T-cell epitopes of the La/SSB autoantigen: prediction based on the homology modeling of HLA-DQ2/DQ7 with the insulin-B peptide/HLA-DQ8 complex

T-cell epitopes are important components of the inappropriate response of the immune system to self-proteins in autoimmune diseases. In this study, the candidate T-cell epitopes of the La/SSB autoantigen, the main target of the autoimmune response in patients with Sjogren's Syndrome (SS), and Systemic Lupus Erythematosus (SLE) were predicted using as a template the HLA-DQ2 and DQ7 molecules, which are genetically linked to patients with SS and SLE. Modeling of DQ2 and DQ7 was based on the crystal structure of HLA-DQ8, an HLA molecule of high risk factor of type I diabetes, which is also an autoimmune disease. The quality and reliability of the modeled DQ2 and DQ7 was confirmed by the Ramachandran plot and the TINKER molecular modeling software. Common and/or similar candidate T-cell epitopes, obtained by comparing three different approaches the Taylor's sequence pattern, the TEPITOPE quantitative matrices, and the MULTIPRED artificial neural network, were subjected to homology modeling with the crystal structure of the insulin-B peptide complexed with HLA-DQ8, and the best superposed candidate epitopes were placed into the modeled HLA-DQ2 and DQ7 binding grooves to perform energy minimization calculations. Six T-cell epitopes were predicted for HLA-DQ7 and nine for HLA-DQ2 covering parts of the amino-terminal and the central regions of the La/SSB autoantigen. Residues corresponding to the P1, P4, and P9 pockets of the HLA-DQ2 and DQ7 binding grooves experience very low SASA because they are less exposed to the microenvironment of the groove. The proposed T-cell epitopes complexed with HLA-DQ2/DQ7 were further evaluated for their binding efficiency according to their potential interaction energy, binding affinity, and IC50 values. Our approach constitutes the ground work for a rapid and reliable experimentation concerning the T-cell epitope mapping of autoantigens, and could lead to the development of T-cell inhibitors as immunotherapeutics in autoimmune diseases.     

Docking of B-cell epitope antigen to specific hepatitis B antibody

The interaction of pres1 region of hepatitis B virus B-cell epitope antigen with specific hepatitis B neutralizing monoclonal antibody was examined by docking study. We modelled the 3D complex structure of B-cell epitope antigen residues CTTPAQGNSMFPSCCCTKPTDGNCY by homology modelling and docked it with the crystal structure of monoclonal antibody specific for the pres1 region of the hepatitis B virus. At the optimized docked conformation, the interactions between the amino acids of antigen and antibody were examined. It is found that the docked complex is stabilized by 59.3 kcal/mol. The stability of the docked antigen-antibody complex is due to hydrogen bonding and van der Waals interactions. The amino acids of the antigen and antibody responsible for the interaction were identified.     

Mass spectrometric and peptide chip characterization of an assembled epitope: analysis of a polyclonal antibody model serum directed against the Sjøgren/systemic lupus erythematosus autoantigen TRIM21

We demonstrate the development of a mass spectrometry‐based epitope‐mapping procedure in combination with Western blot analysis that works also with antigens that are insoluble in nondenaturing buffers consuming minute amounts of antigen (approximately 200 pmol) and antibody (approximately 15 pmol), respectively. A polyclonal anti‐TRIM21 rabbit antibody serum is applied as a model serum for future patient analyses to set up the system. The major epitope that is recognized by the anti‐TRIM21 serum spans the central TRIM21 region LQ‐EE REL GE‐KE, showing that immunization with a 139‐amino acid residue long peptide resulted in a ‘monospecific’ polyclonal antibody repertoire. Protein structure investigations, secondary structure predictions, and surface area calculations revealed that the best matching partial sequence to fulfill all primary and secondary structure requirements was the four amino acid spanning motif ‘L–E–Q–L’, which is present in both the sequential and the α‐helical peptide conformation. Peptide chip analyses confirmed the mass spectrometric results and showed that the peptide chip platform is an appropriate method for displaying secondary structure‐relying epitope conformations. As the same secondary structures are present in vivo, patient antibody screening, e.g., to identify subgroups of patients according to distinct epitope antibody reactivities, is feasible. Copyright © 2013 John Wiley & Sons, Ltd.     

