Conformation of methyl beta-lactoside bound to the ricin B-chain: interpretation of transferred nuclear Overhauser effects facilitated by spin simulation and selective deuteration

Spin simulation and selective deuteration have been used to aid in the interpretation of 1D transferred nuclear Overhauser effect (TRNOE) NMR experiments on ricin B-chain/ligand systems. Application of these methods has revealed a change in the conformation of deuterated methyl beta-lactoside upon binding to the ricin B-chain which results in a slight change in glycosidic torsional angels which appear to dominate in the solution conformation. The combination of simulation and experiment also shows an important sensitivity of TRNOE magnitudes to dissociation rate constants and available spin-diffusion pathways for the ricin B-chain/ligand systems under study. The sensitivity to dissociation rates allows determination of rate constants for methyl beta-lactoside and methyl beta-galactoside of 50 and 300 s-1, respectively.     

Significant conformational changes in an antigenic carbohydrate epitope upon binding to a monoclonal antibody.

Transferred nuclear Overhauser enhancement spectroscopy (TRNOE) was used to observe changes in a ligand's conformation upon binding to its specific antibody. The ligands studied were methyl O-beta-D-galactopyranosyl(1----6)-4-deoxy-4-fluoro-beta-D-galactopyra nos ide (me4FGal2) and its selectively deuteriated analogue, methyl O-beta-D-galactopyranosyl(1----6)-4-deoxy-2-deuterio-4-fluoro-beta -D- galactopyranoside (me4F2dGal2). The monoclonal antibody was mouse IgA X24. The solution conformation of the free ligand me4F2dGal2 was inferred from measurements of vicinal 1H-1H coupling constants, long-range 1H-13C coupling constants, and NOE cross-peak intensities. For free ligand, both galactosyl residues adopt a regular chair conformation, but the NMR spectra are incompatible with a single unique conformation of the glycosidic linkage. Analysis of 1H-1H and 1H-13C constants indicates that the major conformer has an extended conformation: phi = -120 degrees; psi = 180 degrees; and omega = 75 degrees. TRNOE measurements on me4FGal2 and me4F2dGal2 in the presence of the specific antibody indicate that the pyranose ring pucker of each galactose ring remains unchanged, but rotations about the glycosidic linkage occur upon binding to X24. Computer calculations indicate that there are two sets of torsion angles that satisfy the observed NMR constraints, namely, phi = -152 +/- 9 degrees; psi = -128 +/- 7 degrees; and omega = -158 +/- 6 degrees; and a conformer with phi = -53 +/- 6 degrees; psi = 154 +/- 10 degrees; and omega = -173 +/- 6 degrees. Neither conformation is similar to any of the observed conformations of the free disaccharide.(ABSTRACT TRUNCATED AT 250 WORDS)     

The galactose-binding sites of the cytotoxic lectin ricin can be chemically blocked in high yield with reactive ligands prepared by chemical modification of glycopeptides containing triantennary N-linked oligosaccharides.

A glycopeptide containing a triantennary N-linked oligosaccharide from fetuin was modified by a series of chemical and enzymic reactions to afford a reagent that contained a terminal residue of 6-(N-methylamino)-6-deoxy-D-galactose on one branch of the triantennary structure and terminal galactose residues on the other two branches. Binding assays and gel filtration experiments showed that this modified glycopeptide could bind to the sugar-binding sites of ricin. The ligand was activated at the 6-(N-methylamino)-6-deoxy-D-galactose residue by reaction with cyanuric chloride. The resulting dichlorotriazine derivative of the ligand reacts with ricin, forming a stable covalent linkage. The reaction was confined to the B-chain and was inhibited by lactose. Bovine serum albumin and ovalbumin were not modified by the activated ligand under similar conditions, and we conclude, therefore, that the reaction of the ligand with ricin B-chain was dependent upon specific binding to sugar-binding sites. Ricin that had its galactose-binding sites blocked by the covalent reaction with the activated ligand was purified by affinity chromatography. The major species in this fraction was found to contain 2 covalently linked ligands per ricin B-chain, while a minor species contained 3 ligands per B-chain. The cytotoxicity of blocked ricin was at least 1000-fold less than that of native ricin for cultured cells in vitro, even though the activity of the A-chain in a cell-free system was equal to that from native ricin. Modified ricin that contained only 1 covalently linked ligand was also purified. This fraction retained an ability to bind to galactose affinity columns, although with a lower affinity than ricin, and was only 5- to 20-fold less cytotoxic than native ricin.     

