Structural basis for the carbohydrate specificities of artocarpin: variation in the length of a loop as a strategy for generating ligand specificity

Artocarpin, a tetrameric lectin of molecular mass 65 kDa, is one of the two lectins extracted from the seeds of jackfruit. The structures of the complexes of artocarpin with mannotriose and mannopentose reported here, together with the structures of artocarpin and its complex with Me-alpha-mannose reported earlier, show that the lectin possesses a deep-seated binding site formed by three loops. The binding site can be considered as composed of two subsites; the primary site and the secondary site. Interactions at the primary site composed of two of the loops involve mainly hydrogen bonds, while those at the secondary site comprising the third loop are primarily van der Waals in nature. Mannotriose in its complex with the lectin interacts through all the three mannopyranosyl residues; mannopentose interacts with the protein using at least three of the five mannose residues. The complexes provide a structural explanation for the carbohydrate specificities of artocarpin. A detailed comparison with the sugar complexes of heltuba, the only other mannose-specific jacalin-like lectin with known three-dimensional structure in sugar-bound form, establishes the role of the sugar-binding loop constituting the secondary site, in conferring different specificities at the oligosaccharide level. This loop is four residues longer in artocarpin than in heltuba, providing an instance where variation in loop length is used as a strategy for generating carbohydrate specificity.     

Crystal structure of the jacalin-T-antigen complex and a comparative study of lectin-T-antigen complexes

Thomsen-Friedenreich antigen (Galbeta1-3GalNAc), generally known as T-antigen, is expressed in more than 85% of human carcinomas. Therefore, proteins which specifically bind T-antigen have potential diagnostic value. Jacalin, a lectin from jack fruit (Artocarpus integrifolia) seeds, is a tetramer of molecular mass 66kDa. It is one of the very few proteins which are known to bind T-antigen. The crystal structure of the jacalin-T-antigen complex has been determined at 1.62A resolution. The interactions of the disaccharide at the binding site are predominantly through the GalNAc moiety, with Gal interacting only through water molecules. They include a hydrogen bond between the anomeric oxygen of GalNAc and the pi electrons of an aromatic side-chain. Several intermolecular interactions involving the bound carbohydrate contribute to the stability of the crystal structure. The present structure, along with that of the Me-alpha-Gal complex, provides a reasonable qualitative explanation for the known affinities of jacalin to different carbohydrate ligands and a plausible model of the binding of the lectin to T-antigen O-linked to seryl or threonyl residues. Including the present one, the structures of five lectin-T-antigen complexes are available. GalNAc occupies the primary binding site in three of them, while Gal occupies the site in two. The choice appears to be related to the ability of the lectin to bind sialylated sugars. In either case, most of the lectin-disaccharide interactions are at the primary binding site. The conformation of T-antigen in the five complexes is nearly the same.     

Structural basis for the energetics of jacalin-sugar interactions: promiscuity versus specificity

Jacalin, a tetrameric lectin, is one of the two lectins present in jackfruit (Artocarpus integrifolia) seeds. Its crystal structure revealed, for the first time, the occurrence of the beta-prism I fold in lectins. The structure led to the elucidation of the crucial role of a new N terminus generated by post-translational proteolysis for the lectin's specificity for galactose. Subsequent X-ray studies on other carbohydrate complexes showed that the extended binding site of jacalin consisted of, in addition to the primary binding site, a hydrophobic secondary site A composed of aromatic residues and a secondary site B involved mainly in water-bridges. A recent investigation involving surface plasmon resonance and the X-ray analysis of a methyl-alpha-mannose complex, had led to a suggestion of promiscuity in the lectin's sugar specificity. To explore this suggestion further, detailed isothermal titration calorimetric studies on the interaction of galactose (Gal), mannose (Man), glucose (Glc), Me-alpha-Gal, Me-alpha-Man, Me-alpha-Glc and other mono- and oligosaccharides of biological relevance and crystallographic studies on the jacalin-Me-alpha-Glc complex and a new form of the jacalin-Me-alpha-Man complex, have been carried out. The binding affinity of Me-alpha-Man is 20 times weaker than that of Me-alpha-Gal. The corresponding number is 27, when the binding affinities of Gal and Me-alpha-Gal, and those of Man and Me-alpha-Man are compared. Glucose (Glc) shows no measurable binding, while the binding affinity of Me-alpha-Glc is slightly less than that of Me-alpha-Man. The available crystal structures of jacalin-sugar complexes provide a convincing explanation for the energetics of binding in terms of interactions at the primary binding site and secondary site A. The other sugars used in calorimetric studies show no detectable binding to jacalin. These results and other available evidence suggest that jacalin is specific to O-glycans and its affinity to N-glycans is extremely weak or non-existent and therefore of limited value in processes involving biological recognition.     