Synthesis of a trimeric gp120 epitope mimic conjugated to a T-helper peptide to improve antigenicity

A fully synthetic trivalent mimotope of gp120 conjugated to pan allelic HLA DR binding epitope was prepared using solid-phase peptide synthesis and optimized copper-catalyzed azide-alkyne cycloaddition. The methodology efficiently provides chemically uniform heteromultimeric peptide constructs with enhanced binding, avidity, and specificity toward an established HIV-neutralizing human antibody, MAb b12. The versatile synthetic strategy serves as a powerful platform for the development of synthetic peptides as potential HIV-1 vaccine candidates.     

Conformational distribution of a 14-residue peptide in solution: a fluorescence resonance energy transfer study

We demonstrate here that a nitrile-derivatized phenylalanine residue, p-cyanophenylalanine (Phe(CN)), and tryptophan (Trp) constitute a novel donor-acceptor pair for fluorescence resonance energy transfer (FRET). The F?rster distance of this FRET pair was determined to be approximately 16 A and hence is well suited for determining relatively short separation distances. To validate the applicability of this FRET pair in conformational studies, we studied the conformational heterogeneity of a 14-residue amphipathic peptide, Mastoparan X (MPx peptide), in water and 7 M urea solution as well as at different temperatures. Specifically, seven nitrile-derivatized mutants of the MPx peptide, each containing a Phe(CN) residue that replaces different positions along the peptide sequence (i.e., from position 5 to 11) and serves as a resonance energy donor to the native Trp residue at position 3, were studied spectroscopically. The FRET efficiencies obtained from these peptides allowed us to gain a global picture regarding the conformational distribution of the MPx peptide in different environments. Our results suggest that the MPx molecules exist in water as an ensemble of rather compact conformations, with a radius of gyration of approximately 4.2 A, whereas in 7 M urea the radius of gyration increases to approximately 6.5 A, indicating that the peptide conformations become more extended under this condition. However, we found that temperature had only a negligible effect on the size of the MPx peptide, underlining the difference between the thermally and chemically denatured states of polypeptides. The application of the Gaussian chain or the wormlike chain model allowed us to further obtain the probability distribution function of the separation distance between any two residues along the peptide sequence. We found that the effective bond length of the MPx peptide, obtained by using the Gaussian chain model, is 2.78 A in water and 4.28 A in 7 M urea.     

Conformational analysis of a farnesyltransferase peptide inhibitor, CVIM

The conformational states of the peptide Cys-Val-Ile-Met (or CVIM) were computed and characterized. CVIM inhibits farnesylation of the Ras oncogene product, p21^ras, at the cysteine residue of the C-terminal segment. CVIM is active in an extended conformation. A similar peptide (KTKCVFM) appears to bind the enzyme in the Type I bend conformation. In the present study, the conformations of CVIM were computed in an aqueous environment with the peptide in the zwitterionic state. Solvation free energy based on solvent accessible surface area and a distance dependent dielectric were used in the calculations. Final conformations of multiple independent Monte Carlo simulated annealing (MCSA) conformational searches were used as starting points for Metropolis Monte Carlo (MMC) runs. Conformations saved at intervals during MMC runs were analyzed. Conformers were separated by interactive clustering in dihedral angle coordinates. The four lowest energy conformers corresponding to a Type I bend, extended, AB-bend, and BA-bend were within 0.3 kcal/mol of each other, and dominant in terms of population. The Type I bend and extended conformers were supported by the binding studies. The extended conformer was the most populated. In the AB-bend conformer, `A' indicates the -helix conformation of Val, and `B' indicates the -strand conformation of Ile. The AB- and BA-bend conformations differed from the extended conformation in the value of Val and Ile , respectively, and from the Type I bend conformation in the value of Ile and Val , respectively. The four lowest energy conformers were characterized in terms of energy, density of low energy conformations (or entropy), structure, side chain rotamer fraction population, and interatomic distances.     