C-type lectin-like carbohydrate recognition of the hemolytic lectin CEL-III containing ricin-type -trefoil folds

CEL-III is a Ca(2+)-dependent hemolytic lectin, isolated from the marine invertebrate Cucumaria echinata. The three-dimensional structure of CEL-III/GalNAc and CEL-III/methyl alpha-galactoside complexes was solved by x-ray crystallographic analysis. In these complexes, five carbohydrate molecules were found to be bound to two carbohydrate-binding domains (domains 1 and 2) located in the N-terminal 2/3 portion of the polypeptide and that contained beta-trefoil folds similar to ricin B-chain. The 3-OH and 4-OH of bound carbohydrate molecules were coordinated with Ca(2+) located at the subdomains 1alpha, 1gamma, 2alpha, 2beta, and 2gamma, simultaneously forming hydrogen bond networks with nearby amino acid side chains, which is similar to carbohydrate binding in C-type lectins. The binding of carbohydrates was further stabilized by aromatic amino acid residues, such as tyrosine and tryptophan, through a stacking interaction with the hydrophobic face of carbohydrates. The importance of amino acid residues in the carbohydrate-binding sites was confirmed by the mutational analyses. The orientation of bound GalNAc and methyl alpha-galactoside was similar to the galactose moiety of lactose bound to the carbohydrate-binding site of the ricin B-chain, although the ricin B-chain does not require Ca(2+) ions for carbohydrate binding. The binding of the carbohydrates induced local structural changes in carbohydrate-binding sites in subdomains 2alpha and 2beta. Binding of GalNAc also induced a slight change in the main chain structure of domain 3, which could be related to the conformational change upon binding of specific carbohydrates to induce oligomerization of the protein.     

Identification of the tryptophan residue located at the low-affinity saccharide binding site of ricin D

The saccharide binding ability of the low affinity (LA-) binding site of ricin D was abrogated by N-bromosuccinimide (NBS)-oxidation, while in the presence of lactose the number of tryptophan residues eventually oxidized decreased by 1 mol/mol and the saccharide binding ability was retained (Hatakeyama et al., (1986) J. Biochem. 99, 1049-1056). Based on these findings, the tryptophan residue located at the LA-binding site of ricin D was identified. Two derivatives of ricin D which were modified with NBS in the presence and absence of lactose were separated into their constituent polypeptide chains (A- and B-chains), respectively. The modified tryptophan residue or residues was/were found to be contained in the B-chain, but not in the A-chain. From lysylendopeptidase and chymotryptic digests, peptides containing oxidized tryptophan residues were isolated by gel filtration on Bio-Gel P-30 and HPLC. Analysis of the peptides containing oxidized tryptophan revealed that three tryptophan residues at positions 37, 93, and 160 on the B-chain were oxidized in the inactive derivative of ricin D, in which the saccharide binding ability of the LA-binding site was abrogated by NBS-oxidation. On the other hand, the modified residues were determined to be tryptophans at positions 93 and 160 in the active derivative of ricin D which was modified in the presence of lactose, indicating that upon binding with lactose, the tryptophan residue at position 37 of the B-chain was protected from NBS-oxidation. From these results, it is suggested that tryptophan at position 37 on the B-chain is the essential residue for saccharide binding at the LA-binding site of ricin D.     

