Enzymatic synthesis of blood group A and B trisaccharide analogues

Glycosyltransferases A and B utilize the donor substrates UDP-GalNAc and UDP-Gal, respectively, in the biosynthesis of the human blood group A and B trisaccharide antigens from the O(H)-acceptor substrates. These enzymes were cloned as synthetic genes and expressed in Escherichia coli, thereby generating large quantities of enzyme for donor specificity evaluations. The amino acid sequence of glycosyltransferase A only differs from glycosyltransferase B by four amino acids, and alteration of these four amino acid residues (Arg-176-->Gly, Gly-235-->Ser, Leu-266-->Met and Gly-268-->Ala) can change the donor substrate specificity from UDP-GalNAc to UDP-Gal. Crossovers in donor substrate specificity have been observed, i.e., the A transferase can utilize UDP-Gal and B transferase can utilize UDP-GalNAc donor substrates. We now report a unique donor specificity for each enzyme type. Only A transferase can utilize UDP-GlcNAc donor substrates synthesizing the blood group A trisaccharide analog alpha-D-Glcp-NAc-(1-->3)-[alpha-L-Fucp-(1-->2)]-beta-D-Galp-O-(CH2 )7CH3 (4). Recombinant blood group B was shown to use UDP-Glc donor substrates synthesizing blood group B trisaccharide analog alpha-D-Glcp-(1-->3)-[alpha-L-Fucp-(1-->2)]-beta-D-Galp-O-(CH2) 7CH3 (5). In addition, a true hybrid enzyme was constructed (Gly-235-->Ser, Leu-266-->Met) that could utilize both UDP-GlcNAc and UDP-Glc. Although the rate of transfer with UDP-GlcNAc by the A enzyme was 0.4% that of UDP-GalNAc and the rate of transfer with UDP-Glc by the B enzyme was 0.01% that of UDP-Gal, these cloned enzymes could be used for the enzymatic synthesis of blood group A and B trisaccharide analogs 4 and 5.     

Recognition of the blood group H type 2 trisaccharide epitope by 28 monoclonal antibodies and three lectins

The patterns of cross-reaction of 30 monoclonal antibodies and three lectins were determined by ELISA with 21 ABH, Ii or Lewis related synthetic oligosaccharides coupled to bovine serum albumin. At least seven main groups of cross-reactive patterns were identified among the antibodies, plus several intermediate patterns between two of the main antibody groups. The three lectins had different cross-reaction patterns,Galactia tenuiflora was different from all the antibodies,Ulex europaeus lectin 1 andLotus tetragonolobus were similar, but not identical to groups III and V of antibodies respectively. The anti-H antibodies cross-reacting with A type 2 gave similar agglutination scores with all the normal ABO erythrocytes, while the anti-H antibodies not cross-reacting with A type 2 reacted with different scores: O>A₂>A₂B>B>A₁>A₁B>O_h, suggesting that these antibodies react better with the free H epitopes and do not recognize the H in A or B epitopes. Based on the ELISA and agglutination results and the lowest energy conformations of each oligosaccharide obtained by computer modelling, the most probable oligosaccharide surface areas recognized by each antibody main group are illustrated.     

Molecular dynamics simulations and the conformational mobility of blood group oligosaccharides

