NMR and molecular modeling studies on two glycopeptides from the carbohydrate-protein linkage region of connective tissue proteoglycans.

Complete 1H and 13C NMR assignments are reported for two glycopeptides representing the carbohydrate-protein linkage region of connective tissue proteoglycans. These glycopeptides are the octasaccharide hexapeptide, Ser(GlcpAbeta(1-->3) Galpbeta(1-->3)Galpbeta(1-->4)Xylpbeta)-Gly-Ser-Gly-Se r (GlcpAbeta(1-->3)Galpbeta(1-->3)Galpbeta(1-->4)Xylp beta)-Gly (1), and the tetrasaccharide dipeptide, Ser(GlcpAbeta(1-->3)Galpbeta(1-->3)Galpbeta(1-->4)X ylpbeta)-Gly (2). The vicinal coupling constant data show that the monosaccharide residues adopt4 C 1 chair conformations. Distance geometry/simulated annealing calculations using 2D NOESY derived distance constraints yielded a single family of structures for the tetrasaccharide moiety, with well defined interglycosidic linkage conformations. The straight phi torsion angles of the glycosidic C1'-O1 bonds showed a strict preference for the -sc range whereas the psi torsion angles (O1-Cn) exhibited dependence upon the interglycosidic linkage position (-ac for beta(1-->3) linkage, +ac for beta(1-->4) linkage). The predominant conformation about the glycopeptide bond is straight phi = -sc and psi = +ac. The presence of strong daN (i, i+1) NOE contacts, and the general absence of dNN (i, i+1) contacts (except for a weak Ser-5/Gly-6 dNN contact) and the dbN (i, i+1) contacts (except for Ser-1/Gly-2) in the ROESY spectrum, suggest that the backbone for 1 is predominantly in an extended conformation. A comparison of the ROESY data for 1 with those obtained from the unglycosylated hexapeptide (3) of the same sequence suggests that glycosylation has only a marginal influence on the backbone conformation of the hexapeptide.     

Solution conformation of a model hexapeptide containing RGD sequence.

The solution conformation of a model hexapeptide Asp-Arg-Gly-Asp-Ser-Gly (DRGDSG) containing the RGD sequence has been studied in DMSO-d6 as well as in aqueous solution (H2O:D2O/90:10%) by 1H NMR spectroscopy. The unambiguous identification of spin systems of various amino acid residues and sequence specific assignment of all proton resonances was achieved by a combination of two dimensional COSY and NOESY experiments. The temperature coefficient data of the amide proton chemical shifts in conjunction with the vicinal coupling constants, i.e. 3JNH-C alpha H, NOESY and ROESY results indicate that the peptide in both the solvents exists in a blend of conformers with beta-sheet like extended backbone structure and folded conformations. The folded conformers do not appear to be stabilised by intramolecular hydrogen bonding. Our results are consistent with the flexibility of RGD segment observed in the NMR studies on the protein echistatin containing the RGD motif (references 23-25).     

Crystal structure of cyclophilin A complexed with a binding site peptide from the HIV-1 capsid protein.

The cellular protein, cyclophilin A (CypA), is incorporated into the virion of the type 1 human immunodeficiency virus (HIV-1) via a direct interaction with the capsid domain of the viral Gag polyprotein. We demonstrate that the capsid sequence 87His-Ala-Gly-Pro-Ile-Ala92 (87HAGPIA92) encompasses the primary cyclophilin A binding site and present an X-ray crystal structure of the CypA/HAGPIA complex. In contrast to the cis prolines observed in all previously reported structures of CypA complexed with model peptides, the proline in this peptide, Pro 90, binds the cyclophilin A active site in a trans conformation. We also report the crystal structure of a complex between CypA and the hexapeptide HVGPIA, which also maintains the trans conformation. Comparison with the recently determined structures of CypA in complexes with larger fragments of the HIV-1 capsid protein demonstrates that CypA recognition of these hexapeptides involves contacts with peptide residues Ala(Val) 88, Gly 89, and Pro 90, and is independent of the context of longer sequences.     