The role of binding ligand in toxic hybrid proteins: a comparison of EGF-ricin, EGF-ricin A-chain, and ricin

To analyze the influence of ricin B-chain on the toxicity of hybrid-protein conjugates, the rate of cellular uptake of conjugates, and the rate at which ricin A-chain (RTA) is delivered to the cytoplasm, we have constructed toxic hybrid proteins consisting of epidermal growth factor (EGF) coupled in disulfide linkage either to ricin or to RTA. EGF-ricin is no more toxic on A431 cells than EGF-RTA. The two conjugates demonstrate similar kinetics of cellular uptake (defined as antibody irreversible toxicity). EGF-RTA and EGF-ricin, like ricin, required a 2-2 1/2 hour period at 37 degrees before the onset of protein synthesis inhibition occurred. Our results suggest that RTA determines the processes which carry it, either in conjugate or toxin, from the plasma membrane binding site to the cytoplasm following endocytosis, and the ricin B chain is not required for these processes.     

Studies on the molecular recognition of synthetic methyl beta-lactoside analogs by ricin, a cytotoxic plant lectin

The binding of methyl beta-lactoside and of all possible monodeoxy derivatives of methyl beta-lactoside to the galactose-specific highly cytotoxin lectin ricin, has been investigated. The distribution of low-energy conformers of the disaccharide structures has been first determined using molecular-mechanics calculations and high-resolution NMR spectroscopy. The nuclear Overhauser enhancements and specific deshieldings observed are in agreement with a similar distribution of low-energy conformers for all studied compounds which may be described by a major conformer defined by phi (H1'-C1'-O1'-C4) and psi (C1'-O1'-C4-H4) torsion angles of 49 degrees and 5 degrees, respectively, with contribution of conformers with angles phi/psi 24 degrees/-59 degrees, 22 degrees/-32 degrees and 6 degrees/-44 degrees. Assuming that the disaccharides bind to the lectin in these preferred conformations, the apparent dissociation constants for the ricin-disaccharide complexes have been interpreted in terms of specific polar and nonpolar interactions. In agreement with X-ray data, the hydroxyl groups at positions 3, 4 and 6 of the beta-D-galactopyranose moiety appear as key polar groups in the interaction with ricin. These results are in contrast to previous results which have established that position 6 is not involved in lectin binding. An important nonpolar interaction involving position 3 of the beta-D-glucopyranose moiety, seems to be operative. The distribution of low-energy conformers of these disaccharide structures permits this interaction to take place with the hydroxyl group at this position intramolecularly bonded, thus rendering this region of the molecule more lipophylic in character for acceptance into nonpolar regions of the combining site.     

The specific cytotoxicity of immunoconjugates containing blocked ricin is dependent on the residual binding capacity of blocked ricin: evidence that the membrane binding and A-chain translocation activities of ricin cannot be separated.

Recently we have developed blocked ricin, a derivative of native ricin in which the galactose-binding sites of the B-chain are blocked by covalent modification with affinity ligands. This modification impedes the binding function of the B-chain, while sparing its ability to facilitate the entry of the toxic subunit of ricin, the A-chain, into the cytoplasm. Immunotoxins prepared with blocked ricin approach the cytotoxic potency of native ricin with antibody-dependent specificity. Here we report that the high cytotoxic potency of these immunoconjugates, which is attributed to the preserved translocation function of the ricin B-chain, is dependent on the minimal residual lectin activity of blocked ricin. Our findings support the notion that two functions of ricin, membrane binding and translocation, cannot be separated.     

Effect of pH on the conformation and stability of the plant toxin ricin

Intrinsic protein fluorescence of native plant toxin and its isolated subunits were studied. The effect of pH was studied on: conformation of ricin and its A- and R-chains; affinity to galactose of ricin and its binding B-subunit. At two pH 5.0 and 7.0, the structural stability of toxin and subunits was estimated according to denaturational action of guanidine chloride. It was demonstrated that position of maximum and the spectrum shape of fluorescence of native toxin and catalytical A-subunit insignificantly depends on pH in the range of 3-8, whereas sufficient changes of the separameters for the ricin B-chain reveal structural transition at pH 4-5. The affinity of galactose of ricin and its isolated B-chain depends on pH, the maximal binding is observed at pH 7. The structural stability of ricin and isolated chains significantly differs at pH 7.5 and 5.0, thus the structure stability of ricin and A-chain increases, and that of B-chain decreases at pH 5.0.     