Conformational polymorphism of the PrP106-126 peptide in different environments: a molecular dynamics study

Extensive molecular dynamic simulations (approximately 240 ns) have been used to investigate the conformational behavior of PrP106-126 prion peptide in four different environments (water, dimethyl sulfoxide, hexane, and trifluoroethanol) and under both neutral and acidic conditions. The conformational polymorphism of PrP106-126 in solution observed in the simulations supports the role of this fragment in the structural transition of the native to the abnormal form of prion protein in response to changes in the local environmental conditions. The peptide in solution is primarily unstructured. The simulations show an increased presence of helical structure in an apolar solvent, in agreement with the results from circular dichroism spectroscopy. In water solution, beta-sheet elements were observed between residues 108-112 and either residues 115-121 or 121-126. An alpha-beta transition was observed under neutral conditions. In DMSO, the peptide adopted an extended conformation, in agreement with nuclear magnetic resonance experiments.     

Conformational analysis of a stereochemically complete set of cis-enediol peptide analogues

A conformational analysis of a stereochemically complete set of peptide analogues based on a cis-enediol unit is presented. The cis-enediol unit, which can replace a two or a three amino acid segment of a peptide, contains two "side chains", four asymmetrical carbon atoms, and six free dihedral angles. To determine the accessible conformational space, the molecules are divided into three fragments, each containing two free dihedral angles. The energy surfaces are computed for all dihedral angle values, and the possible conformations of the cis-enediol unit analogues are built using all combinations of the surface minima. Such a "build-up" procedure, which is very fast, is able to reproduce 75% of the minima obtained from a full dihedral angle exploration of the conformational space. The cis-enediol unit minima are compared with the corresponding di- and tripeptide minima; all peptide minima can be closely matched by a cis-enediol unit minimum of low energy (less than 2.2 kcal/mol above the lowest energy conformer). However, there are low energy minima of the cis-enediol unit that have no corresponding minima in peptides. The results are shown to depend strongly on the chirality of the analogues. The ability of each of the stereoisomers to mimic natural peptides, evaluated by the present approach, is correlated with its experimental activity in a renin inhibition assay.     

Conformational energy map for a pair of peptide units using the bond polarisability method

A method recently developed for the calculation of intramolecular nonbonded interactions based on experimental bond polarisabilities and atomic charges and transition π-charges obtained from MO calculations has been applied to the alanyl dipeptide. The potential energy contours in the ?, ψ plane obtained by this method compare favourably with those derived from the frequency of occurrence of conformations in globular proteins. An analysis of the various components of the nonbonded interaction energy indicates that the fairly frequent occurrence of conformations around ? = ?80°, ψ=0° is presumably due to a favourable interaction of the π-polarisation.     

Synthesis of branched oxime-linked peptide mimetics of the MUC1 containing a universal T-helper epitope

Our goal was to develop mimics of MUC1, highly immunogenic to induce an efficient immune response against the tumor-associated form of MUC1, and sufficiently different from the natural antigen to bypass the tolerance barrier in humans. With the aim of obtaining a well-defined peptide construct as a means of evoking the precise immune responses required in immunotherapy, we synthesized artificial mimics of the MUC1 protein composed of two MUC1 repeat units of inverse orientation and a universal T-helper epitope. To synthesize these heteromeric peptide constructs, we followed a convergent approach using chemoselective ligation based on oxime chemistry. A stem peptide was first synthesized bearing two orthogonally masked aldehydes. After successive deprotection, two oxime bonds can be specifically generated. The proposed strategy proved to be concise and robust, and allowed the synthesis of the tri-branched protein in a very satisfactory yield. The different constructs were tested for their ability to generate antibodies able to recognize the MUC1 protein.     