Structure of porphobilinogen synthase from Pseudomonas aeruginosa in complex with 5-fluorolevulinic acid suggests a double Schiff base mechanism

The seeds of jack fruit (Artocarpus integrifolia) contain two tetrameric lectins, jacalin and artocarpin. Jacalin was the first lectin found to exhibit the beta-prism I fold, which is characteristic of the Moraceae plant lectin family. Jacalin contains two polypeptide chains produced by a post-translational proteolysis which has been shown to be crucial for generating its specificity for galactose. Artocarpin is a single chain protein with considerable sequence similarity with jacalin. It, however, exhibits many properties different from those of jacalin. In particular, it is specific to mannose. The structures of two crystal forms, form I and form II, of the native lectin have been determined at 2.4 and 2.5 A resolution, respectively. The structure of the lectin complexed with methyl-alpha-mannose, has also been determined at 2.9 A resolution. The structure is similar to jacalin, although differences exist in details. The crystal structures and detailed modelling studies indicate that the following differences between the carbohydrate binding sites of artocarpin and jacalin are responsible for the difference in the specificities of the two lectins. Firstly, artocarpin does not contain, unlike jacalin, an N terminus generated by post-translational proteolysis. Secondly, there is no aromatic residue in the binding site of artocarpin whereas there are four in that of jacalin. A comparison with similar lectins of known structures or sequences, suggests that, in general, stacking interactions with aromatic residues are important for the binding of galactose while such interactions are usually absent in the carbohydrate binding sites of mannose-specific lectins with the beta-prism I fold.     

The monosaccharide binding site of lentil lectin: an X-ray and molecular modelling study

The X-ray crystal structure of lentil lectin in complex with -d-glucopyranose has been determined by molecular replacement and refined to anR-value of 0.20 at 3.0 Å resolution. The glucose interacts with the protein in a manner similar to that found in the mannose complexes of concanavalin A, pea lectin and isolectin I fromLathyrus ochrus. The complex is stabilized by a network of hydrogen bonds involving the carbohydrate oxygens O6, O4, O3 and O5. In addition, the -d-glucopyranose residue makes van der Waals contacts with the protein, involving the phenyl ring of Phe123. The overall structure of lentil lectin, at this resolution, does not differ significantly from the highly refined structures of the uncomplexed lectin.Molecular docking studies were performed with mannose and its 2-O and 3-O-m-nitro-benzyl derivatives to explain their high affinity binding. The interactions of the modelled mannose with lentil lectin agree well with those observed experimentally for the protein-carbohydrate complex. The highly flexible Me-2-O-(m-nitro-benzyl)--d-mannopyranoside and Me-3-O-(m-nitro-benzyl)--d-mannopyranoside become conformationally restricted upon binding to lentil lectin. For best orientations of the two substrates in the combining site, the loss of entropy is accompanied by the formation of a strong hydrogen bond between the nitro group and one amino acid, Gly97 and Asn125, respectively, along with the establishment of van der Waals interactions between the benzyl group and the aromatic amino acids Tyr100 and Trp128.RL and FC are joint first authors.     

