Native crystal structure of a nitric oxide-releasing lectin from the seeds of Canavalia maritima

Here, we report the crystallographic study of a lectin from Canavalia maritima seeds (ConM) and its relaxant activity on vascular smooth muscle, to provide new insights into the understanding of structure/function relationships of this class of proteins. ConM was crystallized and its structure determined by standard molecular replacement techniques. The amino acid residues, previously suggested incorrectly by manual sequencing, have now been determined as I17, I53, S129, S134, G144, S164, P165, S187, V190, S169, T196, and S202. Analysis of the structure indicated a dimer in the asymmetric unit, two metal binding sites per monomer, and loops involved in the molecular oligomerization. These confer 98% similarity between ConM and other previously described lectins, derived from Canavalia ensiformis and Canavalia brasiliensis. Our functional data indicate that ConM exerts a concentration-dependent relaxant action on isolated aortic rings that probably occurs via an interaction with a specific lectin-binding site on the endothelium, resulting in a release of nitric oxide.     

Structural analysis of Canavalia maritima and Canavalia gladiata lectins complexed with different dimannosides: new insights into the understanding of the structure-biological activity relationship in legume lectins

Plant lectins, especially those purified from species of the Leguminosae family, represent the best studied group of carbohydrate-binding proteins. The legume lectins from Diocleinae subtribe are highly similar proteins that present significant differences in the potency/efficacy of their biological activities. The structural studies of the interactions between lectins and sugars may clarify the origin of the distinct biological activities observed in this high similar class of proteins. In this way, this work presents a crystallographic study of the ConM and CGL (agglutinins from Canavalia maritima and Canavalia gladiata, respectively) in the following complexes: ConM/CGL:Man(alpha1-2)Man(alpha1-O)Me, ConM/CGL:Man(alpha1-3)Man(alpha1-O)Me and ConM/CGL:Man(alpha1-4)Man(alpha1-O)Me, which crystallized in different conditions and space group from the native proteins. The structures were solved by molecular replacement, presenting satisfactory values for R(factor) and R(free). Comparisons between ConM, CGL and ConA (Canavalia ensiformis lectin) binding mode with the dimannosides in subject, presented different interactions patterns, which may account for a structural explanation of the distincts biological properties observed in the lectins of Diocleinae subtribe.     

Structure of a lectin from Canavalia gladiata seeds: new structural insights for old molecules

Background

Lectins are mainly described as simple carbohydrate-binding proteins. Previous studies have tried to identify other binding sites, which possible recognize plant hormones, secondary metabolites, and isolated amino acid residues. We report the crystal structure of a lectin isolated from Canavalia gladiata seeds (CGL), describing a new binding pocket, which may be related to pathogen resistance activity in ConA-like lectins; a site where a non-protein amino-acid, α-aminobutyric acid (Abu), is bound.

Results

The overall structure of native CGL and complexed with α-methyl-mannoside and Abu have been refined at 2.3 Å and 2.31 Å resolution, respectively. Analysis of the electron density maps of the CGL structure shows clearly the presence of Abu, which was confirmed by mass spectrometry.

Conclusion

The presence of Abu in a plant lectin structure strongly indicates the ability of lectins on carrying secondary metabolites. Comparison of the amino acids composing the site with other legume lectins revealed that this site is conserved, providing an evidence of the biological relevance of this site. This new action of lectins strengthens their role in defense mechanisms in plants.     

Structure of Dioclea virgata lectin: Relations between carbohydrate binding site and nitric oxide production

The lectin of Dioclea virgata (DvirL), both native and complexed with X-man, was submitted to X-ray diffraction analysis and the crystal structure was compared to that of other Diocleinae lectins in order to better understand differences in biological properties, especially with regard to the ability of lectins to induce nitric oxide (NO) production. An association was observed between the volume of the carbohydrate recognition domain (CRD), the ability to induce NO production and the relative positions of Tyr12, Arg228 and Leu99. Thus, differences in biological activity induced by Diocleinae lectins are related to the configuration of amino acid residues in the carbohydrate binding site and to the structural conformation of subsequent regions capable of influencing site-ligand interactions. In conclusion, the ability of Diocleinae lectins to induce NO production depends on CRD configuration.     