Interaction of ricin and its constituent polypeptides with dipalmitoylphosphatidylcholine vesicles

The interaction of ricin and of its constituent polypeptides, the A- and B-chain, with dipalmitoylphosphatidylcholine (DPPC) vesicles was investigated. The A- and B-chain were individually associated with DPPC vesicles, although the intact ricin was not associated. The maximum binding and association constants were evaluated to be 154 micrograms per mg of DPPC and Ka = 2.30 X 10(5) M-1 for the A-chain, and 87 micrograms per mg of DPPC and Ka = 14.5 X 10(5) M-1 for the B-chain, respectively. The A-chain could induce the phase transition release of carboxyfluorescein from DPPC vesicles to a greater extent than the B-chain, whereas the release induced by the intact ricin was negligible. The evidence indicated that the hydrophobic regions on the A-chain and on the B-chain were buried inside when the two chains constituted the intact ricin molecule through one interchain disulfide bond, and that the A-chain caused perturbation of the DPPC bilayer at the phase transition temperature with consequent leakage of carboxyfluorescein.     

Conformation of MgATP bound to nucleotidyl and phosphoryl transfer enzymes 1H-transferred NOE measurements on complexes of methionyl tRNA synthetase and pyruvate kinase.

The conformations of MgATP bound to a nucleotidyl transfer enzyme, methionyl tRNA synthetase and a phosphoryl transfer enzyme, pyruvate kinase, were studied by transferred NOE (TRNOE) measurements in 1H NMR. The experiments were performed on D2O solutions at 276 MHz and 300 MHz, and 10 degrees C in the presence of approximately a tenfold excess of substrate over the enzyme (sites). Selective inversion of chosen resonances was accomplished with an appropriately tailored DANTE sequence consisting of 100 phase-alternating hard 1.8 degree pulses. NOE measurements were made in terms of difference spectra (with and without inversion) at 6-8 delay times ranging from 10-500 ms following the DANTE sequence. A full complement of ten NOE build-up curves obtained for each enzyme complex was analyzed by using the complete relaxation-matrix method (which includes all the non-exchangeable protons in MgATP) suitably modified to include exchange between bound and free substrate. Molecular mechanics computations were used to examine the energetic implications of the NOE-determined structure. The final structures obtained for MgATP bound to the two enzymes were very similar to each other, with a 3'-endo sugar pucker and an anti conformation with a glycosidic torsional angle (O'4-C'1-N9-C8) of 39 degrees +/- 4 degrees. Both enzymes contain multiple binding sites for MgATP and hence the structure obtained in each case represents an average due to chemical exchange. However, TRNOE experiments performed on a tryptic fragment of methionyl tRNA synthetase which has a single MgATP binding site, show that the same structure fits these measurements as well. This evidence, coupled with the striking similarity of the structures deduced, for the two enzyme complexes, and the reciprocal sixth-power dependence of NOE on interproton distance, strongly suggests that the conformations at the individual binding sites of both the enzymes are virtually identical. This conclusion is in contrast with multiple conformations of MgATP bound to pyruvate kinase, proposed by Rosevear, P.R., Fox, T.L. & Mildvan, A.S. (1987) Biochemistry 26, 3487-3493.     

Effects of Iodination on Cytoagglutination by and Toxicity of Ricinus communis Lectins

Iodinations of two Ricinus communis lectins, ricin D and hemagglutinin (CBH), with potassium iodide at pH 7.0 and 0°C led to inactivation of the cytoagglutinating activity on sarcoma 180 ascites tumor cells as well as the toxicity to HeLa cells of ricin D, whereas the cytoagglutinating activity of CBH was affected slightly. In the presence of lactose, which binds to ricin D, one tyrosyl residue in the B-chain of ricin D was protected from iodination and 40% of the cytoagglutinating activity was retained. This protection against iodination was not observed in the presence of glucose, which does not bind to ricin D. This suggested that the protected tyrosyl residue in the B-chain of ricin D may be situated at or near the saccharide binding site and directly involved in the binding to the saccharide moieties of the cellular receptors.