Stereoselective single-step synthesis and X-ray crystallographic investigation of acetylated aryl 1,2-trans glycopyranosides and aryl 1,2-cis C2-hydroxy-glycopyranosides

Reported is an attractive and environmentally friendly method for the synthesis of the title compounds in moderate yield using inexpensive 1,2,3,4,6-penta-O-acetyl-beta-D-gluco- and galactopyranoses as sugar donors, five different phenols as acceptors and H-beta zeolite as the catalyst. The yield (23-28%) of aryl 3,4,6-tri-O-acetyl-alpha-D-glycopyranosides obtained in this single-step procedure is considerably higher than that obtained using previously reported methods. Treatment of an orthoacetate, 3,4,6-tri-O-acetyl-[1,2-O-(1-p-fluorophenoxyethylidene)]-alpha-D-glucopyranose, with p-fluorophenol under the same solvent-free reaction conditions also led to the formation of the title compounds in similar yield and composition. X-ray crystallographic analysis of phenyl 3,4,6-tri-O-acetyl-alpha-D-glucopyranoside and p-fluorophenyl 3,4,6-tri-O-acetyl-alpha-D-glucopyranoside showed that the molecular packing is stabilized by C-H...O, C-H...pi and C-H...F interactions, in addition to regular hydrogen bonding patterns.     

Structural basis of carbohydrate transfer activity by human UDP-GalNAc: polypeptide alpha-N-acetylgalactosaminyltransferase (pp-GalNAc-T10)

Mucin-type O-glycans are important carbohydrate chains involved in differentiation and malignant transformation. Biosynthesis of the O-glycan is initiated by the transfer of N-acetylgalactosamine (GalNAc) which is catalyzed by UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminyltransferases (pp-GalNAc-Ts). Here we present crystal structures of the pp-GalNAc-T10 isozyme, which has specificity for glycosylated peptides, in complex with the hydrolyzed donor substrate UDP-GalNAc and in complex with GalNAc-serine. A structural comparison with uncomplexed pp-GalNAc-T1 suggests that substantial conformational changes occur in two loops near the catalytic center upon donor substrate binding, and that a distinct interdomain arrangement between the catalytic and lectin domains forms a narrow cleft for acceptor substrates. The distance between the catalytic center and the carbohydrate-binding site on the lectin beta sub-domain influences the position of GalNAc glycosylation on GalNAc-glycosylated peptide substrates. A chimeric enzyme in which the two domains of pp-GalNAc-T10 are connected by a linker from pp-GalNAc-T1 acquires activity toward non-glycosylated acceptors, identifying a potential mechanism for generating the various acceptor specificities in different isozymes to produce a wide range of O-glycans.     

Structural and functional characterization of the GalNAc/Gal-specific lectin from the phytopathogenic ascomycete Sclerotinia sclerotiorum (Lib.) de Bary

The lectin found in mycelium and sclerotes of the phytopathogenic fungus Sclerotinia sclerotiorum is a homodimer consisting of two identical non-covalently bound subunits of 16,000 Da. CD spectra analysis revealed that the S. sclerotiorum agglutinin (SSA) contains predominantly beta-sheet structures. SSA exhibits specificity towards GalNAc whereby the hydroxyls at positions 4 and 6 of the pyranose ring play a key role in the interaction with simple sugars. The carbohydrate-binding site of SSA can also accommodate disaccharides. The N-terminal sequence of SSA shares no significant similarity with any other protein except a lectin from the Sclerotiniaceae species Ciborinia camelliae. A comparison of SSA and the lectins from C. camelliae and some previously characterized lectins indicates that the Sclerotiniaceae lectins form a homogeneous family of fungal lectins. This newly identified lectin family, which is structurally unrelated to any other family of fungal lectins, is most probably confined to the Ascomycota.     