Conformational Analysis of the β-amyloid Peptide Fragment, β(12–28)

Abstract

NMR and CD spectroscopy have been used to examine the conformation of the peptide, β(12–28), (VHHQKLVFFAEDVGSNK) in aqueous and 60% TFE/40% H₂0 solution at pH 2.4. In 60% TFE solution, the peptide is helical as confirmed by the CD spectrum and by the pattern of the NOE cross peaks detected in the NOESY spectrum of the peptide. In aqueous solution, the peptide adopts a more extended and flexible conformation. Broadening of resonances at low temperature, temperature-dependent changes in the chemical shifts of several of the CH_α resonances and the observation of a number of NOE contacts between the hydrophobic side-chain protons of the peptide are indicative of aggregation in aqueous solution. The behavior of β(12–28) in 60% TFE and in aqueous solution are consistent with the overall conformation and aggregation behavior reported for the larger peptide fragment, β(1–28) and the parent β-amyloid peptide.     

Conformation and calcium-binding properties of a bicyclic nonapeptide

The conformation and calcium binding properties of the bicyclic nonapeptide BCP2, cyclo-(Glu(1)-Ala(2)-Pro(3)-Gly(4)-Lys(5)-Ala(6)-Pro(7)-Gly(8))-cyclo-(1gamma --> 5epsilon) Gly(9), have been investigated by means of NMR spectroscopy. Interproton distances, evaluated by nuclear Overhauser effect (NOE) contacts, and straight phi angles, from (3)J(NH-alphaCH), have been used to obtain a feasible model for the BCP2-Ca(2+) (BCP: bicyclic peptide) complex by means of restrained molecular dynamics (RMD). The NMR analysis of the free peptide, carried out in CD(3)CN, shows the presence in solution of at least four conformers in intermediate exchange rate. The addition of calcium ions caused the appearance of a new set of resonances, differing from those observed for the free BCP2. A comparison with published data about the conformational behavior of the closely analogous peptide BCP3, differing from BCP2 for two Leu residues instead of two Ala residues in positions 2 and 6, shows that this simple substitution dramatically increases the peptide flexibility. On the contrary, upon calcium ion addition, both BCP2 and BCP3 reach a strictly close conformation, as strongly testified by the almost identical (1)H-NMR spectra exhibited by both peptides. The RMD molecular model of the BCP2-Ca(2+) complex, here reported, is a quite symmetric structure, presenting a three-dimensional cavity ideal for the binding of spherical cations. Four carbonyls from the main ring (Ala(2), Gly(4), Ala(6) and Gly(8)) point toward it, offering, together with the two carbonyls of the peptide bridge (Gly(9) and gammaGlu(1)), putative coordinations to the cation.     

Conformational Analysis of the β-amyloid Peptide Fragment, β(12-28)

Abstract NMR and CD spectroscopy have been used to examine the conformation of the peptide, β(12-28), (VHHQKLVFFAEDVGSNK) in aqueous and 60% TFE/40% H(2)0 solution at pH 2.4. In 60% TFE solution, the peptide is helical as confirmed by the CD spectrum and by the pattern of the NOE cross peaks detected in the NOESY spectrum of the peptide. In aqueous solution, the peptide adopts a more extended and flexible conformation. Broadening of resonances at low temperature, temperature-dependent changes in the chemical shifts of several of the CH(α) resonances and the observation of a number of NOE contacts between the hydrophobic side-chain protons of the peptide are indicative of aggregation in aqueous solution. The behavior of β(12-28) in 60% TFE and in aqueous solution are consistent with the overall conformation and aggregation behavior reported for the larger peptide fragment, β(1-28) and the parent β-amyloid peptide.     

Efficient synthesis of O-linked glycopeptide by a transglycosylation using endo alpha-N-acetylgalactosaminidase from Streptomyces sp.