Investigation of the binding of a carbohydrate-mimetic peptide to its complementary anticarbohydrate antibody by STD-NMR spectroscopy and molecular-dynamics simulations

Saturation transfer difference (STD)‐NMR spectroscopy was used to probe experimentally the bioactive solution conformation of the carbohydrate mimic MDWNMHAA 1 of the O‐polysaccharide of Shigella flexneri Y when bound to its complementary antibody, mAb SYA/J6. Molecular dynamics simulations using the ZymeCAD? Molecular Dynamics platform were also undertaken to give a more accurate picture of the conformational flexibility and the possibilities for bound ligand conformations. The ligand topology, or the dynamic epitope, was mapped with the CORCEMA‐ST (COmplete Relaxation and Conformational Exchange Matrix Analysis of Saturation Transfer) program that calculates a total matrix analysis of relaxation and exchange effects to generate predicted STD‐NMR intensities from simulation. The comparison of these predicted STD enhancements with experimental data was used to select a representative binding mode. A protocol that employed theoretical STD effects calculated at snapshots during the entire course of a molecular dynamics (MD) trajectory of the peptide bound to the Fv portion of the antibody, and not the averaged atomic positions of receptor–ligand complexes, was also examined. In addition, the R factor was calculated on the basis of STD (fit) to avoid T1 bias, and an effective R factor, R_eff, was defined such that if the calculated STD (fit) for proton k was within error of the experimental STD (fit) for proton k, then that calculated STD (fit) for proton k was not included in the calculation of the R factor. This protocol was effective in deriving the antibody‐bound solution conformation of the peptide which also differed from the bound conformation determined by X‐ray crystallography; however, several discrepancies between experimental and calculated STD (fit) values were observed. The bound conformation was therefore further refined with a simulated annealing refinement protocol known as STD‐NMR intensity‐restrained CORCEMA optimization (SICO) to give a more accurate representation of the bound peptide epitope. Further optimization was required in this case, but a satisfactory correlation between experimental and calculated STD values was obtained. Attempts were also made to obtain STD enhancements with a synthetic pentasaccharide hapten, corresponding to the O‐polysaccharide, while bound to the antibody. However, unfavorable kinetics of binding in this system prevented sufficient STD build‐up, which, in turn, hindered a rigorous analysis via full STD build‐up curves.     

Conformational studies of a bombolitin III-derived peptide mimicking the four-helix bundle structural motif of proteins

Bombolitins are five structurally related heptadecapeptides originally isolated from the venom of a bumblebee. In aqueous solution, bombolitins at sufficiently high concentration form oligomeric aggregates with consequent conformational transition from a random coil to the alpha-helical structure. Previous studies suggested that oligomeric aggregates could mimic the four-helix bundle structural motif of proteins. In the present work, we synthesized the following peptide sequence formed by two bombolitin III sequences linked head-to-tail by the tetrapeptide bridge -Gly-Pro-Val-Asp-: I(1)-K(2)-I(3)-M(4)-D(5)-I(6)-L(7)-A(8)-K(9)-L(10)-G(11)-K(12)-V(13)-L(14)-A(15)-H(16)-V(17)-G(18)-P(19)-V(20)-D(21)-I(22)-K(23)-I(24)-M(25)-D(26)-I(27)-L(28)-A(29)-K(30)-L(31)-G(32)-K(33)-V(3)(4)-L(35)-A(36)-H(37)-V(38)-NH(2). The tetrapeptide GPVD connecting the two helical peptide sequences was chosen to facilitate the formation of the helix-loop-helix structural motif. The conformational properties of the peptide were studied by CD, NMR, and molecular dynamics calculations. The results indicate the presence of a helix-loop-helix conformation at 10(-)(5) M concentration. At higher concentrations, NOESY connectivities were detected which are compatible with the presence of dimers or higher aggregates of peptide molecules in the helix-loop-helix structure packed in an antiparallel fashion. Molecular dynamics simulation were run either with NOE distance restraints or without restraints in explicit solvent for extended time. The results of these simulations support the dimerization of the molecules in the helix-loop-helix structure with formation of the four-helix bundle motif.     