Architecture of the sugar binding sites in carbohydrate binding proteins-a computer modeling study

Different sugars, Gal, GalNAc and Man were docked at the monosaccharide binding sites of Erythrina corallodenron (EcorL), peanut lectin (PNA), Lathyrus ochrus (LOLI), and pea lectin (PSL). To study the lectin-carbohydrate interactions, in the complexes, the hydroxymethyl group in Man and Gal favors, gg and gt conformations respectively, and is the dominant recognition determination. The monosaccharide binding site in lectins that are specific to Gal/GalNAc is wider due to the additional amino acid residues in loop D as compared to that in lectins specific to Man/Glc, and affects the hydrogen bonds of the sugar involving residues from loop D, but not its orientation in the binding site. The invariant amino acid residues Asp from loop A, and Asn and an aromatic residue (Phe or Tyr) in loop C provides the basic architecture to recognize the common features in C4 epimers. The invariant Gly in loop B together with one or two residues in the variable region of loop D/A holds the sugar tightly at both ends. Loss of any one of these hydrogen bonds leads to weak interaction. While the subtle variations in the sequence and conformation of peptide fragment that resulted due to the size and location of gaps present in amino acid sequence in the neighborhood of the sugar binding site of loop D/A seems to discriminate the binding of sugars which differ at C4 atom (galacto and gluco configurations). The variations at loop B are important in discriminating Gal and GalNAc binding. The present study thus provides a structural basis for the observed specificities of legume lectins which uses the same four invariant residues for binding. These studies also bring out the information that is important for the design/engineering of proteins with the desired carbohydrate specificity.     

Structural basis of phospholipase activity of Staphylococcus hyicus lipase

Staphylococcus hyicus lipase differs from other bacterial lipases in its high phospholipase A1 activity. Here, we present the crystal structure of the S. hyicus lipase at 2.86 A resolution. The lipase is in an open conformation, with the active site partly covered by a neighbouring molecule. Ser124, Asp314 and His355 form the catalytic triad. The substrate-binding cavity contains two large hydrophobic acyl chain-binding pockets and a shallow and more polar third pocket that is capable of binding either a (short) fatty acid or a phospholipid head-group. A model of a phospholipid bound in the active site shows that Lys295 is at hydrogen bonding distance from the substrate's phosphate group. Residues Ser356, Glu292 and Thr294 hold the lysine in position by hydrogen bonding and electrostatic interactions. These observations explain the biochemical data showing the importance of Lys295 and Ser356 for phospholipid binding and phospholipase A1 activity.     

Structural basis for the specificity of basic winged bean lectin for the Tn-antigen: a crystallographic, thermodynamic and modelling study

The crystal structure of winged bean basic agglutinin in complex with GalNAc-alpha-O-Ser (Tn-antigen) has been elucidated at 2.35 angstroms resolution in order to characterize the mode of binding of Tn-antigen with the lectin. The Gal moiety occupies the primary binding site and makes interactions similar to those found in other Gal/GalNAc specific legume lectins. The nitrogen and oxygen atoms of the acetamido group of the sugar make two hydrogen bonds with the protein atoms whereas its methyl group is stabilized by hydrophobic interactions. A water bridge formed between the terminal oxygen atoms of the serine residue of the Tn-antigen and the side chain oxygen atom of Asn128 of the lectin increase the affinity of the lectin for Tn-antigen compared to that for GalNAc. A comparison with the available structures reveals that while the interactions of the glyconic part of the antigen are conserved, the mode of stabilization of the serine residue differs and depends on the nature of the protein residues in its vicinity. The structure provides a qualitative explanation for the thermodynamic parameters of the complexation of the lectin with Tn-antigen. Modeling studies indicate the possibility of an additional hydrogen bond with the lectin when the antigen is part of a glycoprotein.     

