-EC to the binary IFNα2-IFNAR2-EC complex.

Fusion of HIV-1 and target cells is mediated by the envelope protein gp41 that undergoes a series of conformational changes during the process of infection. Knowledge of the structural biology of gp41 allows the design of potent peptide inhibitors that prevent the virus from entering lymphocytes and macrophages. The design of such inhibitors is the subject of this review.

The HIV-1 envelope glycoprotein gp41 undergoes a sequence of extensive conformational changes while participating in the fusion of the virus with the host cell. Since the discovery of its postfusion conformation, the structure and function of the protease-resistant six-helix bundle (6-HB) have been the subject of extensive investigation. In this work, we describe additional determinants (S528-Q540 and W666-N677) in the fusion peptide proximal region (FP-PR) and the membrane proximal external region (MPER) that stabilize the six-helix bundle and are involved in the interaction of T-20 (FUZEON, an anti-HIV-1 fusion inhibitor drug) with the gp41 FP-PR. Circular dichroism and sedimentation equilibrium measurements indicate that the 1:1 mixture of N and C peptides comprising residues A541-T569 and I635-K665 from the gp41 first and second helical repeats, HR1 and HR2, respectively, fail to form a stable six-helix bundle. Triglutamic acid and triarginine tags were added to these N and C peptides, respectively, at the termini distant from the FP-PR and the MPER to alter their pi and increase their solubility at pH 3.5. The tagged HR1 and HR2 peptides were elongated by addition of residues S528-Q540 from the FP-PR and residues W666-N677 from the MPER, respectively. A 1:1 complex of the elongated peptides formed a stable six-helix bundle which melted at 60°C. These results underscore the importance of a detailed high-resolution characterization of MPER interactions, the results of which may improve our understanding of the structure-function relationship of gp41 and its role in HIV-1 fusion.

HIV-1 coreceptor usage plays a critical role in virus tropism and pathogenesis. A switch from CCR5- to CXCR4-using viruses occurs during the course of HIV-1 infection and correlates with subsequent disease progression. A single mutation at position 322 within the V3 loop of the HIV-1 envelope glycoprotein gp120, from a negatively to a positively charged residue, was found to be sufficient to switch an R5 virus to an X4 virus. In this study, the NMR structure of the V3 region of an RS strain, HIV-1^JR-FL, in complex with an HIV-1-neutralizing antibody was determined. Positively charged and negatively charged residues at positions 304 and 322, respectively, oppose each other in the β-hairpin structure, enabling a favorable electrostatic interaction that stabilizes the postulated R5 conformation. Comparison of the R5 conformation with the postulated X4 conformation of the V3 region (positively charged residue at position 322) reveals that electrostatic repulsion between residues 304 and 322 in X4 strains triggers the observed one register shift in the N-terminal strand of the V3 region. We posit that electrostatic interactions at the base of the V3 β-hairpin can modulate the conformation and thereby influence the phenotype switch. In addition, we suggest that interstrand cat ion-π interactions between positively charged and aromatic residues induce the switch to the X4 conformation as a result of the S306R mutation. The existence of three pairs of identical (or very similar) amino acids in the V3 C-terminal strand facilitates the switch between the R5 and X4 conformations.

The HIV-1 envelope glycoprotein gp41 is responsible for viral fusion with the host cell. The fusion process, as well as the full structure of gp41, is not completely understood. One of the strongest inhibitors of HIV-1 fusion is a 36-residue peptide named T-20, gp41_(638-673) (Fuzeon, also called Enfuvirtide or DP-178; residues are numbered according to the HXB2 gp160 variant) now used as an and HIV-1 drug. This peptide also contains the immunogenic sequences that represent the full or partial recognition epitope for the broadly neutralizing human monoclonal antibodies 2F5 and 4E10, respectively. Due to its hydrophobicity, T-20 tends to aggregate at high concentrations in water, and therefore the structure of this molecule in aqueous solution has not been previously determined. We expressed a uniformly ¹³C/ ¹⁵N-labeled 42-residue peptide NN-T-20-NITN (gp41_636-677) and used heteronuclear 2D and 3D NMR methods to determine its structure. Due to the additional gp41-native hydrophilic residues, NN-T-20-NITN dissolved in water, enabling for the first time determination of its secondary structure at near physiological conditions. Our results show that the NN-T-20-NITN peptide is composed of a mostly unstructured N-terminal region and a helical region beginning at the center of T-20 and extending toward the C-terminus. The helical region is found under various conditions and has been observed also in a 13-residue peptide gp41_659-671. We suggest that this helical conformation is maintained in most of the different tertiary structures of the gp41 envelope protein that form during the process of viral fusion. Accordingly, an important element of the immunogenicity of gp41 and the inhibitory properties of Fuzeon may be the propensity of specific sequences in these polypeptides to assume helical structures.

