A new strategy for identification of N-glycosylated proteins and unambiguous assignment of their glycosylation sites using HILIC enrichment and partial deglycosylation

Characterization of glycoproteins using mass spectrometry ranges from determination of carbohydrate-protein linkages to the full characterization of all glycan structures attached to each glycosylation site. In a novel approach to identify N-glycosylation sites in complex biological samples, we performed an enrichment of glycosylated peptides through hydrophilic interaction liquid chromatography (HILIC) followed by partial deglycosylation using a combination of endo-beta-N-acetylglucosaminidases (EC 3.2.1.96). After hydrolysis with these enzymes, a single N-acetylglucosamine (GlcNAc) residue remains linked to the asparagine residue. The removal of the major part of the glycan simplifies the MS/MS fragment ion spectra of glycopeptides, while the remaining GlcNAc residue enables unambiguous assignment of the glycosylation site together with the amino acid sequence. We first tested our approach on a mixture of known glycoproteins, and subsequently the method was applied to samples of human plasma obtained by lectin chromatography followed by 1D gel-electrophoresis for determination of 62 glycosylation sites in 37 glycoproteins.     

Exploiting sequence and structure homologs to identify protein-protein binding sites

A rapid increase in the number of experimentally derived three-dimensional structures provides an opportunity to better understand and subsequently predict protein-protein interactions. In this study, structurally conserved residues were derived from multiple structure alignments of the individual components of known complexes and the assigned conservation score was weighted based on the crystallographic B factor to account for the structural flexibility that will result in a poor alignment. Sequence profile and accessible surface area information was then combined with the conservation score to predict protein-protein binding sites using a Support Vector Machine (SVM). The incorporation of the conservation score significantly improved the performance of the SVM. About 52% of the binding sites were precisely predicted (greater than 70% of the residues in the site were identified); 77% of the binding sites were correctly predicted (greater than 50% of the residues in the site were identified), and 21% of the binding sites were partially covered by the predicted residues (some residues were identified). The results support the hypothesis that in many cases protein interfaces require some residues to provide rigidity to minimize the entropic cost upon complex formation.     

The action of inhibitors of sugar transport phlorizin, phloretin and cytochalasin B in model systems

Phlorizin, phloretin and cytochalasin B are known to be specific sugar transport inhibitors. A study was made of their effects on the carbohydrate-protein interaction in solution as a model system for examining the initial steps of sugar membrane transport. Glycogen precipitation by concanavalin A is inhibited only by alpha-methylmannoside, whereas both phlorizin and phloretin inhibit interactions between hexokinase and glucose, and between glucose-6-phosphate dehydrogenase and glucose-6-phosphate. Cytochalasin B was found to exert no effect on both the concanavalin A--glycogen interaction and the enzyme reactions investigated. The data obtained in the model system examination may suggest that the sites of glucose and cytochalasin binding are, respectively, spatially uncoupled.     

Scoring optimisation of unbound protein-protein docking including protein binding site predictions

The prediction of the structure of the protein-protein complex is of great importance to better understand molecular recognition processes. During systematic protein-protein docking, the surface of a protein molecule is scanned for putative binding sites of a partner protein. The possibility to include external data based on either experiments or bioinformatic predictions on putative binding sites during docking has been systematically explored. The external data were included during docking with a coarse-grained protein model and on the basis of force field weights to bias the docking search towards a predicted or known binding region. The approach was tested on a large set of protein partners in unbound conformations. The significant improvement of the docking performance was found if reliable data on the native binding sites were available. This was possible even if data for single key amino acids at a binding interface are included. In case of binding site predictions with limited accuracy, only modest improvement compared with unbiased docking was found. The optimisation of the protocol to bias the search towards predicted binding sites was found to further improve the docking performance resulting in approximately 40% acceptable solutions within the top 10 docking predictions compared with 22% in case of unbiased docking of unbound protein structures.     