Gal beta-(1-->3)-GalNAc-linked hexapeptide was synthesized by a transglycosylation using Gal beta-(1-->3)-GalNAc beta-pNP as a donor and a serine-containing hexapeptide as an acceptor using endo GalNAc-ase from Streptomyces sp.. The Gal beta-(1-->3)-GalNAc residue was transferred to the hydroxyl group of the serine residue of the peptide. The total yield of the glycopeptide via this process was better than that of the chemoenzymatic method. This process was confirmed to be a versatile method for the synthesis of O-linked glycopeptides.     

Structural details of HIV-1 recognition by the broadly neutralizing monoclonal antibody 2F5: epitope conformation, antigen-recognition loop mobility, and anion-binding site

2F5 is a monoclonal antibody with potent and broadly neutralizing activity against HIV-1. It targets the membrane-proximal external region (MPER) of the gp41 subunit of the envelope glycoprotein and interferes with the process of fusion between viral and host cell membranes. This study presents eight 2F5 F_ab′ crystal structures in complex with various gp41 peptide epitopes. These structures reveal several key features of this antibody-antigen interaction. (1) Whenever free of contacts caused by crystal artifacts, the extended complementarity-determining region H3 loop is mobile; this is true for ligand-free and epitope-bound forms. (2) The interaction between the antibody and the gp41 ELDKWA epitope core is absolutely critical, and there are also close and specific contacts with residues located N-terminal to the epitope core. (3) Residues located at the C-terminus of the gp41 ELDKWA core do not interact as tightly with the antibody. However, in the presence of a larger peptide containing the gp41 fusion peptide segment, these residues adopt a conformation consistent with the start of an α-helix. (4) At high sulfate concentrations, the electron density maps of 2F5 F_ab′-peptide complexes contain a peak that may mark a binding site for phosphate groups of negatively charged lipid headgroups. The refined atomic-level details of 2F5 paratope-epitope interactions revealed here should contribute to a better understanding of the mechanism of 2F5-based virus neutralization, in general, and prove important for the design of potential vaccine candidates intended to elicit 2F5-like antibody production.     

Solid state and solution conformations of a hybrid alphagammaalphaalphagammaalpha hexapeptide. Characterization of a backbone expanded analog of the alpha-polypeptide 3(10)-helix

The stereochemically constrained gamma amino acid residue gabapentin (1-(aminomethyl)cyclohexaneacetic acid, Gpn) has been incorporated into a host alpha-peptide sequence. The structure of a hybrid alphagammaalphaalphagammaalpha peptide, Boc-Leu-Gpn-Aib-Leu-Gpn-Aib-OMe in crystals reveals a continuous helical conformation stabilized by three intramolecular 4 --> 1 C(12) hydrogen bonds across the alphagamma/alphagamma segments and one C(10) hydrogen bond across the central alphaalpha segment. This conformation corresponds to an expanded analog of the canonical all-alpha polypeptide 3(10)-helix, with insertion of two additional backbone atoms at each gamma residue. Solvent dependence of NH chemical shifts in CDCl(3) solution are consistent with conformation in which the NH groups of Aib (3), Leu (4), Gpn (5), and Aib (6) are hydrogen bonded, a feature observed in the solid state. The nonsequential NOEs between Gpn (2) NH <--> Leu (4) NH and Gpn (2) NH <--> Gpn (5) NH support the presence of additional conformations in solution. Temperature-dependent line broadening of NH resonances confirms the occurrence of rapid exchange between multiple conformations at room temperature. Two conformational models which rationalize the observed nonsequential NOEs are presented, both of which contain three hydrogen bonds and are consistent with the known stereochemical preferences of the Gpn residue.     

Conformational studies of N-Tyr-MIF-1 in aqueous solution by 1H nuclear magnetic resonance spectroscopy.

