Biomedical subjects

Robert J Woods

Publications and source records attributed to Robert J Woods.

12 recordsLinked to original sources

PubMed2007

Fourier transform mass spectrometry to monitor hyaluronan-protein interactions: use of hydrogen/deuterium amide exchange.

The use of Fourier transform mass spectrometry (FTMS) to monitor noncovalent complex formation in the gas phase under native conditions between the Link module from human tumor necrosis factor stimulated gene-6 (Link_TSG6) and hyaluronan (HA) oligosaccharides is reported. In particular, a titration experiment with increasing concentrations of octasaccharide (HA(8)) to protein produced a noncovalent complex with 1:1 stoichiometry when the oligosaccharide was in molar excess. However, in the presence of a molar excess of tetrasaccharide (HA(4)) nearly all proteins and oligosaccharides were observed in their unbound charge states. These results are consistent with solution-phase properties for this interaction in which HA(8), but not HA(4), supports high affinity Link_TSG6 binding. Hydrogen/deuterium amide exchange mass spectrometry (H/D-EX MS) was also utilized to investigate the level of global deuterium incorporation, over time, for Link_TSG6 in both the absence and presence of HA(8). After dilution into quenching conditions, deuterium incorporation reached limiting asymptotic values of 37 and 26 deuterons for the free and bound protein at 240 and 480 min, respectively, indicating that the oligosaccharide interferes with amide exchange on binding. To detect sequence-specific deuterium incorporation, pepsin digestion of Link_TSG6 in both the absence and presence of HA(8) was performed. A level of deuterium incorporation of 10-30% was observed for peptides analyzed in free Link_TSG6. Interestingly, HA(8) blocked some sites of proteolysis in Link_TSG6 compared to the free protein. Molecular modeling indicated that amino acids proximal to the ligand correlated with regions of the protein that were resistant to enzymatic digestion. Of the peptides that could be analyzed by H/D-EX MS in the presence of the ligand, a 30-60% reduction in deuterium incorporation, relative to the free protein, was observed, even for those sequences not directly involved in HA binding. These results support the utility of FTMS as a method for the characterization of protein-carbohydrate interactions.

Binding Sites↗

PubMed2006

Structural basis of the inhibition of Golgi alpha-mannosidase II by mannostatin A and the role of the thiomethyl moiety in ligand-protein interactions.

The X-ray crystal structures of mannose trimming enzyme drosophila Golgi alpha-mannosidase II (dGMII) complexed with the inhibitors mannostatin A (1) and an N-benzyl analogue (2) have been determined. Molecular dynamics simulations and NMR studies have shown that the five-membered ring of mannostatin A is rather flexible occupying pseudorotational itineraries between 2T3 and 5E, and 2T3 and 4E. In the bound state, mannostatin A adopts a 2T1 twist envelope conformation, which is not significantly populated in solution. Possible conformations of the mannosyl oxacarbenium ion and an enzyme-linked intermediate have been compared to the conformation of mannostatin A in the cocrystal structure with dGMII. It has been found that mannostatin A best mimics the covalent linked mannosyl intermediate, which adopts a 1S5 skew boat conformation. The thiomethyl group, which is critical for high affinity, superimposes with the C-6 hydroxyl of the covalent linked intermediate. This functionality is able to make a number of additional polar and nonpolar interactions increasing the affinity for dGMII. Furthermore, the X-ray structures show that the environment surrounding the thiomethyl group of 1 is remarkably similar to the arrangements around the methionine residues in the protein. Collectively, our studies contradict the long held view that potent inhibitors of glycosidases must mimic an oxacarbenium ion like transition state.

Binding Sites↗

PubMed2006

Understanding the bacterial polysaccharide antigenicity of Streptococcus agalactiae versus Streptococcus pneumoniae.