Structural study of the carbohydrate moieties of two human immunoglobulin subclasses (IgG2 and IgG4)

Asparagine-linked sugar chains were quantitatively released as oligosaccharides from human IgG2 and IgG4 myeloma proteins by hydrazinolysis followed by N-acetylation and NaB3H4 reduction. Each oligosaccharide was isolated by serial lectin column chromatography. Study of their structures by sequential exoglycosidase digestion and methylation analysis, revealed that all of them were of the bi-antennary complex-type containing Man alpha 1-6(+/- GlcNAc beta 1-4)(Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4(+/- Fuc alpha 1-6)GlcNAc as core structures, and GlcNAc beta 1-, Gal beta 1-4GlcNAc beta 1- and Sia alpha 2-6Gal beta 1- in their outer chain moieties. However, the molar ratio of each oligosaccharide was different in each IgG sample, indicating that clonal variation is included in the sugar chain moieties of IgG molecules. One of the IgG2 contained four asparagine-linked sugar chains in one molecule, two on the Fc fragment and the remainder on the Fab fragment. The sugar chains in the Fc fragment contained much less galactose as compared with the Fab fragment.     

Structural modeling of SoxF protein from Chlorobium tepidum: an approach to understand the molecular basis of thiosulfate oxidation

Microbial redox reactions of inorganic sulfur compounds play a vital role in balancing the turnover of this element in the environment. These vital reactions are carried out by the enzyme system encoded by the sox operon. The central player of the sulfur oxidation biochemistry is the SoxY–Z protein complex. Another protein called SoxF having sulfide dehydrogenase activity has the ability to reactivate the inactivated SoxY–Z protein complex. This SoxF protein is obtained from the sox operon of Chlorobium tepidium. In the present work an attempt has been made to understand the structural details of the activity of SoxF protein. A plausible biochemical mechanism has been predicted regarding the involvement of the SoxF protein in biological sulfur anion oxidation process. Since this is the first report regarding the structural biology of SoxF protein this study may shed light in the hitherto unknown molecular biochemistry of sulfur anion oxidation by sox operon.     

Species-specificity of amphibia carbohydrate chains: the Bufo viridis case study

The jelly coat surrounding the eggs of amphibia is composed of oviducal mucins and plays an important role in the fertilization process. From a structural and chemical point of view, these jellies are very different from one species to another. Bufo viridis is the 13th amphibia species studied in term of carbohydrate structural analysis. The oligosaccharides have been released from the oviducal mucins by reductive beta elimination, purified by various chromatography procedures and analyzed by (1)H and (13)C 1D-2D NMR spectroscopy. Among the 15 compounds, ten have novel structures, although they possess some well-known structural patterns as blood group epitopes (Le(x), Le(y)) or other sequences already observed in other amphibia species. These results reinforce our hypothesis about the strict species-specificity of these carbohydrate chains. It must be noted that such species-specificity does not depend on one particular monosaccharide but it is rather due to a set of particular tri- or tetrasaccharide sequences. Hence, B. viridis species could be characterized by the simultaneous presence of a 2,3,6-trisubstituted galactosyl residue, the GlcNAc(beta 1-3)[Fuc(alpha 1-4)]GlcNAc beta sequence and the Le(x), Le(y) or Cad determinants. The anionic charge of the oligosaccharides is carried only by sialic acid alpha-(2-->6)-linked to GalNAc-ol residue as in Bufo bufo or in Bufo arenarum.     

Structural basis for catalytic racemization and substrate specificity of an N-acylamino acid racemase homologue from Deinococcus radiodurans

N-acylamino acid racemase (NAAAR) catalyzes the racemization of N-acylamino acids and can be used in concert with an aminoacylase to produce enantiopure alpha-amino acids, a process that has potential industrial applications. Here we have cloned and characterized an NAAAR homologue from a radiation-resistant ancient bacterium, Deinococcus radiodurans. The expressed NAAAR racemized various substrates at an optimal temperature of 60 degrees C and had Km values of 24.8 mM and 12.3 mM for N-acetyl-D-methionine and N-acetyl-L-methionine, respectively. The crystal structure of NAAAR was solved to 1.3 A resolution using multiwavelength anomalous dispersion (MAD) methods. The structure consists of a homooctamer in which each subunit has an architecture characteristic of enolases with a capping domain and a (beta/alpha)7 beta barrel domain. The NAAAR.Mg2+ and NAAAR.N-acetyl-L-glutamine.Mg2+ structures were also determined, allowing us to define the Lys170-Asp195-Glu220-Asp245-Lys269 framework for catalyzing 1,1-proton exchange of N-acylamino acids. Four subsites enclosing the substrate are identified: catalytic site, metal-binding site, side-chain-binding region, and a flexible lid region. The high conservation of catalytic and metal-binding sites in different enolases reflects the essentiality of a common catalytic platform, allowing these enzymes to robustly abstract alpha-protons of various carboxylate substrates efficiently. The other subsites involved in substrate recognition are less conserved, suggesting that divergent evolution has led to functionally distinct enzymes.     