Multiple e-Pharmacophore modeling to identify a single molecule that could target both streptomycin and paromomycin binding sites for 30S ribosomal subunit inhibition

The bacterial ribosome is an established target for anti-bacterial therapy since decades. Several inhibitors have already been developed targeting both defined subunits (50S and 30S) of the ribosome. Aminoglycosides and tetracyclines are two classes of antibiotics that bind to the 30S ribosomal subunit. These inhibitors can target multiple active sites on ribosome that have a complex structure. To screen putative inhibitors against 30S subunit of the ribosome, the crystal structures in complex with various known inhibitors were analyzed using pharmacophore modeling approach. Multiple active sites were considered for building energy-based three-dimensional (3D) pharmacophore models. The generated models were validated using enrichment factor on decoy data-set. Virtual screening was performed using the developed 3D pharmacophore models and molecular interaction towards the 30S ribosomal unit was analyzed using the hits obtained for each pharmacophore model. The hits that were common to both streptomycin and paromomycin binding sites were identified. Further, to predict the activity of these hits a robust 2D-QSAR model with good predictive ability was developed using 16 streptomycin analogs. Hence, the developed models were able to identify novel inhibitors that are capable of binding to multiple active sites present on 30S ribosomal subunit.     

Automatic generation of 3D motifs for classification of protein binding sites

Background  

Since many of the new protein structures delivered by high-throughput processes do not have any known function, there is a need for structure-based prediction of protein function. Protein 3D structures can be clustered according to their fold or secondary structures to produce classes of some functional significance. A recent alternative has been to detect specific 3D motifs which are often associated to active sites. Unfortunately, there are very few known 3D motifs, which are usually the result of a manual process, compared to the number of sequential motifs already known. In this paper, we report a method to automatically generate 3D motifs of protein structure binding sites based on consensus atom positions and evaluate it on a set of adenine based ligands.     

Structural glycobiology: a game of snakes and ladders

Oligo- and polysaccharides are infamous for being extremely flexible molecules, populating a series of well-defined rotational isomeric states under physiological conditions. Characterization of this heterogeneous conformational ensemble has been a major obstacle impeding high-resolution structure determination of carbohydrates and acting as a bottleneck in the effort to understand the relationship between the carbohydrate structure and function. This challenge has compelled the field to develop and apply theoretical and experimental methods that can explore conformational ensembles by both capturing and deconvoluting the structural and dynamic properties of carbohydrates. This review focuses on computational approaches that have been successfully used in combination with experiment to detail the three-dimensional structure of carbohydrates in a solution and in a complex with proteins. In addition, emerging experimental techniques for three-dimensional structural characterization of carbohydrate-protein complexes and future challenges in the field of structural glycobiology are discussed. The review is divided into five sections: (1) The complexity and plasticity of carbohydrates, (2) Predicting carbohydrate-protein interactions, (3) Calculating relative and absolute binding free energies for carbohydrate-protein complexes, (4) Emerging and evolving techniques for experimental characterization of carbohydrate-protein structures, and (5) Current challenges in structural glycoscience.     

EmPLiCS: an empirical approach for structure-based design of natural peptide drugs

The computer implementation of a peptide drug-design strategy has been developed. The system is named EmPLiCS (Empirical Peptide Ligand Construction System) according to the strategy of the system, which searches for peptide-ligand structures by referring to empirical rules that are derived from known protein 3D structures. The system was tested on several known peptide-protein complexes. The results demonstrated the ability of this system to detect key residues of peptides that are crucial for interaction with their specific proteins. The system also showed the ability to detect the main chain trace of these peptides. Some of the main chain atoms were detected even though the complete primary structures were not reproduced, suggesting that main chain structure is important in peptide-protein recognition. The results of the present study demonstrated that the empirical rules-based system can generate significant information for use in the design of natural peptide drugs.     