Crystal structure of fungal lectin: six-bladed beta-propeller fold and novel fucose recognition mode for Aleuria aurantia lectin

Aleuria aurantia lectin is a fungal protein composed of two identical 312-amino acid subunits that specifically recognizes fucosylated glycans. The crystal structure of the lectin complexed with fucose reveals that each monomer consists of a six-bladed beta-propeller fold and of a small antiparallel two-stranded beta-sheet that plays a role in dimerization. Five fucose residues were located in binding pockets between the adjacent propeller blades. Due to repeats in the amino acid sequence, there are strong similarities between the sites. Oxygen atoms O-3, O-4, and O-5 of fucose are involved in hydrogen bonds with side chains of amino acids conserved in all repeats, whereas O-1 and O-2 interact with a large number of water molecules. The nonpolar face of each fucose residue is stacked against the aromatic ring of a Trp or Tyr amino acid, and the methyl group is located in a highly hydrophobic pocket. Depending on the precise binding site geometry, the alpha- or beta-anomer of the fucose ligand is observed bound in the crystal. Surface plasmon resonance experiments conducted on a series of oligosaccharides confirm the broad specificity of the lectin, with a slight preference for alphaFuc1-2Gal disaccharide. This multivalent carbohydrate recognition fold is a new prototype of lectins that is proposed to be involved in the host recognition strategy of several pathogenic organisms including not only the fungi Aspergillus but also the phytopathogenic bacterium Ralstonia solanacearum.     

Interactions between indole-3-acetic acid (IAA) with a lectin from Canavalia maritima seeds reveal a new function for lectins in plant physiology

Indole-3-acetic acid (IAA) bound is considered a storage molecule and is inactive. However, some studies have proposed an additional possible regulatory mechanism based on the ability of lectins to form complexes with IAA. We report the first crystal structure of ConM in complex with IAA at 2.15 Å resolution. Based on a tetrameric model of the complex, we hypothesize how the lectin controls the availability of IAA during the early seedling stages, indicating a possible new physiological role for these proteins. A free indole group is also bound to the protein. The ConM interaction with different forms of IAA is a strategy to render the phytohormone unavailable to the cell. Thus, this new physiological role proposed for legume lectins might be a novel mechanism by which IAA levels are decreased in addition to the destruction and formation of new complexes in the later stages of seed germination.     

Interaction between trehalose and alkaline-earth metal ions

We investigated the interaction between trehalose and alkaline-earth metal ions. The nuclear relaxation times of carbon atoms of trehalose were shortened by addition of the alkaline-earth chloride salts, MgCl2, CaCl2, and SrCl2, indicating that trehalose formed metal-complexes with the alkaline-earth metal chlorides. From the data of the 1H-1H coupling constants of trehalose in the presence of the alkaline-earth chlorides, it appeared that trehalose formed complexes with MgCl2, and CaCl2 at the various complexing sites: Mg2+ was coordinated to O-4 and O-4' of trehalose, and Ca2+ to O-2 and O-3. We succeeded in the preparation of two types of crystals of the trehalose/CaCl2. One was a crystal consisting of trehalose, CaCl2, and water in a ratio of 1:1:1. The other was an anhydrous crystal containing trehalose and CaCl2 in a ratio of 1:2. Several applications of the complexing between trehalose and the metal ions for food processing are proposed.     

The effect of changing the hydrophobic S1' subsite of thermolysin-like proteases on substrate specificity.

The hydrophobic S1' subsite is one of the major determinants of the substrate specificity of thermolysin and related M4 family proteases. In the thermolysin-like protease (TLP) produced by Bacillus stearothermophilus (TLP-ste), the hydrophobic S1' subsite is mainly formed by Phe130, Phe133, Val139 and Leu202. In the present study, we have examined the effects of replacing Leu202 by smaller (Gly, Ala, Val) and larger (Phe, Tyr) hydrophobic residues. The mutational effects showed that the wild-type S1' pocket is optimal for binding leucine side chains. Reduction of the size of residue 202 resulted in a higher efficiency towards substrates with Phe in the P1' position. Rather unexpectedly, the Leu202-->Phe and Leu202-->Tyr mutations, which were expected to decrease the size of the S1' subsite, resulted in a large increase in activity towards dipeptide substrates with Phe in the P1' position. This is probably due to the fact that 202Phe and 202Tyr adopt a second possible rotamer that opens up the subsite compared to Leu202, and also favours interactions with the substrate. To validate these results, we constructed variants of thermolysin with changes in the S1' subsite. Thermolysin and TLP-ste variants with identical S1' subsites were highly similar in terms of their preference for Phe vs. Leu in the P1' position.     