Adsorption of the iodinated ricin D to Sepharose 4B indicated that one of the two saccharide binding sites in ricin D is still intact and participates in the binding to saccharide: ricin D was altered from divalent to monovalent by the iodination.

We found from binding experiments with ¹²⁵I-labeled iodinated ricin D to HeLa cells, that the low toxicity of the iodinated ricin D may be attributed mainly to its decreased internalization into the cells and that the divalent binding of ricin D to the cellular receptors is important for this internalization.     

Kinetics of binding of the toxic lectins abrin and ricin to surface receptors of human cells.

Kinetic parameters of the interaction of the toxic lectins abrin and ricin with human erythrocytes and HeLa cells have been measured. The binding of 125I-labeled abrin and ricin to human erythrocytes and to HeLa cells at 37 degrees was maximal around pH 7, whereas at 0 degrees the binding was similar over a broad pH range. The binding occurred at similar rates at 0 degrees and 37 degrees with rate constants in the range 0.9 to 3.0 X 10(5) M-1 s-1. The dissociation was strongly temperature-dependent with rate constants in the range 3.4 to 45 X 10(-4) s-1 at 0 degrees and 3.9 to 18 X 10(-3) s-1 at 37 degrees. The presence of unlabeled lectins as well as lactose increased the rate of dissociation. The association constants measured at equilibrium or calculated from the rate constants were between 0.64 X 10(8) M-1 and 8.2 X 10(8) M-1 for abrus lectins, and between 8.0 X 10(6) M-1 and 4.2 X 10(8) M-1 for ricinus lectins. The association constants for the toxins were lower at 37 degrees than at 0 degrees. Isolated ricin B chain appeared to bind with similar affinity as intact ricin. The number of binding sites was estimated to be 2 to 3 X 10(6) per erythrocyte and 1 to 3 X 10(7) per HeLa cell. The binding sites of HeLa cells all displayed a uniform affinity towards abrin and ricin, both at 0 degrees and at 37 degrees. The same was the case with the binding sites of erythrocytes at 0 degrees. However, the data indicated that at 20 degrees erythrocytes possessed binding sites with two different affinities. Only a fraction of the cell-bound toxin appeared to be irreversibly bound and could not be removed by washing with 0.1 M lactose. The fraction of the total amount of bound toxin which became irreversibly bound to HeLa cells was for both toxins about 2 X 10(-3)/min at 37 degrees, whereas no toxin was irreversibly bound at 0 degrees. In the case of erythrocytes no toxin became irreversibly bound, either at 0 degrees or 37 degrees, indicating that the toxins are unable to penetrate into these cells.     

The lower cytotoxicity of cinnamomin (a type II RIP) is due to its B-chain

The cytotoxicity of intact cinnamomin (a type II ribosome-inactivating protein, RIP) and the RNA N-glycosidase activity of cinnamomin A-chain have been studied and compared with those of ricin. Cinnamomin A-chain exhibits a similar RNA N-glycosidase activity in inhibiting in vitro protein synthesis compared with that of ricin, whereas the cytotoxicity to BA/F3beta cells of intact cinnamomin is markedly lower than intact ricin. In order to demonstrate that it is the B-chains of the two RIPs that bear the difference in cytotoxicity, two hybrid RIPs are prepared from the purified A-/B-chains of cinnamomin and ricin by the disulfide exchange reaction. It has been found that hybrid RIP constructed from cinnamomin A-chain and ricin B-chain is more toxic to BA/F3beta cells than the native cinnamomin, and equivalent to the native ricin. However, the cytotoxicity to BA/F3beta cells of the hybrid RIP constructed from the ricin A-chain and cinnamomin B-chain is lower than ricin, equivalent to the native cinnamomin. Furthermore, the bound amounts of two B-chains on the cell surface are determined by the method of direct cellular ELISA and Scatchard analysis of the binding of the two B-chains indicates that cinnamomin and ricin share similar binding sites with different affinity.     