N-Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) is an endogenous brain peptide with multiple effects on animal behavior. However, there have been no studies on the conformation of this tetrapeptide. In this report, we studied the conformation of N-Tyr-MIF-1 in aqueous solution by conventional one-dimensional and two-dimensional (COSY and NOESY) 1H nuclear magnetic resonance spectroscopy at 300 MHz. A complete set of assignments for the resolved resonances and approximate assignments for the overlapping resonances were made. The results demonstrate that N-Tyr-MIF-1 is in slow exchange between two conformers, most likely determined by the cis and trans states of the proline residue. The minor conformation represents 30 +/- 3% of the population over the temperature range from 3 degrees to 73 degrees. In the major conformation, the tyrosine aromatic ring appears to be close enough to interact directly with the proline pyrrolidine ring, as indicated by a strong temperature dependence of the proline C beta H, C delta H and C delta H' chemical shifts. In contrast, this interaction of the tyrosine and proline rings is not present in the minor conformation.     

Monoclonal antibodies against different epitopes of peptide hormones. Use of photoreactive analogues in studies on vasopressin.

In order to produce monoclonal antibodies directed against different epitopes of the neurohypophyseal hormone vasopressin, the hormone was coupled to carrier proteins via photoreactive groups at different positions in the vasopressin sequence: [2-(4-azidophenylalanine), 8-arginine]vasopressin (peptide P1, photoreactive group at position 2) and desamino-[8-N6-(4-azidophenylamidino)lysine]vasopressin (peptide P2, photoreactive group at position 8) were conjugated to thyroglobulin by flash photolysis. Monoclonal antibodies against these conjugates bound ([3H]8-arginine]vasopressin with dissociation constants ranging over 40-400 nM. Epitope analysis by means of competitive ELISA showed that the monoclonal antibody obtained with peptide P1 as hapten was directed against the C-terminal acyclic tripeptide when its conformation was stabilized by interaction with the disulphide-linked cyclic hexapeptide. In contrast, the epitope analysis of three monoclonal anti-(peptide P2) antibodies demonstrated that they recognized antigenic determinants in the cyclic hexapeptide ring, mainly the hydrophobic surface formed by Tyr2 and Phe3. Our results suggest that monoclonal antibodies against different epitopes in small peptide hormones can be generated selectively by using photoreactive peptides in such a way that different antigenic sites are exposed in the hapten-carrier conjugate.     

Conformation and mobility of the RNA-binding N-terminal part of the intact coat protein of cowpea chlorotic mottle virus. A two-dimensional proton nuclear magnetic resonance study

Two-dimensional proton nuclear magnetic resonance (n.m.r.) experiments were performed on the coat protein of cowpea chlorotic mottle virus (molecular mass: 20.2 kDa) present as dimer (pH 7.5) or as capsid consisting of 180 protein monomers (pH 5.0). The spectra of both dimers and capsids showed resonances originating from the flexible N-terminal region of the protein. The complete resonance assignment of a synthetic pentacosapeptide representing this N terminus made it possible to interpret the spectra in detail. The capsid spectrum showed backbone amide proton resonances arising from the first eight residues having a flexible random coil conformation, and side-chain resonances arising from the first 25 N-terminal amino acids. The dimer spectrum showed also side-chain resonances of residues 26 to 33, which are flexible in the dimer but immobilized in the capsid. The n.m.r. experiments indicated that the conformation of the first 25 amino acids of the protein in dimers and capsids is comparable to the conformation of the synthetic peptide, which alternates among extended and helical conformations on the n.m.r. time-scale. It is suggested that the alpha-helical region, situated in the region between residues 10 and 20, binds to the RNA during assembly of the virus particle.     

Epitope mapping of sialyl Lewis(x) bound to E-selectin using saturation transfer difference NMR experiments