Bacterial surface capsular polysaccharides (CPS) that are similar in carbohydrate sequence may differ markedly in immunogenicity and antigenicity. The structural origin of these phenomena is poorly understood. Such a case is presented by the Gram-positive bacteria Streptococcus agalactiae (Group B Streptococcus; GBS) type III (GBSIII) and Streptococcus pneumoniae (Pn) type 14 (Pn14), which share closely related CPS sequences. Nevertheless, antibodies (Abs) against GBSIII rarely cross-react with the CPS from Pn14. To establish the origin for the variation in CPS antigenicity, models for the immune complexes of CPS fragments from GBSIII and Pn14, with the variable fragment (Fv) of a GBS-specific mAb (mAb 1B1), are presented. The complexes are generated through a combination of comparative Ab modeling and automated ligand docking, followed by explicitly solvated 10-ns molecular dynamics simulations. The relationship between carbohydrate sequence and antigenicity is further quantified through the computation of interaction energies using the Molecular Mechanics-Generalized Born Surface Area (MM-GBSA) method, augmented by conformational entropy estimates. Despite the electrostatic differences between Pn14 and GBSIII CPS, analysis indicates that entropic penalties are primarily responsible for the loss of affinity of the highly flexible Pn14 CPS for mAb 1B1. The similarity of the solution conformation of the relatively rigid GBSIII CPS with that in the immune complex characterizes the previously undescribed 3D structure of the conformational epitope. The analysis provides a comprehensive interpretation for a large body of biochemical and immunological data related to Ab recognition of bacterial polysaccharides and should be applicable to other Ab-carbohydrate interactions.

Amino Acid Sequence↗

PubMed2005

Structural elucidation of type III group B Streptococcus capsular polysaccharide using molecular dynamics simulations: the role of sialic acid.

The conformational properties of the capsular polysaccharide (CPS) from group B Streptococcus serotype III (GBS III) are derived from 50 ns explicitly solvated molecular dynamics simulations of a 25-residue fragment of the CPS. The results from the simulations are shown to be consistent with experimental NMR homo- and heteronuclear J-coupling and NOE data for both the sialylated native CPS and for the chemically desialylated polysaccharide. A helical structure is predicted with a diameter of 29.3 A and a pitch 89.5 A, in which the sialylated side chains are arrayed on the exterior surface of the helix. The results provide an explanation for the observation that CPS antigenicity varies with carbohydrate chain length up to approximately 4 pentasaccharide repeat units. The conformation of the immunodominant region is established and shown to be independent of the presence of sialic acid. The data provide an explanation for the observation that the specificity of the determinant, associated with the major population of antibodies raised upon immunization of rabbits with GBS III, is dependent on the presence of sialic acid. In the sialylated native CPS, the antibody response is largely directed against the immunodominant core of the helix. From simulations of the desialylated CPS, a model emerges which suggests that the minor population of antibodies, whose determinant is not sialic acid dependent, recognizes the same immunodominant region, but that in the disordered CPS this region is not presented in a regular repeating motif.

Carbohydrate Conformation↗

PubMed2005

The Amber biomolecular simulation programs.

We describe the development, current features, and some directions for future development of the Amber package of computer programs. This package evolved from a program that was constructed in the late 1970s to do Assisted Model Building with Energy Refinement, and now contains a group of programs embodying a number of powerful tools of modern computational chemistry, focused on molecular dynamics and free energy calculations of proteins, nucleic acids, and carbohydrates.

Algorithms↗

PubMed2004

Inhibition of Golgi mannosidase II with mannostatin A analogues: synthesis, biological evaluation, and structure-activity relationship studies.

Mannostatin and aminocyclopentitetrol analogues with various substitutions at the amino function were synthesized. These compounds were tested as inhibitors of human Golgi and lysosomal alpha-mannosidases. Modification of the amine of mannostatin had only marginal effects, whereas similar modifications of aminocyclopentitetrol led to significantly improved inhibitors. Ab initio calculations and molecular docking studies were employed to rationalize the results. It was found that mannostatin and aminocyclopentitretrol could bind to Golgi alpha-mannosidase II in a similar mode to that of the known inhibitor swainsonine. However, due to the flexibility of the five-membered rings of these compounds, additional low-energy binding modes could be adopted. These binding modes may be relevant for the improved activities of the benzyl-substituted compounds. The thiomethyl moiety of mannostatin was predicted to make favorable hydrophobic interactions with Arg228 and Tyr727 that would possibly account for its greater inhibitory activity.

Amines↗

PubMed2004

Effects of glycosylation on peptide conformation: a synergistic experimental and computational study.