The potent antiviral and antiproliferative activities of human type I interferons (IFNs) are mediated by a single receptor comprising two subunits, IFNAR1 and IFNAR2. The structure of the IFNAR2 IFN binding ectodomain (IFNAR2-EC), the first helical cytokine receptor structure determined in solution, reveals the molecular basis for IFN binding. The atypical perpendicular orientation of its two fibronectin domains explains the lack of C domain involvement in ligand binding. A model of the IFNAR2-EC/IFNα2 complex based on double mutant cycle-derived constraints uncovers an extensive and predominantly aliphatic hydrophobic patch on the receptor that interacts with a matching hydrophobic surface of IFNα2. An adjacent motif of alternating charged side chains guides the two proteins into a tight complex. The binding interface may account for crossreactivity and ligand specificity of the receptor. This molecular description of IFN binding should be invaluable for study and design of IFN-based biomedical agents.

The peptide gp41_659-671 (ELLELDKWASLWN) comprises the entire epitope for one of the three known antibodies capable of neutralizing a broad spectrum of primary HIV-1 isolates and is the only such epitope that is sequential. Here we present the NMR structure of gp41_659-671 in water. This peptide forms a monomeric 3₁₀-helix stabilized by i,i+3 side chain-side chain interactions favored by its primary sequence. In this conformation the peptide presents an exposed surface, which is mostly hydrophobic and consists of conserved HIV-1 residues. The presence of the 3₁₀-helix is confirmed by its characteristic CD pattern. Studies of the 3₁₀-helix have been hampered by the absence of a model peptide adopting this conformation, gp41_659-671 can serve as such a model to investigate the spectral characteristics of the 3₁₀-helix, the factors that influence its stability, and the propensity of different amino acids to form a 3₁₀-helix. The observation that the 3₁₀-helical conformation is highly populated in the peptide gp41_659-671 indicates that the corresponding segment in the cognate protein is an autonomous folding unit. As such, it is very likely that the helical conformation is maintained in gp41 throughout the different tertiary structures of the envelope protein that form during the process of viral fusion. However, the exposure of the gp41_659-671 segment may vary, leading to changes in the reactivity of anti-gp41 antibodies in the different stages of viral fusion. Since gp41_659-671 is an autonomous folding unit, peptide immunogens consisting of the complete gp41_659-671 sequence are likely to induce antibodies highly cross-reactive with HIV-1.

Continued use of non-specific chemical insecticides poses potential risks to the environment and to human health resulting from non-target toxicity and increased insect resistance to these agents. Scorpions produce anti-insect selective polypeptide toxins that bind to and modulate voltage- sensitive ion channels in excitable tissues, thus offering alternative, environmentally safer means for insect pest control. Despite this potential, little is known about their structural elements dictating anti-insect preference, which may be useful for the design of selective insecticides. We used a bacterial system for expression and genetic dissection of two pharmacologically distinct scorpion toxins: alpha and excitatory. By exploiting a multi-disciplinary approach consisting of mutagenesis, protein chemistry, electrophysiology, binding and toxicity assays, and structural studies, we elucidated the bioactive surface of two anti-insect toxins, LqhαIT and Bj-xtrIT. In both polypeptides the bioactive surface is composed of residues surrounding the C-terminal region. In addition, a direct, immediate approach in using the toxin genes was demonstrated by engineering baculoviruses with cDNAs encoding LqhIT2 (depressant toxin), and LqhIT1 (excitatory toxin) resulting in viral vectors with significantly improved insecticidal efficacy. (C) 2000 Society of Chemical Industry.

'Melittin, a 26 residue, non-cell-selective cycolytic peptide, is the major component of the venom of the honey bee Apis mellifera. In a previous study, a diastereomer ([D]-V-5,V-8,I-17,K-21-melittin, D-amino acids at positions V-5,V-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,V-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,V-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,V-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,V-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,V-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.

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- l(IIIB) bound to the Fab fragment of an anti-gp 120 HIV neutralizing antibody, 0.5/β, was studied by ¹H NMR spectroscopy. This 18-residue peptide represents the epitope recognized by 0.5β 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 → 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 Å 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 β- strands. This loop conformation could be observed due to the unusual large size (17 residues) of the antigenic determinant recognized by 0.5β.

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 (K_D ∼4 × 10^-7 M). Although calcineurin B aggregates at NMR concentrations of ∼ 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.

To increase our understanding of the molecular basis for antibody specificity and for the cross-reactivity of anti-peptide 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. We have developed an NMR methodology based on two-dimensional difference spectrum measurements which extract the information concerning antibody-peptide interactions and intramolecular interactions in the bound ligand from the crowded and unresolved spectrum of the Fab complex. These measurements yield restraints on interproton distances in the complex which are used to dock the peptide into calculated models for the antibodies' combining sites. Comparison of the interactions of three antibodies against a cholera toxin peptide (CTP3), which differ in their cross-reactivity with the toxin, yields information about the size and conformation of antigenic determinants recognized by antibodies, the structure of their combining sites and relationships between antibodies' primary structure, and their interactions with pep tide antigens.

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 β-turn formed by the sequence VPGS. The third antibody, TE34, recognizes a different epitope within the same peptide and a β-turn formed by the sequence IDSQ. Neither of these two turns was observed in the free peptide. The formation of a β-turn in the bound peptide gives a compact conformation that maximizes the contact with the antibody and that has greater conformational freedom than a-helix or β-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 3le 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.