Complete lipopolysaccharide of Plesiomonas shigelloides O74:H5 (strain CNCTC 144/92). 2. Lipid A, its structural variability, the linkage to the core oligosaccharide, and the biological activity of the lipopolysaccharide

Plesiomonas shigelloides is a Gram-negative bacterium associated with waterborne infections, which is common in tropical and subtropical habitats. Contrary to the unified antigenic classification of P. shigelloides, data concerning the structure and activity of their lipopolysaccharides (LPS and endotoxin) are limited. This study completes the structural investigation of phenol- and water-soluble fractions of P. shigelloides O74 (strain CNCTC 144/92) LPS with the emphasis on lipid A heterogeneity, describing the entire molecule and some of its biological in vitro activities. Structures of the lipid A and the affinity-purified decasaccharide obtained by de-N,O-acylation of P. shigelloides O74 LPS were elucidated by chemical analysis combined with electrospray ionization multiple-stage mass spectrometry (ESI-MS(n)), MALDI-TOF MS, and NMR spectroscopy. Lipid A of P. shigelloides O74 is heterogeneous, and three major forms have been identified. They all were asymmetric, phosphorylated, and hexaacylated, showing different acylation patterns. The beta-GlcpN4P-(1-->6)-alpha-GlcpN1P disaccharide was substituted with the primary fatty acids: (R)-3-hydroxytetradecanoic acid [14:0(3-OH)] at N-2 and N-2' and (R)-3-hydroxydodecanoic acid [12:0(3-OH)] at O-3 and O-3'. The heterogeneity among the three forms (I-III) of P. shigelloides O74 lipid A was attributed to the substitution of the acyl residues at N-2' and O-3' with the secondary acyls: (I) cis-9-hexadecenoic acid (9c-16:1) at N-2' and 12:0 at O-3', (II) 14:0 at N-2' and 12:0 at O-3', and (III) 12:0 at N-2' and 12:0 at O-3'. The pro-inflammatory cytokine-inducing activities of P. shigelloides O74 LPS were similar to those of Escherichia coli O55 LPS.     

Development of specific inhibitors for heparin-binding proteins based on the cobra cardiotoxin structure: an effective synthetic strategy for rationally modified heparin-like disaccharides and a trisaccharide

Recently, a new heparin disaccharide-binding site on the convex side of cobra cardiotoxin (CTX) was identified by NMR spectroscopy and molecular modeling. To further characterize this site two heparin-like disaccharides were synthesized for binding studies with CTX, and a trisaccharide was synthesized for testing the sequence of the disaccharide binding to CTX. Thus six differentially protected monosaccharide building blocks (three l-iduronic acids and three d-glucosamines) were prepared. These include a l-iduronic acid elongation building block namely methyl 2-O-acetyl-4-O-levulinoyl-3-O-pivaloyl-alpha-l-idopyranosyluronate trichloroacetimidate for which a single-crystal X-ray structure was determined to have M(r)=576.79, a=9.3098(11)A alpha=90 degrees , b=10.3967(12)A beta=90 degrees , c=28.026(3)A gamma=90 degrees , V=2712.7(6)A(3), P2(1)2(1)2(1), Z=4, mu=0.71073A, and R=0.0378 for 7586 observed reflections. It shows that the molecular structure of the donor is in the (1)C(4) conformation with significant 1,3-diaxial interactions between O-1 and O-3 as well as O-2 and O-4. The disaccharides and trisaccharide vary in the degree and position of O- and N-sulfation. The pivaloyl group was used as permanent protecting group of hydroxyl. The levulinoyl group was used as the temporary protecting group to protect the hydroxyl for elongation.     