Sugar Chips immobilized with synthetic sulfated disaccharides of heparin/heparan sulfate partial structure

Carbohydrate chip technology has a great potential for the high-throughput evaluation of carbohydrate-protein interactions. Herein, we report syntheses of novel sulfated oligosaccharides possessing heparin and heparan sulfate partial disaccharide structures, their immobilization on gold-coated chips to prepare array-type Sugar Chips, and evaluation of binding potencies of proteins by surface plasmon resonance (SPR) imaging technology. Sulfated oligosaccharides were efficiently synthesized from glucosamine and uronic acid moieties. Synthesized sulfated oligosaccharides were then easily immobilized on gold-coated chips using previously reported methods. The effectiveness of this analytical method was confirmed in binding experiments between the chips and heparin binding proteins, fibronectin and recombinant human von Willebrand factor A1 domain (rh-vWf-A1), where specific partial structures of heparin or heparan sulfate responsible for binding were identified.     

A new protein binding pocket similarity measure based on comparison of clouds of atoms in 3D: application to ligand prediction

Background  

Predicting which molecules can bind to a given binding site of a protein with known 3D structure is important to decipher the protein function, and useful in drug design. A classical assumption in structural biology is that proteins with similar 3D structures have related molecular functions, and therefore may bind similar ligands. However, proteins that do not display any overall sequence or structure similarity may also bind similar ligands if they contain similar binding sites. Quantitatively assessing the similarity between binding sites may therefore be useful to propose new ligands for a given pocket, based on those known for similar pockets.     

Solution structure of a cyanovirin-N:Man alpha 1-2Man alpha complex: structural basis for high-affinity carbohydrate-mediated binding to gp120

BACKGROUND: Cyanovirin-N (CVN) is a novel, 11 kDa cyanobacterial protein that potently inhibits viral entry by diverse strains of HIV through high-affinity carbohydrate-mediated interactions with the viral envelope glycoprotein gp120. CVN contains two symmetry-related carbohydrate binding sites of differing affinities that selectively bind to Man(8) D1D3 and Man(9) with nanomolar affinities, the carbohydrates that also mediate CVN:gp120 binding. High-resolution structural studies of CVN in complex with a representative oligosaccharide are desirable for understanding the structural basis for this unprecedented specificity. RESULTS: We have determined by multidimensional heteronuclear NMR spectroscopy the three-dimensional solution structure of CVN in complex with two equivalents of the disaccharide Manalpha1-2Manalpha, a high-affinity ligand which represents the terminal-accessible disaccharide present in Man(8) D1D3 and Man(9). The structure reveals that the bound disaccharide adopts the stacked conformation, thereby explaining the selectivity for Man(8) D1D3 and Man(9) over other oligomannose structures, and presents two novel carbohydrate binding sites that account for the differing affinities of the two sites. The high-affinity site comprises a deep pocket that nearly envelops the disaccharide, while the lower-affinity site comprises a semicircular cleft that partially surrounds the disaccharide. The approximately 40 A spacing of the two binding sites provides a simple model for CVN:gp120 binding. CONCLUSIONS: The CVN:Manalpha1-2Manalpha complex provides the first high-resolution structure of a mannose-specific protein-carbohydrate complex with nanomolar affinity and presents a new carbohydrate binding motif, as well as a new class of carbohydrate binding protein, that facilitates divalent binding via a monomeric protein.     

Three-Dimensional Models of Immune Cell Surface Proteins and Identification of Binding Sites

The extracellular regions of many cell surface proteins of the immune system contain distinct domains that may be linked in many different ways and are often only loosely tethered to the transmembrane segment. In efforts to identify regions critical for binding, molecular models of these domains are used to select residues for mutagenesis and to map binding sites. Many immune cell surface proteins belong to protein superfamilies and display only limited sequence identity compared to proteins of known three-dimensional (3D) structure, often 30% or less. Therefore, detailed 3D structures are difficult to predict, and structure-based sequence analysis and model assessment are particularly important components of the model building process. In some cases, experimentally determined structures have made it possible to assess the accuracy of predictions, which illustrates the opportunities and shortcomings of the approach. Herein the model-based identification of binding sites in cell surface proteins is described and representative examples are discussed.     