Molecular dynamics simulations were carried out without explicit consideration of solvent to explore the conformational mobility of blood group A and H oligosaccharides. The potential energy force field of Rasmussen and co-workers was used with the CHARMM program on a number of disaccharide and trisaccharide models composed of fucose, galactose, glucose, N-acetyl glucosamine, and N-acetyl galactosamine chosen to represent various fragments of blood group oligosaccharides. In agreement with results of earlier studies, stable chair conformations were found for each pyranoside from which no transitions were detected in simulations as long as 800 ps. Exocyclic dihedral angles, including that of C5-C6, generally show numerous transitions on a time scale of approximately 5-30 ps. The dihedral angles of some but not all glycosidic linkages of blood group oligosaccharides show transitions on the time scale of 30-50 ps, implying that the extent of internal motion in blood group oligosaccharides depends strongly on linkage stereochemistry. For certain blood group A and H oligosaccharides that show limited internal motion in these simulations, we argue that the calculations are consistent with our previous analysis of 1H nuclear Overhauser enhancement (NOE) data that imply single conformations over a wide range of temperature and solvent conditions. While the trajectories are consistent with 13C T1 values that have been interpreted as indicating rigid conformations, measurements of 13C-NOE and T1 as a function of magnetic field strength are proposed as an improved method for experimental detection of the internal motion that is suggested for certain oligosaccharides in these simulations. The results of these simulations differ substantially from those of peptides of a similar molecular weight in that the oligosaccharides show much less internal motion.     

Molecular dynamics simulation of Lewis blood groups and related oligosaccharides.

Molecular dynamics simulations without explicit inclusion of solvent molecules have been performed to study the motions of Lewisa and Lewisb blood group oligosaccharides, and two blood group A tetrasaccharides having type I and type II core chains. The blood group H trisaccharide has also been studied and compared with the blood group A type II core chain. The potential energy surface developed by Rasmussen and co-workers was used with the molecular mechanics code CHARMM. The lowest energy minima of the component disaccharide fragments were obtained from conformational energy mapping. The lowest energy minima of these disaccharide fragments were used to build the tri- and tetrasaccharides that were further minimized before the actual heating/equilibration and dynamics simulations. The trajectories of the disaccharide fragments, e.g., Fuc alpha- (1----4)GlcNAc, Gal beta-(1----4)GlcNAc, etc., show transitions among various minima. However, the oligosaccharides were found to be dynamically stable and no transitions to other minimum energy conformations were observed in the time series of the glycosidic dihedral angles even during trajectories as long as 300 ps. The stable conformations of the glycosidic linkages in the oligosaccharides are not necessarily the same as the minimum energy conformation of the corresponding isolated disaccharides. The average fluctuations of the glycosidic angles in the oligosaccharides were well within the range of +/- 15 degrees. The results of these trajectory calculations were consistent with the relatively rigid single-conformation models derived for these oligosaccharides from 1H-nmr data.     

Molecular dynamics simulation of a phosphatidylglycerol membrane

Although molecular dynamics simulations are an important tool for studying membrane systems, relatively few simulations have used anionic lipids. This paper reports the first simulation of a pure phosphatidylglycerol (PG) bilayer. The properties of this equilibrated palmitoyloleoylphosphatidylglycerol membrane agree with experimental observations of PG membranes and with previous simulations of monolayers and mixed bilayers containing PG lipids. These simulations also provide interesting insights into hydrogen bonding interactions in PG membranes. This equilibrated membrane will be a useful starting point for simulations of membrane proteins interacting with PG lipids.     

Molecular dynamics simulation of a phospholipid membrane

We present the results of molecular dynamics (MD) simulations of a phospholipid membrane in water, including full atomic detail. The goal of the simulations was twofold: first we wanted to set up a simulation system which is able to reproduce experimental results and can serve as a model membrane in future simulations. This goal being reached it is then further possible to gain insight in to those properties that are experimentally more difficult to access. The system studied is dipalmitoylphosphatidylcholine/water, consisting of 5408 atoms. Using original force field parameters the membrane turned out to approach a gel-like state. With slight changes of the parameters, the system adopted a liquid-crystalline state. Separate 80 ps runs were performed on both the gel and liquid-crystalline systems. Comparison of MD results with reliable experimental data (bilayer repeat distance, surface area per lipid, tail order parameters, atom distributions) showed that our simulations, especially the one in the liquid-crystalline phase, can serve as a realistic model for a phospholipid membrane. Further analysis of the trajectories revealed valuable information on various properties. In the liquid-crystalline phase, the interface turns out to be quite diffuse, with water molecules penetrating into the bilayer to the position of the carbonyl groups. The 10–90% width of the interface turns out to be 1.3 nm and the width of the hydrocarbon interior 3.0 nm. The headgroup dipoles are oriented at a small angle with respect to the bilayer plane. The resulting charge distribution is almost completely cancelled by the water molecules. The electron density distribution shows a large dip in the middle of the membrane. In this part the tails are more flexible. The mean life time between dihedral transitions is 20 ps. The average number of gauche angles per tail is 3.5. The occurrence of kinks is not a significant feature.Abbreviations MD molecular dynamics - DPPC dipalmitoylphosphatidylcholine - SPC simple point charges - DPPE dipalmitoylphosphatidylethanolamine Correspondence to: H. J. C. Berendsen     