Burkholderia cenocepacia lectin A binding to heptoses from the bacterial lipopolysaccharide

Bacteria from the Burkholderia cepacia complex (Bcc) cause highly contagious pneumonia among cystic fibrosis (CF) patients. Among them, Burkholderia cenocepacia is one of the most dangerous in the Bcc and is the most frequent cause of morbidity and mortality in CF patients. Indeed, it is responsible of "cepacia syndrome", a deadly exacerbation of infection, that is the main cause of poor outcomes in lung transplantation. Burkholderia cenocepacia produces several soluble lectins with specificity for fucosylated and mannosylated glycoconjugates. These lectins are present on the bacterial cell surface and it has been proposed that they bind to lipopolysaccharide epitopes. In this work, we report on the interaction of one B. cenocepacia lectin, BC2L-A, with heptose and other manno configured sugar residues. Saturation transfer difference NMR spectroscopy studies of BC2L-A with different mono- and disaccharides demonstrated the requirement of manno configuration with the hydroxyl or glycol group at C6 for the binding process. The crystal structure of BC2L-A complexed with the methyl-heptoside confirmed the location of the carbohydrate ring in the binding site and elucidated the orientation of the glycol tail, in agreement with NMR data. Titration calorimetry performed on monosaccharides, heptose disaccharides and bacterial heptose-containing oligosaccharides and polysaccharides confirmed that bacterial cell wall contains carbohydrate epitopes that can bind to BC2L-A. Additionally, the specific binding of fluorescent BC2L-A lectin on B. cenocepacia bacterial surface was demonstrated by microscopy.     

X-ray crystal structure of galabiose, O-alpha-D-galactopyranosyl-(1---4)-D-galactopyranose

O-alpha-D-Galactopyranosyl-(1---4)-D-galactopyranose, C12H22O11, Mr = 342.30, crystallises in the orthorhombic space group P2(1)2(1)2(1), and has alpha = 5.826(1), b = 13.904(3), c = 17.772(4) A, Z = 4, and Dx = 1.579 g.cm-3. Intensity data were collected with a CAD4 diffractometer. The structure was solved by direct methods and refined to R = 0.063 and Rw = 0.084 for 2758 independent reflections. The glycosidic linkage is of the type 1-axial-4-axial with torsion angles phi O-5' (O-5'-C-1'-O-1'-C-4) = 98.1(2) degrees, psi C-3 (C-3-C-4-O-1'-C-1') = -81.9(3) degrees, phi H (H-1'-C-1'-O-1'-C-4) = -18 degrees, and psi H (H-4-C-4-O-1'-C-1') = 35 degrees. The conformation is stabilised by an O-3 . . . O-5' intramolecular hydrogen-bond with length 2.787(3) A and O-3-H . . . O-5' = 162 degrees. The glycosidic linkage causes a folding of the molecule with an angle of 117 degrees between the least-square planes through the pyranosidic rings. The crystal investigated contained 56(1)% of alpha- and 44(1)% of beta-galabiose as well as approximately 70% of the gauche-trans and approximately 30% of the trans-gauche conformers about the exocyclic C-5'-C-6' and C-5-C-6 bonds. The crystal packing is governed by hydrogen bonding that engages all oxygen atoms except the intramolecular acceptor O-5' and the glycosidic O-1' oxygen atoms.     

Effect of disaccharides on the stabilization of bovine trypsin against detergent and autolysis

Osmolytes have been reported to stabilize biomolecules and even whole organisms against exposure to adverse environmental conditions. In this work, we report for the first time the use of some of these osmolytes, viz., the disaccharides trehalose and sucrose, in the stabilization of bovine trypsin against exposure to the anionic detergent sodium dodecyl sulfate and autolysis. Exposure of trypsin to SDS at a molar ratio of 1:45 led to decrease in trypsin activity by 61%. In the presence of 1 M sucrose and 1 M trehalose, the residual trypsin activity was found to increase to that of original enzyme activity. These two disaccharides were also found to slow down the rate of autolysis, resulting in residual activities of 80 and 88%, respectively, after incubation for 24 h. Active site titration showed retention of the fraction of active sites in the presence of trehalose. Fluorescence and CD spectroscopies were used to decipher the probable mechanism of this protective role of the disaccharides. Although complete resumption of secondary structure was not seen in the presence of the two disaccharides, the spectra of trypsin in the presence of stabilizers resembled the spectrum of native trypsin and were significantly different from the spectrum of detergent‐denatured enzyme. Correlating the data obtained from spectroscopy with those obtained from activity assay, we propose that the retention of secondary structure of the enzyme is largely responsible for the retention of the functionally active form of trypsin. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Substrate binding mode and its implication on drug design for botulinum neurotoxin A