Theoretical investigation on the glycan‐binding specificity of Agrocybe cylindracea galectin using molecular modeling and molecular dynamics simulation studies

Galectins are β‐galactoside binding proteins which have the ability to serve as potent antitumor, cancer biomarker, and induce tumor cell apoptosis. Agrocybe cylindracea galectin (ACG) is a fungal galectin which specifically recognizes α(2,3)‐linked sialyllactose at the cell surface that plays extensive roles in the biological recognition processes. To investigate the change in glycan‐binding specificity upon mutations, single point and double point site‐directed in silico mutations are performed at the binding pocket of ACG. Molecular dynamics (MD) simulation studies are carried out for the wild‐type (ACG) and single point (ACG1) and double point (ACG2) mutated ACGs to investigate the dynamics of substituted mutants and their interactions with the receptor sialyllactose. Plausible binding modes are proposed for galectin–sialylglycan complexes based on the analysis of hydrogen bonding interactions, total pair‐wise interaction energy between the interacting binding site residues and sialyllactose and binding free energy of the complexes using molecular mechanics–Poisson–Boltzmann surface area. Our result shows that high contribution to the binding in different modes is due to the direct and water‐mediated hydrogen bonds. The binding specificity of double point mutant Y59R/N140Q of ACG2 is found to be high, and it has 26 direct and water‐mediated hydrogen bonds with a relatively low‐binding free energy of −47.52 ± 5.2 kcal/mol. We also observe that the substituted mutant Arg59 is crucial for glycan‐binding and for the preference of α(2,3)‐linked sialyllactose at the binding pocket of ACG2 galectin. When compared with the wild‐type and single point mutant, the double point mutant exhibits enhanced affinity towards α(2,3)‐linked sialyllactose, which can be effectively used as a model for biological cell marker in cancer therapeutics. Copyright © 2015 John Wiley & Sons, Ltd.     

Structural basis for high substrate-binding affinity and enantioselectivity of 3-quinuclidinone reductase AtQR

(R)-3-Quinuclidinol, a useful compound for the synthesis of various pharmaceuticals, can be enantioselectively produced from 3-quinuclidinone by 3-quinuclidinone reductase. Recently, a novel NADH-dependent 3-quinuclidionone reductase (AtQR) was isolated from Agrobacterium tumefaciens, and showed much higher substrate-binding affinity (>100 fold) than the reported 3-quinuclidionone reductase (RrQR) from Rhodotorula rubra. Here, we report the crystal structure of AtQR at 1.72 Å. Three NADH-bound protomers and one NADH-free protomer form a tetrameric structure in an asymmetric unit of crystals. NADH not only acts as a proton donor, but also contributes to the stability of the α7 helix. This helix is a unique and functionally significant part of AtQR and is related to form a deep catalytic cavity. AtQR has all three catalytic residues of the short-chain dehydrogenases/reductases family and the hydrophobic wall for the enantioselective reduction of 3-quinuclidinone as well as RrQR. An additional residue on the α7 helix, Glu197, exists near the active site of AtQR. This acidic residue is considered to form a direct interaction with the amine part of 3-quinuclidinone, which contributes to substrate orientation and enhancement of substrate-binding affinity. Mutational analyses also support that Glu197 is an indispensable residue for the activity.     