Molecular dynamics simulation study and hybrid pharmacophore model development in human LTA4H inhibitor design

Human leukotriene A4 hydrolase (hLTA4H) is a bi-functional enzyme catalyzes the hydrolase and aminopeptidase functions upon the fatty acid and peptide substrates, respectively, utilizing the same but overlapping binding site. Particularly the hydrolase function of this enzyme catalyzes the rate-limiting step of the leukotriene (LT) cascade that converts the LTA4 to LTB4. This product is a potent pro-inflammatory activator of inflammatory responses and thus blocking this conversion provides a valuable means to design anti-inflammatory agents. Four structurally very similar chemical compounds with highly different inhibitory profile towards the hydrolase function of hLTA4H were selected from the literature. Molecular dynamics (MD) simulations of the complexes of hLTA4H with these inhibitors were performed and the results have provided valuable information explaining the reasons for the differences in their biological activities. Binding mode analysis revealed that the additional thiophene moiety of most active inhibitor helps the pyrrolidine moiety to interact the most important R563 and K565 residues. The hLTA4H complexes with the most active compound and substrate were utilized in the development of hybrid pharmacophore models. These developed pharmacophore models were used in screening chemical databases in order to identify lead candidates to design potent hLTA4H inhibitors. Final evaluation based on molecular docking and electronic parameters has identified three compounds of diverse chemical scaffolds as potential leads to be used in novel and potent hLTA4H inhibitor design.     

Solution conformation of a cyclic neurokinin antagonist: A nmr and molecular dynamics study

The solution structure of a hexapeptide, cyclo(Gln-Trp-Phe-Gly-Leu-Met), which is a selective NK-2 antagonist, has been studied by a combination of two-dimensional nmr and molecular dynamics (MD) techniques. The simulation based on nmr and MD data resulted in the convergence to a family of structures. Free molecular dynamics for 50 ps in the presence of DMSO solvent molecules shows that the structure is energetically stable. One intramolecular hydrogen bond between the amide proton of Gin and the carbonyl oxygen of Gly was revealed. This result is consistent with the results from the measurement of the temperature coefficient of the amide protons. The extent of intermolecular hydrogen bonding between the amide protons of the peptide and DMSO was also revealed by the free MD simulation. The resulting structure of the cyclic peptide contains a variation type I′ β-turn in the Gly-Leu-Met-Gln segment. Comparison of the structure of this peptide with that of other NK-2 antagonist cyclic hexapeptides was made, and the activity of cyclic antagonists appears to be inversely related to the conformational rigidity of the cyclic peptides. © 1994 John Wiley & Sons, Inc.     

Molecular dynamics simulation and steered molecular dynamics simulation on irisin dimers

Irisin is found closely associated with promoting the browning of beige fat cells in white adipose tissue. The crystal structure reveals that irisin forms a continuous inter-subunit β-sheet dimer. Here, molecular dynamics (MD) simulation and steered molecular dynamics (SMD) simulation were performed to investigate the dissociation process and the intricate interactions between the two irisin monomers. In the process of MD, the interactions between the monomers were roughly analyzed through the average numbers of both hydrophobic contacts and H-bonds. Then, SMD was performed to investigate the accurate interaction energy between the monomers. By the analysis of dissociation energy, the van der Waals (vdW) force was identified as the major energy to maintain the dimer structure, which also verified the results of MD simulation. Meanwhile, 11 essential residues were discovered by the magnitude of rupture force during dissociation. Among them, residues Arg75, Glu79, Ile77, Ala88, and Trp90 were reported in a previous study using the method of mutagenesis and size exclusion chromatography, and several new important residues (Arg72, Leu74, Phe76, Gln78, Val80, and Asp91) were also identified. Interestingly, the new important residues that we discovered and the important residues that were reported are located in the opposite side of the β-sheet of the dimer.     