Estimation of the amount of internalized ricin that reaches the trans- Golgi network

We have used a protocol for internalization of ricin, a ligand binding to plasma membrane glycoproteins and glycolipids with terminal galactosyl residues, and infection with the vesicular stomatitis virus ts 045 mutant in BHK-21 cells to determine whether internalized plasma membrane molecules tagged by ricin reach distinct compartments of the biosynthetic-exocytic pathway. At 39.5 degrees C newly synthesized G protein of ts 045 was largely prevented from leaving the endoplasmic reticulum. At the same temperature ricin was endocytosed and reached, in addition to endosomes and lysosomes, elements of the Golgi complex. When the temperature was lowered to 19.5 degrees C, no more ricin was delivered to the Golgi complex, but now G protein accumulated in the Golgi stacks and the trans-Golgi network (TGN). Double-labeling immunogold cytochemistry on ultracryosections was used to detect G protein and ricin simultaneously. These data, combined with stereological and biochemical methods, showed that approximately 5% of the total amount of ricin within the cells, corresponding to 6-8 X 10(4) molecules per cell, colocalized with G protein in the Golgi complex after 60 min at 39.5 degrees C. Of this amount approximately 70-80% was present in the TGN. Since most of the ricin molecules remain bound to their binding sites at the low pH prevailing in compartments of the endocytic pathway, the results indicate that a fraction of the internalized plasma membrane molecules with terminal galactose are not recycled directly from endosomes or delivered to lysosomes, but are routed to the Golgi complex. Also, the results presented here, in combination with other recent studies on ricin internalization, suggest that translocation of the toxic ricin A-chain to the cytosol occurs in the TGN.     

The binding of the B-chain of ricin to Burkitt lymphoma cells. A new approach to ligand-receptor interaction studies

It is shown that conformational changes of receptor proteins brought about by binding of a ligand induce changes in the lipid environment of the receptor that can be monitored by fluorescent lipid probes. On this basis a new approach to studies of ligand-receptor binding is proposed. Using the interaction of the ricin B-chain with Burkitt lymphoma cells as an example and fluorescent labelled sphingomyelin as a probe, the ligand-induced changes of fluorescence anisotropy were shown to be concentration-dependent and to permit determination of the binding constant and the number of receptor-binding sites. The method was found to be specific and highly sensitive, allowing detection of the action of one RB molecule per cell. Scatchard analysis of the binding of 125I-RB demonstrated the presence on the cell surface of two binding sites with Kd approximately 10(-10) and approximately 10(-8) M, respectively. Only the high-affinity sites were detected by the fluorescence technique. Saturation of these sites resulted in maximum inhibition of protein synthesis.     

Nuclear-magnetic-resonance studies on the conformation of membrane-bound alpha-mating factor. Transferred nuclear Overhauser effect analysis

The C-H proton resonances of alpha-mating factor, yeast pheromone, in 2H2O solution were assigned. The phase transition temperature of perdeuterated dipalmitoylglycerophosphocholine (suspension) was found to be 35.5 degrees C. In the presence of vesicles of this phospholipid, the exchange broadening and transferred nuclear Overhauser effect (TRNOE) of peptide proton resonances (at 50 degrees C) were analyzed. The mode of binding of this peptide with the phospholipid bilayer was elucidated. The N-terminal nine residues (Trp1-Gly9) are tightly bound to the bilayer, while the C-terminal four residues (Gln10-Tyr13) are left free in aqueous phase. This is consistent with the previous observation that the C-terminal three residues (Pro11-Tyr13) are not essential for the activity of this pheromone [Masui, Y. et al. (1977) Biochem. Biophys. Res. Commun. 78, 534-538]. Furthermore, from the TRNOE analyses, the conformation of the membrane-bound N-terminal part of alpha-mating factor was elucidated; the residues Trp1-Gln5 form a compact helical structure while the residues Lys7-Gly9 form an extended structure. A similar TRNOE was also observed for an active decapeptide analog Trp1-Gln10. This confirms the previous conclusion that the physiological activities of this pheromone and analog peptides are correlated with the conformations of membrane-bound peptide molecules [Higashijima, T. et al. (1983) FEBS Lett. 159, 229-232].     