Large-scale preparation and characterization of N-linked glycopeptides from bovine fetuin.

A preparative scheme has been developed to purify asialo-glycopeptides from each of the three N-linkage sites of bovine fetuin, allowing the isolation of 100-mumols quantities of asialo-glycopeptides from 20 g of fetuin. The procedure yields seven asialo-glycopeptides which were determined to be 95% homogenous in peptide and oligosaccharide structure. The isolation scheme uses two high-capacity reverse-phase eluant systems. The primary RP-HPLC purification performed with boric acid buffered to pH 7 with triethylamine resolved sialylated tryptic glycopeptides simultaneously on the basis of glycosylation site and degree of sialylation. A second RP-HPLC purification was performed eluting isocratically with dilute phosphoric acid which resolved residual peptide and oligosaccharide heterogeneity from asialo-glycopeptides containing short peptides. Structural characterization of the products was performed utilizing 400-MHz proton NMR spectroscopy and amino acid and monosaccharide analysis. The glycopeptides contain two previously identified variant triantennary oligosaccharides which possess either Gal beta(1----4) or Gal beta(1----3) linkages to N-acetylglucosamine at one terminal branch or a biantennary oligosaccharide. These compounds should prove to be invaluable in studying carbohydrate-protein interactions, such as binding by the Gal/GalNAc lectin of mammalian hepatocytes, in the detailed three-dimensional structural analysis of complex oligosaccharides, and as purified substrates for the study of the action of glycoconjugate-modifying enzymes.     

Colloidal gold-labeled insulin complex

Summary Biologically active insulin gold complex was used as an ultrastructural marker to study insulin binding sites, uptake, and internalization in isolated rat adipocytes. The preparation conditions for monodispersed particles, ca. 16 nm in diameter and loaded with approximately 100 insulin molecules, are reported. The complex is stable for at least six weeks. Single particles or small clusters were scattered across the cell membrane. The distribution of unbound receptors seemed to be independent of the extensive system of pre-existing surface connected vesicles in adipocytes. The uptake of particles took place predominantly via non-coated pinocytotic invaginations; clathrin-coated pits did not seem to be important for this process. Lysosome-like structures contained aggregates of 10–15 particles. These data suggest that insulin gold complex is a useful marker for the specific labeling of insulin binding sites.Supported by Deutsche Forschungsgemeinschaft D3 SFB 87     

Spermadhesin AQN1 is a candidate receptor molecule involved in the formation of the oviductal sperm reservoir in the pig

Sperm are stored in the isthmic region of the oviduct under conditions that maintain viability and suppress early capacitation steps until ovulation occurs. The initial contact between sperm and oviductal epithelium is mediated by carbohydrate-protein interactions. In the pig, the carbohydrate recognition system has been shown to involve oligomannosyl structures. The spermadhesins AWN and AQN1 are the dominant porcine carbohydrate-binding sperm proteins. The objective of this study was to demonstrate that AQN1 contributes to sperm binding to the oviductal epithelium. AQN1 showed a broad carbohydrate-binding pattern as it recognizes both alpha- and beta-linked galactose as well as Manalpha1-3(Manalpha1-6)Man structures, whereas AWN bound only the galactose species. Binding of ejaculated sperm to oviductal epithelium was inhibited by addition of AQN1 but not by AWN. Mannose-binding sites were localized over the rostral region of the sperm head. Flow cytometry showed that, under capacitating conditions, the population of live sperm was shifted within 30 min toward an increase in the proportion of cells with low mannose- and high galactose-binding. The loss of mannose-binding sites was accompanied by the loss of AQN1 in sperm extracts and the significant reduction in the sperm-oviduct binding. The oviductal epithelium was shown by GNA-lectin histochemistry and by SDS-PAGE and lectin blotting of the apical membrane fraction to express mannose components that could be recognized by AQN1. These results demonstrate that the sperm lectin AQN1 fulfils the criteria for an oviduct receptor in the pig and may play a role in the formation of the oviductal sperm reservoir.     