Molecular dynamics simulation study of a pulmonary surfactant film interacting with a carbonaceous nanoparticle

This article reports an all-atom molecular dynamics simulation to study a model pulmonary surfactant film interacting with a carbonaceous nanoparticle. The pulmonary surfactant is modeled as a dipalmitoylphosphatidylcholine monolayer with a peptide consisting of the first 25 residues from surfactant protein B. The nanoparticle model with a chemical formula C₁₈₈H₅₃ was generated using a computational code for combustion conditions. The nanoparticle has a carbon cage structure reminiscent of the buckyballs with open ends. A series of molecular-scale structural and dynamical properties of the surfactant film in the absence and presence of nanoparticle are analyzed, including radial distribution functions, mean-square displacements of lipids and nanoparticle, chain tilt angle, and the surfactant protein B peptide helix tilt angle. The results show that the nanoparticle affects the structure and packing of the lipids and peptide in the film, and it appears that the nanoparticle and peptide repel each other. The ability of the nanoparticle to translocate the surfactant film is one of the most important predictions of this study. The potential of mean force for dragging the particle through the film provides such information. The reported potential of mean force suggests that the nanoparticle can easily penetrate the monolayer but further translocation to the water phase is energetically prohibitive. The implication is that nanoparticles can interact with the lung surfactant, as supported by recent experimental data by Bakshi et al.     

Molecular dynamics simulation of a high-affinity antibody-protein complex

One nanosecond molecular dynamic (MD) simulation of anti-hen egg white lysozyme (HEL) antibody HyHEL63 (HH63) complexed with HEL reveals rigid and flexible regions of the HH63 binding site. Fifty conformations, extracted from the MD trajectory at regular time intervals were superimposed on HH63-HEL X-ray crystal structure, and the root mean squared deviations (RMSDs) and deviations in Calpha atom positions between the X-ray structure and the MD conformer were measured. Residue positions showing the large deviations in both light chain and heavy chain of the antibody were same in all the MD conformers. The residue positions showing smallest deviations were same for all the conformers in the case of light chain, whereas relatively variable in the heavy chain. Positions of large and small deviations fell in the complementarity determining regions (CDRs), for both heavy and light chains. The larger deviations were in CDR-2 of light and CDR-1 of heavy chain. Smaller deviations were in CDR-3 of light and CDR-2 and CDR-3 of heavy chains. The large and small deviating regions highlight flexible and rigid regions of HH63 binding site and suggest a mosaic binding mechanism, including both "induced fit" and preconfigured "lock-and-key" type of binding. Combined "induced fit" and "lock-and-key" binding would be a better definition for the formation of large complexes, which bury larger surface area on binding, as in the case of antibody-HEL complex. We further show that flexible regions, comprising mostly charged and polar residues, form intermolecular interactions with HEL, whereas rigid regions do not. Electrostatic complementarity between HH63 and HEL also imply optimized binding affinity. Flexible and rigid regions of a high-affinity antibody are selected during the affinity maturation of the antibody and have specific functional significance. The functional importance of local inherently flexible regions is to establish intermolecular contacts or they play a key role in molecular recognition, whereas local rigid regions provide the structural framework.     