Formation of γ-BTC in Flooded Rice Field Soils Treated with γ-BHC

The amino acids of the B-chains of two abrins (designated as abrin-a and abrin-b) from the seeds of Abrus precatorius have been sequenced. The sequence of the B-chain of abrin-a was solved by analysis of peptides derived by enzymatic digestions with trypsin, Iysylendopeptidase, and chymotrypsin, as well as by chemical cleavage with cyanogen bromide. The sequence of the B-chain of abrin-b was analyzed by sequence analysis of tryptic peptides and comparing these sequences with those of corresponding peptides of the B-chain of abrin-a. The B-chains of abrin-a and abrin-b consist of 268 amino acid residues and share 256 identical residues. Comparison of their sequences with that of the ricin B-chain shows that 60% of the residues of both abrin B-chains are identical to those of the ricin B-chain and that two saccharide-binding sites in ricin B-chain identified by a crystallographic study are highly conserved in both abrin B-chains.     

Conformation of a trimannoside bound to mannose-binding protein by nuclear magnetic resonance and molecular dynamics simulations

A model of the carbohydrate recognition domain of the serum form of mannose-binding protein (MBP) from rat complexed with methyl 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside is presented. Allowed conformations for the bound sugar were derived from simulated annealing protocols incorporating distance restraints computed from transferred NOESY spectra. The resulting sugar conformations were then modeled into the MBP binding site, and these models of the complex were refined using molecular dynamics (MD) simulations in the presence of solvent water. These studies indicate that only one of the two major conformations of the alpha(1-->6) linkage found in solution is significantly populated in the bound state (omega = 60 degrees ), whereas the alpha(1-->3) linkage samples at least two states, similar to its behavior in free solution. The bound conformation allows direct hydrogen bonds to form between the sugar and K182 of MBP, in addition to other water-mediated hydrogen bonds. Estimates of binding constants of candidate complexes based on changes in solvent-accessible surface areas upon binding support the NMR and MD results. These estimates further suggest that the enthalpic gains of the additional sugar-MBP interactions in a trisaccharide as opposed to a monosaccharide are offset by entropic penalties, offering an explanation for previous binding data.     

Synthesis, crystal structure, and molecular conformation of N-Boc-L-Phe-dehydro-Leu-L-Val-OCH3

The peptide N-Boc-L-Phe-dehydro-Leu-L-Val-OCH3 was synthesized by the usual workup procedure and finally by coupling the N-Boc-L-Phe-dehydro-Leu-OH to valine methyl ester. It was crystallized from its solution in methanol-water mixture at 4 degrees C. The crystals belong to the triclinic space group P1 with a = 5.972(5) A, b = 9.455(6) A, c = 13.101(6) A, alpha = 103.00(4) degrees, beta = 97.14(5) degrees, gamma = 102.86(5) degrees, V = 690.8(8) A, Z = 1, dm = 1.179(5) Mg m-3 and dc = 1.177(5) Mg m-3. The structure was determined by direct methods using SHELXS86. It was refined by block-diagonal least-squares procedure to an R value of 0.060 for 1674 observed reflections. The C alpha 2-C beta 2 distance of 1.323(9) A in dehydro-Leu is an appropriate double bond length. The bond angle C alpha-C beta-C gamma in the dehydro-Leu residue is 129.4(8) degrees. The peptide backbone torsion angles are theta 1 = -168.6(6) degrees, omega 0 = 170.0(6) degrees, phi 1 = -44.5(9) degrees, psi 1 = 134.5(6) degrees, omega 1 = 177.3(6) degrees, phi 2 = 54.5(9) degrees, psi 2 = 31.1(10) degrees, omega 2 = 171.7(6) degrees, phi 3 = 51.9(8) degrees, psi T3 = 139.0(6) degrees, theta T = -175.7(6) degrees. These values show that the backbone adopts a beta-turn II conformation. As a result of beta-turn, an intramolecular hydrogen bond is formed between the oxygen of the ith residue and NH of the (i + 3)th residue at a distance of 3.134(6) A.(ABSTRACT TRUNCATED AT 250 WORDS)