Comparison of the catalytic parameters and reaction specificities of a phage and an archaeal flap endonuclease

Flap endonucleases (FENs) catalyse the exonucleolytic hydrolysis of blunt-ended duplex DNA substrates and the endonucleolytic cleavage of 5'-bifurcated nucleic acids at the junction formed between single and double-stranded DNA. The specificity and catalytic parameters of FENs derived from T5 bacteriophage and Archaeoglobus fulgidus were studied with a range of single oligonucleotide DNA substrates. These substrates contained one or more hairpin turns and mimic duplex, 5'-overhanging duplex, pseudo-Y, nicked DNA, and flap structures. The FEN-catalysed reaction properties of nicked DNA and flap structures possessing an extrahelical 3'-nucleotide (nt) were also characterised. The phage enzyme produced multiple reaction products of differing length with all the substrates tested, except when the length of duplex DNA downstream of the reaction site was truncated. Only larger DNAs containing two duplex regions are effective substrates for the archaeal enzyme and undergo reaction at multiple sites when they lack a 3'-extrahelical nucleotide. However, a single product corresponding to reaction 1 nt into the double-stranded region occurred with A. fulgidus FEN when substrates possessed a 3'-extrahelical nt. Steady-state and pre-steady-state catalytic parameters reveal that the phage enzyme is rate-limited by product release with all the substrates tested. Single-turnover maximal rates of reaction are similar with most substrates. In contrast, turnover numbers for T5FEN decrease as the size of the DNA substrate is increased. Comparison of the catalytic parameters of the A. fulgidus FEN employing flap and double-flap substrates indicates that binding interactions with the 3'-extrahelical nucleotide stabilise the ground state FEN-DNA interaction, leading to stimulation of comparative reactions at DNA concentrations below saturation with the single flap substrate. Maximal multiple turnover rates of the archaeal enzyme with flap and double flap substrates are similar. A model is proposed to account for the varying specificities of the two enzymes with regard to cleavage patterns and substrate preferences.     

Structural basis of calcium and galactose recognition by the lectin PA-IL of Pseudomonas aeruginosa

The structure of the tetrameric Pseudomonas aeruginosa lectin I (PA-IL) in complex with galactose and calcium was determined at 1.6 A resolution, and the native protein was solved at 2.4 A resolution. Each monomer adopts a beta-sandwich fold with ligand binding site at the apex. All galactose hydroxyl groups, except O1, are involved in a hydrogen bond network with the protein and O3 and O4 also participate in the co-ordination of the calcium ion. The stereochemistry of calcium galactose binding is reminiscent of that observed in some animal C-type lectins. The structure of the complex provides a framework for future design of anti-bacterial compounds.     

Structural basis of oncogenic activation caused by point mutations in the kinase domain of the MET proto-oncogene: modeling studies

Missense mutations in the tyrosine kinase domain of the MET proto-oncogene occur in selected cases of papillary renal carcinoma. In biochemical and biological assays, these mutations produced constitutive activation of the MET kinase and led to tumor formation in nude mice. Some mutations caused transformation of NIH 3T3 cells. To elucidate the mechanism of ligand-independent MET kinase activation by point mutations, we constructed several 3D models of the wild-type and mutated MET catalytic core domains. Analysis of these structures showed that some mutations (e.g., V1110I, Y1248H/D/C, M1268T) directly alter contacts between residues from the activation loop in its inhibitory conformation and those from the main body of the catalytic domain; others (e.g., M1149T, L1213V) increase flexibility at the critical points of the tertiary structure and facilitate subdomain movements. Mutation D1246N plays a role in stabilizing the active form of the enzyme. Mutation M1268T affects the S+1 and S+3 substrate-binding pockets. Models implicate that although these changes do not compromise the affinity toward the C-terminal autophosphorylation site of the MET protein, they allow for binding of the substrate for the c-Abl tyrosine kinase. We provide biochemical data supporting this observation. Mutation L1213V affects the conformation of Tyr1212 in the active form of MET. Several somatic mutations are clustered at the surface of the catalytic domain in close vicinity of the probable location of the MET C-terminal docking site for cytoplasmic effectors.