Asparagine-linked glycosylation, the co-translational covalent attachment of carbohydrates to asparagine side chains, has a major effect on the folding, stability, and function of many proteins. The carbohydrate composition in mature glycoproteins is heterogeneous due to modification of the initial oligosaccharide by glycosidases and glycosyltransferases during the glycoprotein passage through the endoplasmic reticulum and Golgi apparatus. Despite the diversity of carbohydrate structures, the core beta-D-(GlcNAc)(2) remains conserved in all N-linked glycoproteins. Previously, results from our laboratory showed that the molecular composition of the core disaccharide has a critical and unique conformational effect on the peptide backbone. Herein, we employ a synergistic experimental and computational approach to study the effect of the stereochemistry of the carbohydrate--peptide linkage on glycopeptide structure. A glycopeptide derived from a hemagglutinin protein fragment was synthesized, with the carbohydrate attached to the peptide with an alpha-linked stereochemistry. Computational and biophysical analyses reveal that the conformations of the peptide and alpha- and beta-linked glycopeptides are uniquely influenced by the attached saccharide. The value of computational approaches for probing the influence of attached saccharides on polypeptide conformation is highlighted.

Biophysical Phenomena↗

PubMed2004

The structure and conformational behavior of sulfonium salt glycosidase inhibitors in solution: a combined quantum mechanical NMR approach.

We present the conformational analysis of an inhibitor of alpha-mannosidase, based on a novel sulfonium salt structure (1) that mimics the mannosyl cation intermediate. Because of the number of possible isomeric structures for 1, as well as its complex molecular structure, traditional conformational analysis by NMR was not applicable. Instead, a single experimentally consistent structure was obtained from finite perturbation quantum mechanical calculations of the NMR J-couplings at the B3LYP/6-311G** level. Using a full relaxation matrix analysis, we showed that the quantum-predicted NMR structure was the only isomer that was consistent with the experimental NOE intensities. The results illustrate the potential for finite perturbation calculations to be useful in the analysis of complex charged molecules.

Enzyme Inhibitors↗

PubMed2004

A hydration study of (1-->4) and (1-->6) linked alpha-glucans by comparative 10 ns molecular dynamics simulations and 500-MHz NMR.

The hydration behavior of two model disaccharides, methyl-alpha-D-maltoside (1) and methyl-alpha-D-isomaltoside (2), has been investigated by a comparative 10 ns molecular dynamics study. The detailed hydration of the two disaccharides was described using three force fields especially developed for modeling of carbohydrates in explicit solvent. To validate the theoretical results the two compounds were synthesized and subjected to 500 MHz NMR spectroscopy, including pulsed field gradient diffusion measurements (1: 4.0. 10(-6) cm(2). s(-1); 2: 4.2. 10(-6) cm(2). s(-1)). In short, the older CHARMM-based force field exhibited a more structured carbohydrate-water interaction leading to better agreement with the diffusional properties of the two compounds, whereas especially the alpha-(1-->6) linkage and the primary hydroxyl groups were inaccurately modeled. In contrast, the new generation of the CHARMM-based force field (CSFF) and the most recent version of the AMBER-based force field (GLYCAM-2000a) exhibited less structured carbohydrate-water interactions with the result that the diffusional properties of the two disaccharides were underestimated, whereas the simulations of the alpha-(1-->6) linkage and the primary hydroxyl groups were significantly improved and in excellent agreement with homo- and heteronuclear coupling constants. The difference between the two classes of force field (more structured and less structured carbohydrate-water interaction) was underlined by calculation of the isotropic hydration as calculated by radial pair distributions. At one extreme, the radial O em leader O pair distribution function yielded a peak density of 2.3 times the bulk density in the first hydration shell when using the older CHARMM force field, whereas the maximum density observed in the GLYCAM force field was calculated to be 1.0, at the other extreme.

Algorithms↗

PubMed2003

Molecular dynamics simulations of galectin-1-oligosaccharide complexes reveal the molecular basis for ligand diversity.

Galectin-1 is a member of a protein family historically characterized by its ability to bind carbohydrates containing a terminal galactosyl residue. Galectin-1 is found in a variety of mammalian tissues as a homodimer of 14.5-kDa subunits. A number of developmental and regulatory processes have been attributed to the ability of galectin-1 to bind a variety of oligosaccharides containing the Gal-beta-(1,4)-GlcNAc (LacNAc(II)) sequence. To probe the origin of this permissive binding, solvated molecular dynamics (MD) simulations of several representative galectin-1-ligand complexes have been performed. Simulations of structurally defined complexes have validated the computational approach and expanded upon data obtained from X-ray crystallography and surface plasmon resonance measurements. The MD results indicate that a set of anchoring interactions between the galectin-1 carbohydrate recognition domain (CRD) and the LacNAc core are maintained for a diverse set of ligands and that substituents at the nonreducing terminus of the oligosaccharide extend into the remainder of a characteristic surface groove. The anionic nature of ligands exhibiting relatively high affinities for galectin-1 implicates electrostatic interactions in ligand selectivity, which is confirmed by a generalized Born analysis of the complexes. The results suggest that the search for a single endogenous ligand or function for this lectin may be inappropriate and instead support a more general role for galectin-1, in which the lectin is able to crosslink heterogeneous oligosaccharides displayed on a variety of cell surfaces. Such binding promiscuity provides an explanation for the variety of adhesion phenomena mediated by galectin-1.