Fluorine-18 radiolabelling, biodistribution studies and preliminary PET evaluation of a new memantine derivative for imaging the NMDA receptor

A synthetic method has been established for preparing [18F]1-amino-3-fluoromethyl-5-methyl-adamantane ([18F]AFA). Biodistribution of the radiotracer in mice showed high brain uptake. The peak uptake (3.7% I.D/g organ) for the brain occurred at 30 min after injection. Accumulation of radioactivity in mouse brain was consistent with the known distribution of the NMDA receptors. The binding of [18F]AFA to the phencyclidine (PCP) binding sites of the NMDA receptor complex and the sigma recognition sites in a Rhesus monkey was also examined using positron emission tomography (PET). The regional brain distribution of [18F]AFA was changed by memantine and by (+)-MK-801, indicating competition for the same binding sites. Treatment with haloperidol caused a marked reduction of radioactivity uptake in all the brain regions examined. (-)-Butaclamol, which has pharmacological specificity for sigma sites, did not have any significant effects.     

A novel sulfated structure in the carbohydrate-protein linkage region isolated from porcine intestinal heparin.

A preparation of porcine stage 14 intestinal heparin, which contains Ser as a predominant amino acid, was used for isolation of the carbohydrate-protein linkage region of heparin. Two glycoserines were isolated in a molar ratio of 96:4 after an exhaustive digestion with a mixture of bacterial heparinase and heparitinases. Their structures were determined by composition analysis, heparitinase digestion, co-chromatography with an authentic glycoserine on high performance liquid chromatography, and by 500-MHz one- and two-dimensional 1H NMR spectroscopy. The structure of the major one is delta GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser and that of the minor is delta GlcA beta 1-4GlcNAc(6-O-sulfate) alpha 1-4GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser. The novel 6-O-sulfated GlcNAc residue was demonstrated to occur in the vicinity of the carbohydrate-protein linkage region. The Gal residues were nonsulfated, in contrast to the sulfated Gal structures recently discovered in the carbohydrate-protein linkage region of chondroitin sulfate proteoglycans. The structural features are discussed in relation to biosynthetic mechanisms of the heparin glycosaminoglycans.     

The carbohydrate-binding site in galectin-3 is preorganized to recognize a sugarlike framework of oxygens: ultra-high-resolution structures and water dynamics

The recognition of carbohydrates by proteins is a fundamental aspect of communication within and between living cells. Understanding the molecular basis of carbohydrate-protein interactions is a prerequisite for the rational design of synthetic ligands. Here we report the high- to ultra-high-resolution crystal structures of the carbohydrate recognition domain of galectin-3 (Gal3C) in the ligand-free state (1.08 ? at 100 K, 1.25 ? at 298 K) and in complex with lactose (0.86 ?) or glycerol (0.9 ?). These structures reveal striking similarities in the positions of water and carbohydrate oxygen atoms in all three states, indicating that the binding site of Gal3C is preorganized to coordinate oxygen atoms in an arrangement that is nearly optimal for the recognition of β-galactosides. Deuterium nuclear magnetic resonance (NMR) relaxation dispersion experiments and molecular dynamics simulations demonstrate that all water molecules in the lactose-binding site exchange with bulk water on a time scale of nanoseconds or shorter. Nevertheless, molecular dynamics simulations identify transient water binding at sites that agree well with those observed by crystallography, indicating that the energy landscape of the binding site is maintained in solution. All heavy atoms of glycerol are positioned like the corresponding atoms of lactose in the Gal3C complexes. However, binding of glycerol to Gal3C is insignificant in solution at room temperature, as monitored by NMR spectroscopy or isothermal titration calorimetry under conditions where lactose binding is readily detected. These observations make a case for protein cryo-crystallography as a valuable screening method in fragment-based drug discovery and further suggest that identification of water sites might inform inhibitor design.     