The seven antigenically distinct serotypes of Clostridium botulinum neurotoxins, the causative agents of botulism, block the neurotransmitter release by specifically cleaving one of the three SNARE proteins and induce flaccid paralysis. The Centers for Disease Control and Prevention (CDC) has declared them as Category A biowarfare agents. The most potent among them, botulinum neurotoxin type A (BoNT/A), cleaves its substrate synaptosome-associated protein of 25 kDa (SNAP-25). An efficient drug for botulism can be developed only with the knowledge of interactions between the substrate and enzyme at the active site. Here, we report the crystal structures of the catalytic domain of BoNT/A with its uncleavable SNAP-25 peptide (197)QRATKM(202) and its variant (197)RRATKM(202) to 1.5 A and 1.6 A, respectively. This is the first time the structure of an uncleavable substrate bound to an active botulinum neurotoxin is reported and it has helped in unequivocally defining S1 to S5' sites. These substrate peptides make interactions with the enzyme predominantly by the residues from 160, 200, 250 and 370 loops. Most notably, the amino nitrogen and carbonyl oxygen of P1 residue (Gln197) chelate the zinc ion and replace the nucleophilic water. The P1'-Arg198, occupies the S1' site formed by Arg363, Thr220, Asp370, Thr215, Ile161, Phe163 and Phe194. The S2' subsite is formed by Arg363, Asn368 and Asp370, while S3' subsite is formed by Tyr251, Leu256, Val258, Tyr366, Phe369 and Asn388. P4'-Lys201 makes hydrogen bond with Gln162. P5'-Met202 binds in the hydrophobic pocket formed by the residues from the 250 and 200 loop. Knowledge of interactions between the enzyme and substrate peptide from these complex structures should form the basis for design of potent inhibitors for this neurotoxin.     

Anhydrobiosis without trehalose in bdelloid rotifers

Eukaryotes able to withstand desiccation enter a state of suspended animation known as anhydrobiosis, which is thought to require accumulation of the non-reducing disaccharides trehalose (animals, fungi) and sucrose (plants), acting as water replacement molecules and vitrifying agents. We now show that clonal populations of bdelloid rotifers Philodina roseola and Adineta vaga exhibit excellent desiccation tolerance, but that trehalose and other disaccharides are absent from carbohydrate extracts of dried animals. Furthermore, trehalose synthase genes (tps) were not found in rotifer genomes. This first observation of animal anhydrobiosis without trehalose challenges our current understanding of the phenomenon and calls for a re-evaluation of existing models.     

The unique extracellular disulfide loop of the glycine receptor is a principal ligand binding element.

A loop structure, formed by the putative disulfide bridging of Cys198 and Cys209, is a principal element of the ligand binding site in the glycine receptor (GlyR). Disruption of the loop's tertiary structure by Ser mutations of these Cys residues either prevented receptor assembly on the cell surface, or created receptors unable to be activated by agonists or to bind the competitive antagonist, strychnine. Mutation of residues Lys200, Tyr202 and Thr204 within this loop reduced agonist binding and channel activation sensitivities by up to 55-, 520- and 190-fold, respectively, without altering maximal current sizes, and mutations of Lys200 and Tyr202 abolished strychnine binding to the receptor. Removal of the hydroxyl moiety from Tyr202 by mutation to Phe profoundly reduced agonist sensitivity, whilst removal of the benzene ring abolished strychnine binding, thus demonstrating that Tyr202 is crucial for both agonist and antagonist binding to the GlyR. Tyr202 also influences receptor assembly on the cell surface, with only large chain substitutions (Phe, Leu and Arg, but not Thr, Ser and Ala) forming functional receptors. Our data demonstrate the presence of a second ligand binding site in the GlyR, consistent with the three-loop model of ligand binding to the ligand-gated ion channel superfamily.     

Hydration of oligosaccharides: anomalous hydration ability of trehalose.