Computer Simulation↗

PubMed2003

Quantifying the role of water in protein-carbohydrate interactions.

Water-mediated interactions play a key role in carbohydrate-lectin binding, where the interactions involve a conserved water that is separated from the bulk solvent and present a bridge between the side chains of the protein and the carbohydrate ligand. To apply quantum mechanical methods to examine the role of conserved waters, we present an analysis in which the relevant carbohydrate atoms are modeled by methanol, and in which the protein is replaced by a limited number of amino acid side chains. Clusters containing a conserved water and a representative amino acid fragment were also examined to determine the influence of amino acid side chains on interaction energies. To quantify the differential binding energies of methanol versus water, quantum mechanical calculations were performed at the B3LYP/6-311++G(3df,3pd)//B3LYP/6-31+G(d) level in which either a methanol molecule was bound to the conserved water (liganded state) or in which a water molecule replaces the methanol (unliganded state). Not surprisingly, the binding of a water to clusters containing charged amino acid side chains was more favorable by 1.55 to 7.23 kcal/mol than that for the binding of a water to the corresponding pure water clusters. In contrast, the binding energy of water to clusters containing polar-uncharged amino acid side chains ranged from 4.35 kcal/mol less favorable to 4.72 kcal/mol more favorable than for binding to the analogous pure water clusters. The overall trend for the binding of methanol versus water, in any of the clusters, favored methanol by an average value of 1.05 kcal/mol. To extend these studies to a complex between a protein (Concanavalin A) and its carbohydrate ligand, a cluster was examined that contained the side chains of three key amino acids, namely asparagine, aspartate, and arginine, as well as a key water molecule, arranged as in the X-ray diffraction structure of Con A. Again, using methanol as a model for the endogenous carbohydrate ligand, energies of -5.94 kcal/mol and -5.70 kcal/mol were obtained for the binding of methanol and water, respectively, to the Con A-water cluster. The extent to which cooperativity enhanced the binding energies has been quantified in terms of nonadditive three-body contributions. In general, the binding of water or methanol to neutral dimers formed cooperative clusters; in contrast, the cooperativity in charged clusters depended on the overall geometry as well as the charge.

Journal Article↗

PubMed2001

The xenograft antigen bound to Griffonia simplicifolia lectin 1-B(4). X-ray crystal structure of the complex and molecular dynamics characterization of the binding site.

The shortage of organs for transplantation into human patients continues to be a driving force behind research into the use of tissues from non-human donors, particularly pig. The primary barrier to such xenotransplantation is the reaction between natural antibodies present in humans and Old World monkeys and the Gal alpha(1-3)Gal epitope (xenograft antigen, xenoantigen) found on the cell surfaces of the donor organ. This hyperacute immune response leads ultimately to graft rejection. Because of its high specificity for the xenograft antigen, isolectin 1-B(4) from Griffonia simplicifolia (GS-1-B(4)) has been used as an immunodiagnostic reagent. Furthermore, haptens that inhibit natural antibodies also inhibit GS-1-B(4) from binding to the xenoantigen. Here we report the first x-ray crystal structure of the xenograft antigen bound to a protein (GS-1-B(4)). The three-dimensional structure was determined from orthorhombic crystals at a resolution of 2.3 A. To probe the influence of binding on ligand properties, we report also the results of molecular dynamics (MD) simulations on this complex as well as on the free ligand. The MD simulations were performed with the AMBER force-field for proteins augmented with the GLYCAM parameters for glycosides and glycoproteins. The simulations were performed for up to 10 ns in the presence of explicit solvent. Through comparison with MD simulations performed for the free ligand, it has been determined that GS-1-B(4) recognizes the lowest energy conformation of the disaccharide. In addition, the x-ray and modeling data provide clear explanations for the reported specificities of the GS-1-B(4) lectin. It is anticipated that a further understanding of the interactions involving the xenograft antigen will help in the development of therapeutic agents for application in the prevention of hyperacute xenograft rejection.

Animals↗