Use of a structural alphabet to find compatible folds for amino acid sequences

The structural annotation of proteins with no detectable homologs of known 3D structure identified using sequence‐search methods is a major challenge today. We propose an original method that computes the conditional probabilities for the amino‐acid sequence of a protein to fit to known protein 3D structures using a structural alphabet, known as “Protein Blocks” (PBs). PBs constitute a library of 16 local structural prototypes that approximate every part of protein backbone structures. It is used to encode 3D protein structures into 1D PB sequences and to capture sequence to structure relationships. Our method relies on amino acid occurrence matrices, one for each PB, to score global and local threading of query amino acid sequences to protein folds encoded into PB sequences. It does not use any information from residue contacts or sequence‐search methods or explicit incorporation of hydrophobic effect. The performance of the method was assessed with independent test datasets derived from SCOP 1.75A. With a Z‐score cutoff that achieved 95% specificity (i.e., less than 5% false positives), global and local threading showed sensitivity of 64.1% and 34.2%, respectively. We further tested its performance on 57 difficult CASP10 targets that had no known homologs in PDB: 38 compatible templates were identified by our approach and 66% of these hits yielded correctly predicted structures. This method scales‐up well and offers promising perspectives for structural annotations at genomic level. It has been implemented in the form of a web‐server that is freely available at http://www.bo‐protscience.fr/forsa .     

Specific protection of DNA by distamycin A, netropsin and bis-netropsins against the action of DNAse I

Interaction of netropsin, distamycin A and a number of bis-netropsins with DNA fragments of definite nucleotide sequence was studied by footprinting technique. The nuclease protection experiments were made at fixed DNA concentration and varying ligand concentrations. The affinity of ligand for a DNA site was estimated from measurements of ligand concentration that causes 50% protection of the DNA site. Distribution pattern of the protected and unprotected regions along the DNA fragment was compared with the theoretically expected arrangement of the ligand along the same DNA. The comparison led us to the following conclusions: 1. Footprinting experiments show that at high levels of binding the arrangement of netropsin molecules along the DNA corresponds closely to the distribution pattern expected from theoretical calculations based on the known geometry of netropsin--DNA complex. However, the observed differences in the affinity of netropsin for various DNA sequences is markedly greater than that expected from theoretical calculations. 2. Netropsin exhibits a greater selectivity of binding than that expected for a ligand with three specific reaction centers associated with the antibiotic amide groups. It binds preferentially to DNA regions containing four or more successive AT pairs. Among 13 putative binding sites for netropsin with four or more successive AT pairs there are 11 strong binding sites and two weaker sites which are occupied at 2 D/P less than or equal to 1/9 and 2 D/P = 1/4, respectively. 3. The extent of specificity manifested by distamycin A is comparable to that shown by netropsin although the molecule of distamycin A contains four rather than three amide groups. At high levels of binding distamycin A occupies the same binding sites on DNA as netropsin does. 4. The binding specificity of bis-netropsins is greater than that of netropsin. Bis-netropsins can bind to DNA in such a way that the two netropsin-like fragments are implicated in specific interaction with DNA base pairs. However, the apparent affinity of bis-netropsins estimated from footprinting experiments is comparable with that of netropsin for the same DNA region. 5. At high levels of binding bis-netropsins and distamycin A (but not netropsin) can occupy any potential site on DNA irrespectively of the DNA sequence. 6. Complex formation with netropsin increases sensitivity to DNase I at certain DNA sites along with the protection effect observed at neighboring sites.