Conformational properties of peptide fragments homologous to the 106-114 and 106-126 residues of the human prion protein: a CD and NMR spectroscopic study

Two peptide fragments, corresponding to the amino acid residues 106-126 (PrP[Ac-106-126-NH(2)]) and 106-114 (PrP[Ac-106-114-NH(2)]) of the human prion protein have been synthesised in the acetylated and amide form at their N- and C-termini, respectively. The conformational preferences of PrP[Ac-106-126-NH(2)] and PrP[Ac-106-114-NH(2)] were investigated using CD and NMR spectroscopy. CD results showed that PrP[Ac-106-126-NH(2)] mainly adopts an alpha-helical conformation in TFE-water mixture and in SDS micelles, while a predominantly random structure is observed in aqueous solution. The shorter PrP[Ac-106-114-NH(2)] fragment showed similar propensities when investigated under the same experimental conditions as those employed for PrP[Ac-106-126-NH(2)]. From CD experiments at different SDS concentrations, an alpha-helix/beta-sheet conformational transition was only observed in the blocked PrP[Ac-106-126-NH(2)] sequence. The NMR analysis confirmed the helical nature of PrP[Ac-106-126-NH(2)] in the presence of SDS micelles. The shorter PrP[Ac-106-114-NH(2)] manifested a similar behaviour. The results as a whole suggest that both hydrophobic effects and electrostatic interactions play a significant role in the formation and stabilisation of ordered secondary structures in PrP[Ac-106-126-NH(2)].     

Conformational features of peptide fragments of the C-end of histone H1

The conformational possibilities of four synthesized oligopeptides with the amino acid sequences 165–172, 173–184, 152–172, and 152–184 of the C-end of histone H1 of calf thymus above been studied in solution under various conditions by the method of circular dichroism. V. I. Nikitin Institute of Chemistry, Academy of Sciences of the Tadchik SSR, Dushanbe. Institute of Cytology, Academy of Sciences of the USSR, Leningrad. Translated from Khimiya Prirodnykh Soedinenii, No. 6, pp. 793–798, November–December, 1990.     

Conformational study of the cembranoid sarcophytol A, a potent anti-tumor-promoter

Conformational analysis of the marine cembranoid sarcophytol A (1a), a potent anti-tumor promoter, was carried out using a newly introduced molecular mechanic and molecular dynamic program Discover. Four minimum-energy conformations were derived, in accordance with a previous results of the epoxidation of 1a, which afforded 7R,8R/7S,8S- and 11R,12R/11S,12S-epoxide pairs. The most stable conformation was the one having C-19 and C-20 directed opposite to C-18, with respect to the average plane of the fourteen-membered ring. X-Ray crystallography of sarcophytol A alpha-methoxy-alpha-trifluoromethylphenylacetate (1c) was carried out simultaneously. This confirmed the 14S absolute configuration of 1a but the conformation of crystalline 1c did not correspond to any of the four minimum-energy conformers of 1a.     

Conformational features of peptide fragments of the C-end of histone H1

The conformational possibilities of four synthesized oligopeptides with the amino acid sequences 165–172, 173–184, 152–172, and 152–184 of the C-end of histone H1 of calf thymus above been studied in solution under various conditions by the method of circular dichroism.V. I. Nikitin Institute of Chemistry, Academy of Sciences of the Tadchik SSR, Dushanbe. Institute of Cytology, Academy of Sciences of the USSR, Leningrad. Translated from Khimiya Prirodnykh Soedinenii, No. 6, pp. 793–798, November–December, 1990.     

Conformational sampling of bioactive molecules: a comparative study

The necessity to generate conformations that sample the entire conformational space accessible to a given molecule is ubiquitous in the field of computer-aided drug design. Protein-ligand docking, 3D database searching, and 3D QSAR are three commonly used techniques that depend critically upon the quality and diversity of the generated conformers. Although there are a wide range of conformational search algorithms available, the extent to which they sample conformational space is often unclear. To address this question, we conducted a robust comparison of the search algorithms implemented in several widely used molecular modeling packages, including Catalyst, Macromodel, Omega, MOE, and Rubicon as well as our own method, stochastic proximity embedding (SPE). We found that SPE used in conjunction with conformational boosting, a heuristic for biasing conformational search toward more extended or compact geometries, along with Catalyst, are significantly more effective in sampling the full range of conformational space compared to the other methods, which show distinct preferences for either more extended or more compact geometries.