The disaccharide trehalose extensively exists in anhydrobiotic organism and is considered to play an important role in preserving the integrity of biomembrane. However, the preserving mechanism remains unclear. In this report, we examine the hydration abilities of trehalose and several oligosaccharides composed of alpha-D-glucopyranosyl residues. The unfrozen water fraction per molecule was determined from differential scanning calorimetry measurements of their aqueous solutions. Further, the NMR relaxation time of the natural abundance 17O of water is measured for several saccharide solutions. These results indicate that trehalose has the highest hydration ability among the saccharides studied. In other words, trehalose can effectively lower the mobility of water molecules hydrogen-bonded with saccharides. It is thus reasonable that, among the disaccharides studied, trehalose exhibits the maximum stabilizing effect on the bilayer structure of lipid whose acyl chains are bonded with each other by the apolar interaction, because the apolar interaction is strengthened with the stabilization of the surrounding water structure.     

Changes in vibrational modes of water and bioprotectants in solution

Inelastic neutron scattering (INS) measurements have been performed on trehalose and sucrose/H(2)O mixtures at very low temperature as a function of concentration by using the TOSCA spectrometer at the ISIS Facility (DRAL, UK). The aim of this work is to investigate by INS the vibrational behaviour of water in presence of trehalose and sucrose in order to characterize the changes induced by these disaccharides on the H(2)O hydrogen-bonded network. In particular, we obtained information about the effects of the two disaccharides in the translational, librational and bending spectral regions of ice. The disaccharide bioprotective effectiveness can be linked by the high destructuring effect emphasised by the analysis of the librational modes region. On the other hand, the analysis of the vibrational region corresponding to the ice bending modes show a high "crystallinity" degree which can justify the cryptobiotic action of disaccharides.     

Mass spectrometric characterization of isoform variants of peanut (Arachis hypogaea) stem lectin (SL-I)

Matrix assisted laser desorption/ionization–time-of-flight (MALDI–TOF) mass spectrometric (MS) analysis of purified Arachis hypogaea stem lectin (SL-I) and its tryptic digests suggested it to be an isoformic glucose/mannose binding lectin. Two-dimensional gel electrophoresis of SL-I indicated six isoforms (A1–A6), which were confirmed by Western blotting and MALDI–TOF MS analysis. Comparative analysis of peptide mass spectra of the isoforms matched with A. hypogaea lectins with three different accession numbers (Q43376_ARAHY, Q43377_ARAHY, Q70DJ5_ARAHY). Tandem mass spectrometric (MS/MS) analysis of tryptic peptides revealed these to be isoformic variants with altered amino acid sequences. Among the peptides, the peptide T12 showed major variation. The ¹⁹⁹Val–Ser–Tyr–Asn²⁰² sequence in peptide T12 of A1 and A2 was replaced by ¹⁹⁹Leu–Ser–His–Glu²⁰² in A3 and A4 (T12′) while in A5 and A6 this sequence was ¹⁹⁹Val–Ser–Tyr–Val²⁰² (T12″). Peptide T1 showed the presence of ¹⁰Asn in the isoforms A1–A5 while in A6 this amino acid was replaced by ¹⁰Lys (T1′). Overall amino acid sequence as identified by MS/MS showed a high degree of similarity between A1, A2 and among A3, A4, A5. Carbohydrate binding domain and adenine binding site seem to be conserved.     

Strain specificity of outer membrane proteins in Escherichia coli.

Outer membrane proteins of various strains of Escherichia coli were compared using three different systems of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The outer membranes of E. coli K-12, E. coli B, and E. coli J-5 had distinctive protein compositions. As regards proteins which interact with peptidoglycan, E. coli K-12 contained O-8 and O-9, while E. coli B possessed one protein which migrated to the position of O-9. Although E. coli J-5 possessed two such proteins, O-8' and O-9', their positions on polyacrylamide gel were different from those of O-8 and O-9. Protein O-7, which migrates slightly more slowly than O-8, was found specifically in E. coli K-12. Proteins O-10 and O-11 were found in all strains tested, although the relative amounts were different depending on the strain. Strains of E. coli K-12 and E. coli J-5 gave three major bands, O-2a, O-2b, and O-3, in the region of high molecular weight. These proteins were repressed by iron in the cultivation media. Strains of E. coli B, on the other hand, gave only O-2b and O-3. E. coli J-5 gave two other major bands in this region, but the amounts were not controlled by iron in the cultivation media.