The TE34 monoclonal antibody against cholera toxin peptide 3 (CTP3; VEVPGSQHIDSQKKA) was sequenced and investigated by two-dimensional transferred NOE difference spectroscopy and molecular modeling. The V_H sequence of TE34, which does not bind cholera toxin, shares remarkable homology to that of TE32 and TE33, which are both anti-CTP3 antibodies that bind the toxin. However, due to a shortened heavy chain CDR3, TE34 assumes a radically different combining site structure. The assignment of the combining site interactions to specific peptide residues was completed by use of AcIDSQRKA, a truncated peptide analogue in which lysine-13 was substituted by arginine, specific deuteration of individual polypeptide chains of the antibody, and a computer model for the Fv fragment of TE34. NMR-derived distance restraints were then applied to the calculated model of the Fv to generate a three-dimensional structure of the TE34/CTP3 complex. The combining site was found to be a very hydrophobic cavity composed of seven aromatic residues. Charged residues are found in the periphery of the combining site. The peptide residues HIDSQKKA form a β-turn inside the combining site. The contact area between the peptide and the TE34 antibody is 388 Ų, about half of the contact area observed in protein-antibody complexes.

The interactions between a peptide of cholera toxin and the aromatic amino acids of the TE33 antipeptide antibody, cross-reactive with the toxin, have been studied by NOESY difference spectroscopy. The 2D difference between the NOESY spectrum of the Fab with a 4-fold excess of the peptide and that of the peptide-saturated Fab reveals cross-peaks growing with excess of the peptide. These cross-peaks are due to magnetization transfer between the Fab and neighboring bound peptide protons, and a further transfer to the free peptide protons by exchange between bound and free peptide (transferred NOE), Additional cross-peaks appearing in the difference spectrum are due to a combination of intramolecular interactions between bound peptide protons and exchange between bound and free peptide. Assignment of cross-peaks is attained by specific deuteration of antibody aromatic amino acids using also the resonance assignment of the free peptide, deduced from the COSY spectrum of the peptide solution. The antibody combining site is found to be highly aromatic. We have identified one or two histidine, two tyrosine, and two tryptophan residues and one phenylalanine residue of the antibody interacting with valine-3, proline-4, glycine-5, glutamine-7, histidine-8, and aspartate-10 of the peptide. The 2D TRNOE difference spectroscopy can be used to study protein-ligand interactions, given that the ligand off rate is fast relative to the spin-lattice relaxation time of the protein and ligand protons (about 1 s). The resolution obtained in the difference spectra implies that the technique is equally applicable for studying proteins having a molecular weight larger than 50000.

Specifically deuteriated Fab fragments of the anti-spin-label antibody AN02 were prepared. NMR difference spectra were obtained, in which the spectrum of Fab with some fraction of the binding sites occupied with spin-label hapten was subtracted from the spectrum of Fab with no spin-label. the peak heights were analyzed as a function of the fractional occupation of the binding site, using a computer program that calculates a best fit to the observed spectra. This method treats all of the peaks in the spectra simultaneously. Analyzing all peaks at once allows for the interdependencies in the spectra arising from overlap of positive and negative signals from different peaks. the fitting program calculates line widths for the peaks arising from protons in the binding site region. Almost all of the line widths calculated for the spectrum of the Fab complex with diamagnetic hapten dinitrophenyldiglycine were found to be narrower than the line widths of the corresponding resonances in the spectrum of Fab with an empty binding site. the distances of the binding site region protons from the unpaired electron of the hapten were also obtained from this calculation. Two tyrosine protons were found to be close (

Resonance Rayleigh light scattering was measured at the Soret absorption band of meso-tetraphenyl porphine (in the free-base form), chlorophyll a, and meso-tetra(4-pyridyl) porphine (in the fully prolonated form). The last compound, unlike the first two, showed unexpected fluorescence on the red side of, but close to, its Soret absorption band, and scattering experiments were not possible in the spectral range where fluorescence was detectable. The depolarization ratios measured for the Rayleigh scattered light of all three compounds were close to the value 1 8 expected for a planar oscillator, in line with previous conclusions that even when the in-plane degeneracy is lifted, the splitting of the in-plane bands at the Soret frequencies is small and the two transitions overlap considerably. Very good agreement was found between the measured scattering intensities and those predicted theoretically at the red edge of the Soret bands. Good agreement was also observed in the blue part of the Soret band in the case where measurements were possible (meso-tetra(4-pyridyl) porphine). However, in the intermediate spectral range the measured intensities exceeded appreciably the theoretically expected ones. It may thus be concluded that the enhanced scattering intensity that had been observed before in the red part of the last electronic absorption band of many compounds is not limited to such bands. The observed discrepancy between the experimentally determined intensities and those calculated from the absorption spectra may be due to momentary heterogeneity in the absorption coefficients, ???, in the population of absorbing molecules, the absorption and scattering depending each on different averages of ???. This heterogeneity is accentuated in the red part of the bands, where transitions from thermally excited molecules are more pronounced than in other parts of the absorption bands.