A complex between sialyl Lewisx (alpha-D-Neu5Ac-[2-->3]- beta-D-Gal-[1-->4]-[alpha-L-Fuc-(1-->3)]-beta-D-GlcNAc-O-[CH2]8 COOMe) and E-selectin was studied using saturation transfer difference (STD) nuclear magnetic resonance (NMR) experiments. These experiments allow the identification of the binding epitope of a ligand at atomic resolution. A semiquantitative analysis of STD total correlation spectroscopy spectra provides clear evidence that the galactose residue receives the largest saturation transfer. The protons H4 and H6 of the galactose residue are in especially close contact to the amino acids of the E-selectin binding pocket. The fucose residue also receives a significant saturation transfer. The GlcNAc and Neu5Ac residues, with the exception of H3 and H3' of Neu5Ac, were found to interact weakly with the protein surface. These findings are in excellent agreement with a recently published X-ray structure and with the earlier findings from syntheses and activity assays. To further characterize the binding pocket of E-selectin, an inhibitory peptide, Ac-TWDQLWDLMK-CONH2, was synthesized and the binding to E-selectin studied utilizing transfer nuclear Overhauser effect spectroscopy (trNOESY) experiments. Finally, competitive trNOESY experiments were performed, showing that the synthetic peptide is a competitive inhibitor of sialyl Lewisx.     

Computational modelling and molecular dynamics simulations of a cyclic peptide mimotope of the CD52 antigen complexed with CAMPATH-1H antibody

A peptide mimotope of the CD52 antigen with the sequence T₁SSPSAD₇ has been co-crystallised with the CAMPATH-1H antibody. Molecular dynamics (MD) simulations in explicit water of the T₁SSPSAD₇ peptide in both antibody free and bound states showed that the peptide's β-turn remained stable in the bound state but it was eliminated in the free state. Based on the observation that Thr₁ and Ala₆ residues made close contacts through their side chain, a new peptide mimotope is proposed: (S,S)-C₁SSPSCD₇. Thr₁ and Ala₆ residues have been mutated in Cys residues and a disulphide bond has been imposed. The new analogue has been simulated in both antibody bound and free states with MD in explicit water. It was found that the peptide remained in the stable β-turn conformation, both in complexed and free states. The difference in configurational entropy was estimated to be 0.15 kJ/K/mol. However, despite the structural similarity, the cyclic analogue lost more than 25% of its buried surface area contact with the antibody and a couple of critical hydrogen bond interactions were broken. It is concluded that design of cyclic analogues that mimic the bound conformation of peptides should be carefully performed and conformational ‘freezing’ does not necessarily guarantee better binding.     

Crystal structure of a human rhinovirus neutralizing antibody complexed with a peptide derived from viral capsid protein VP2.

The three-dimensional structure of the complex between the Fab fragment of an anti-human rhinovirus neutralizing antibody (8F5) and a cross-reactive synthetic peptide from the viral capsid protein VP2 has been determined at 2.5 A resolution by crystallographic methods. The refinement is presently at an R factor of 0.18 and the antigen-binding site and viral peptide are well defined. The peptide antigen adopts a compact fold by two tight turns and interacts through hydrogen bonds, some with ionic character, and van der Waals contacts with antibody residues from the six hypervariable loops as well as several framework amino acids. The conformation adopted by the peptide is closely related to the corresponding region of the viral protein VP2 on the surface of human rhinovirus 1A whose three-dimensional structure is known. Implications for the cross-reactivity between peptides and the viral capsid are discussed. The peptide-antibody interactions, together with the analysis of mutant viruses that escape neutralization by 8F5 suggest two different mechanisms for viral escape. The comparison between the complexed and uncomplexed antibody structures shows important conformational rearrangements, especially in the hypervariable loops of the heavy chain. Thus, it constitutes a clear example of the 'induced fit' molecular recognition mechanism.     

Solution structure of endothelin-3 determined using NMR spectroscopy.

The aqueous solution structure of the 21-residue vasoactive peptide hormone endothelin-3 has been determined using high-resolution NMR spectroscopy. A total of 177 proton-proton distance measurements and 5 chi 1 dihedral angle constraints derived from NMR spectra were used to calculate the structure using a combination of distance geometry and dynamical simulated annealing calculations. The calculations reveal a highly ordered, compact conformation in which a helical region extending from K9 to C15 lies in close apposition with the C-terminal hexapeptide; this interaction seems to be largely driven by hydrophobic interactions. Structure-activity studies are interpreted in terms of the conformational features of the calculated endothelin-3 structure.     