Molecular dynamics simulation study on the structures of fascin mutants

Fascin is a filamentous actin (F-actin) bundling protein, which cross-links F-actin into bundles and becomes an important component of filopodia on the cell surface. Fascin is overexpressed in many types of cancers. The mutation of fascin affects its ability to bind to F-actin and the progress of cancer. In this paper, we have studied the effects of residues of K22, K41, K43, K241, K358, K399, and K471 using molecular dynamics (MD) simulation. For the strong-effect residues, that is, K22, K41, K43, K358, and K471, our results show that the mutation of K to A leads to large values of root mean square fluctuation (RMSF) around the mutated residues, indicating those residues are important for the flexibility and thermal stability. On the other hand, based on residue cross-correlation analysis, alanine mutations of these residues reinforce the correlation between residues. Together with the RMSF data, the local flexibility is extended to the entire protein by the strong correlations to influence the dynamics and function of fascin. By contrast, for the mutants of K241A and K399A those do not affect the function of fascin, the RMSF data do not show significant differences compared with wild-type fascin. These findings are in a good agreement with experimental studies.     

Molecular dynamics simulation study of tubulin dimer interaction with cytostatics

Colchicine, podophylotoxin, and indibulin are natural cytostatics that are used in the treatment of neoplasms. However, application of the compounds is restricted due to their high toxicity and low specificity. Computational experiments modeling tubulin interactions with the cytostatics seem a promising approach to design new analogues of the above-mentioned drugs with higher cytostatic activity and lower toxicity. Therefore, the CHARMM software was used to examine the macromolecules using molecular dynamics and mechanics methods. Particularly, a procedure was applied according to which molecules of each studied cytostatics were placed at several various random positions around the predicted binding site on tubulin. As a result, cytostatic binding regions were identified on the tubulin molecule. It was shown that, during the interaction, structural alterations occurred in these regions that may be responsible for tubulin polymerization. Thus, alterations have been revealed for the first time in the structure of tubulin in the regions of cytostatic binding that can substantially affect its function.     

Molecular dynamics simulation study of probe diffusion in liquid n-alkanes

We performed molecular dynamics simulations for the probe diffusion and friction dynamics of Lennard-Jones (LJ) particles modelled for methyl yellow (MY) in liquid n-alkanes of C₁₂–C₂₀₀ at temperatures of 318, 418, 518 and 618 K. Two LJ particles are chosen: MY1 with a mass of 114 g/mol, LJ parameters of σ = 4.0 Å and ? = 0.4 kJ/mol, and MY2 with a mass of 225 g/mol, σ = 6.0 Å and ? = 0.6 kJ/mol. We observed a clear transition in the power law dependence of MY2 diffusion on the molecular weight of n-alkanes at lower temperatures of 318 and 418 K. The sharp transitions occur near n-dotriacontane (C₃₂). However, no such transition is found for MY1 at all the temperatures and for MY2 at higher temperatures of 518 and 618 K. We also calculated the friction constants of both MY probe molecules in liquid n-alkanes. For the larger probe molecule (MY2), at lower temperatures, a large deviation of slope from the linear dependence of the friction of MY2 on the chain length of n-alkane is observed, which indicates a large reduction of friction in longer chains when compared with the shorter chains, enhancing the diffusion of the probe molecules (MY2).     

Molecular dynamics simulation study of interaction between a class IIa bacteriocin and its immunity protein

Molecular dynamics (MD) simulations of carnobacteriocin B2 (CbnB2), a structurally well-characterized class IIa bacteriocin, and its immunity protein (ImB2) in lipid bilayer environment have been conducted to explore the interaction between them. Six 30-ns simulations were conducted in DPPC or POPG bilayer systems. In these simulations, ImB2 was placed in the aqueous layer with different orientations facing CbnB2 to sample all the faces of ImB2. The MD results indicate that (i) while CbnB2 remained embedded in the bilayer, it tends to move toward the interface, and (ii) the presence of CbnB2 in the DPPC bilayer attracts ImB2 toward the bilayer. In one of the orientations in DPPC bilayer system (simulation 1), ImB2 penetrates the bilayer and interacts with CbnB2 by ion-pair interaction. At several instances toward the later half of the simulation (15-30 ns), ImB2 and CbnB2 were found to form salt-bridge between Arg95 of ImB2 and Glu24 of CbnB2. Simulation in POPG bilayer displayed strong interaction between the positively charged ImB2 and the negatively charged polar head groups of the POPG molecules at the lipid-water interface. However, ImB2 was not able to penetrate the bilayer thereby preventing any interaction between ImB2 and CbnB2.     