Atomic mapping of the interactions between the antiviral agent cyanovirin-N and oligomannosides by saturation-transfer difference NMR

The minimum oligosaccharide structure required for binding to the potent HIV-inactivating protein cyanovirin-N (CV-N) was determined by saturation-transfer difference (STD) NMR spectroscopy. Despite the low molecular mass of the protein (11 kDa), STD-NMR spectroscopy allowed the precise atomic mapping of the interactions between CV-N and various di- and trimannosides, substructures of Man-9, the predominant oligosaccharide on the HIV viral surface glycoprotein gp120. Contacts with mannosides containing the terminal Manalpha(1-->2)Manalpha unit of Man-9 were observed, while (1-->3)- and (1-6)-linked di- and trimannosides showed no interactions, demonstrating that the terminal Manalpha(1-->2)Manalpha structure plays a key role in the interaction. Precise epitope mapping revealed that, for Manalpha(1-->2)ManalphaOMe, Manalpha(1-->2)Manalpha(1-->3)ManalphaOMe, and Manalpha(1-->2)Manalpha(1-->6)ManalphaOMe, the protein is in close contact with H2, H3, and H4 of the nonreducing terminal mannose unit. In contrast, the STD-NMR spectrum of the CV-N/trisaccharide Manalpha(1-->2)Manalpha(1-->2)ManalphaOMe complex was markedly different, with resonances on all sugar units displaying equal enhancements, suggesting that CV-N is able to discriminate between the three structurally related trisaccharides.     

Comparative molecular dynamics analysis of tapasin-dependent and -independent MHC class I alleles

MHC class I molecules load antigenic peptides in the endoplasmic reticulum and present them at the cell surface. Efficiency of peptide loading depends on the class I allele and can involve interaction with tapasin and other proteins of the loading complex. Allele HLA-B*4402 (Asp at position 116) depends on tapasin for efficient peptide loading, whereas HLA-B*4405 (identical to B*4402 except for Tyr116) can efficiently load peptides in the absence of tapasin. Both alleles adopt very similar structures in the presence of the same peptide. Comparative unrestrained molecular dynamics simulations on the alpha(1)/alpha(2) peptide binding domains performed in the presence of bound peptides resulted in structures in close agreement with experiments for both alleles. In the absence of peptides, allele-specific conformational changes occurred in the first segment of the alpha(2)-helix that flanks the peptide C-terminal binding region (F-pocket) and contacts residue 116. This segment is also close to the proposed tapasin contact region. For B*4402, a shift toward an altered F-pocket structure deviating significantly from the bound form was observed. Subsequent free energy simulations on induced F-pocket opening in B*4402 confirmed a conformation that deviated significantly from the bound structure. For B*4405, a free energy minimum close to the bound structure was found. The simulations suggest that B*4405 has a greater tendency to adopt a peptide receptive conformation in the absence of peptide, allowing tapasin-independent peptide loading. A possible role of tapasin could be the stabilization of a peptide-receptive class I conformation for HLA-B*4402 and other tapasin-dependent alleles.     

1H NMR study of the interaction of bacteriophage lambda Cro protein with the OR3 operator. Evidence for a change of the conformation of the OR3 operator on binding.

The specific complex between the lambda phage OR3 operator and the Cro protein has been studied by proton NMR spectroscopy at 500 MHz. The DNA imino proton resonances of this complex have been assigned to specific base pairs using the known assignments of these resonances for the free operator. Increase of the protein/DNA ratio to complete saturation of the OR3 operator with the Cro protein made it possible to follow the shift changes of the resonances. Ambiguities were resolved by nuclear Overhauser effect measurements on the complex. The shifts of the imino proton resonance positions provide information on the changes induced in the conformation of the operator upon complex formation with a dimer of the Cro protein. The most striking shift occurs for the central (GC 9) base pair, which is known to have no direct contacts with the Cro protein. This shift may be induced by a bend in the OR3 operator DNA at the GC 9 base pair to accommodate the operator for the binding of the Cro protein dimer. The imino proton resonances of two additional base pairs can be observed in the complex, demonstrating an overall stabilization of the DNA structure by the binding of the Cro protein.