Fine specificities of two lectins from Cymbosema roseum seeds: a lectin specific for high-mannose oligosaccharides and a lectin specific for blood group H type II trisaccharide

The legume species of Cymbosema roseum of Diocleinae subtribe produce at least two different seed lectins. The present study demonstrates that C. roseum lectin I (CRL I) binds with high affinity to the "core" trimannoside of N-linked oligosaccharides. Cymbosema roseum lectin II (CRL II), on the other hand, binds with high affinity to the blood group H trisaccharide (Fucα1,2Galα1-4GlcNAc-). Thermodynamic and hemagglutination inhibition studies reveal the fine binding specificities of the two lectins. Data obtained with a complete set of monodeoxy analogs of the core trimannoside indicate that CRL I recognizes the 3-, 4- and 6-hydroxyl groups of the α(1,6) Man residue, the 3- and 4-hydroxyl group of the α(1,3) Man residue and the 2- and 4-hydroxyl groups of the central Man residue of the trimannoside. CRL I possesses enhanced affinities for the Man5 oligomannose glycan and a biantennary complex glycan as well as glycoproteins containing high-mannose glycans. On the other hand, CRL II distinguishes the blood group H type II epitope from the Lewis(x), Lewis(y), Lewis(a) and Lewis(b) epitopes. CRL II also distinguishes between blood group H type II and type I trisaccharides. CRL I and CRL II, respectively, possess differences in fine specificities when compared with other reported mannose and fucose recognizing lectins. This is the first report of a mannose-specific lectin (CRL I) and a blood group H type II-specific lectin (CRL II) from seeds of a member of the Diocleinae subtribe.     

Molecular dynamics simulation study of the vanillate transport channel of Opdk

Most water-soluble and small molecules are taken up by substrate-specific channels belonging to the Gram-negative bacteria. The protein named OpdK, a member of OprD family, plays an important role in transporting the vanillate as the only carbon source of Pseudomonas aeruginosa. P. aeruginosa infections can be serious due to the high intrinsic antibiotic resistance owing to the present of the OprD family. We applied standard molecular dynamics, steered molecular dynamics and umbrella sampling, to investigate the thermodynamics of vanillate passing through the pore of OpdK protein at physiological temperature. The results indicate that hydrogen bonds of vanillate-L3 (L3: Gly92-Gln111) and hydrophobic interactions of vanillate-L7 (Gly252-Asn278) are crucial to the transport of vanillate. Compared to L7, L3 can hardly change the shape of the pore, but its amino acids can effectively affect the transport process. The important role of charged residues in the barrel of the protein for the substrate transport has been proved in the experiment researches and our simulations also determinate that these residues may prevent the vanillate from entering the cell. These results provide detailed information that will facilitate the development of effective drugs.     

Molecular dynamics simulation of a dipalmitoylphosphatidylcholine bilayer with NaCl

Molecular dynamics simulations are performed on two hydrated dipalmitoylphosphatidylcholine bilayer systems: one with pure water and one with added NaCl. Due to the rugged nature of the membrane/electrolyte interface, ion binding to the membrane surface is characterized by the loss of ion hydration. Using this structural characterization, binding of Na(+) and Cl(-) ions to the membrane is observed, although the binding of Cl(-) is seen to be slightly weaker than that of Na(+). Dehydration is seen to occur to a different extent for each type of ion. In addition, the excess binding of Na(+) gives rise to a net positive surface charge density just outside the bilayer. The positive density produces a positive electrostatic potential in this region, whereas the system without salt shows an electrostatic potential of zero.