RESUMO
Crystal structures of jacalin in complex with GlcNAc ß-(1,3) Gal-ß-OMe and Gal ß-(1,3) Gal-ß-OMe have been determined. The binding of the ligands to jacalin is similar to that of analogous α-substituted disaccharides. However, the ß-substituted ß-(1,3) linked disaccharides get distorted at the anomeric center and the glycosidic linkage. The distortion results in higher internal energies of the ligands leading to lower affinity to the lectin. This confirms the possibility of using ligand distortion as a strategy for modulating binding affinity. Unlike in the case of ß-substituted monosaccharides bound to jacalin, where a larger distortion at the anomeric center was observed, smaller distortions are distributed among two centers in the structures of the two ß-substituted ß-(1,3) linked disaccharides presented here. These disaccharides, like the unsubstituted and α-substituted counterparts, bind jacalin with the reducing Gal at the primary binding site, indicating that the lower binding affinity of ß-substituted disaccharides is not enough to overcome the intrinsic propensity of Gal ß-(1,3) Gal-based disaccharides to bind jacalin with the reducing sugar at the primary site. © 2017 IUBMB Life, 69(2):72-78, 2017.
Assuntos
Dissacarídeos/química , Lectinas/química , Lectinas/metabolismo , Lectinas de Plantas/química , Sítios de Ligação , Cristalografia por Raios X , Dissacarídeos/metabolismo , Galactanos/química , Galactanos/metabolismo , Ligantes , Modelos Moleculares , Lectinas de Plantas/metabolismo , Ligação ProteicaRESUMO
Jacalin is among the most thoroughly studied lectins. Its carbohydrate-binding site has also been well characterized. It has been postulated that the lower affinity of ß-galactosides for jacalin compared with α-galactosides is caused by steric interactions of the substituents in the former with the protein. This issue has been explored energetically and structurally using different appropriate carbohydrate complexes of jacalin. It turns out that the earlier postulation is not correct. The interactions of the substituent with the binding site remain essentially the same irrespective of the anomeric nature of the substitution. This is achieved through a distortion of the sugar ring in ß-galactosides. The difference in energy, and therefore in affinity, is caused by a distortion of the sugar ring in ß-galactosides. The elucidation of this unprecedented distortion of the ligand as a strategy for modulating affinity is of general interest. The crystal structures also provide a rationale for the relative affinities of the different carbohydrate ligands for jacalin.
Assuntos
Artocarpus/química , Galactosídeos/metabolismo , Lectinas de Plantas/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Galactosídeos/química , Modelos Moleculares , Lectinas de Plantas/química , Conformação ProteicaRESUMO
17 independent crystal structures of family I uracil-DNA glycosylase from Mycobacterium tuberculosis (MtUng) and its complexes with uracil and its derivatives, distributed among five distinct crystal forms, have been determined. Thermodynamic parameters of binding in the complexes have been measured using isothermal titration calorimetry. The two-domain protein exhibits open and closed conformations, suggesting that the closure of the domain on DNA binding involves conformational selection. Segmental mobility in the enzyme molecule is confined to a 32-residue stretch which plays a major role in DNA binding. Uracil and its derivatives can bind to the protein in two possible orientations. Only one of them is possible when there is a bulky substituent at the 5' position. The crystal structures of the complexes provide a reasonable rationale for the observed thermodynamic parameters. In addition to providing fresh insights into the structure, plasticity and interactions of the protein molecule, the results of the present investigation provide a platform for structure-based inhibitor design.
Assuntos
Mycobacterium tuberculosis/enzimologia , Uracila-DNA Glicosidase/química , Uracila-DNA Glicosidase/metabolismo , Uracila/metabolismo , Sítios de Ligação , Ácido Cítrico/metabolismo , Cristalografia por Raios X , Humanos , Modelos Moleculares , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/metabolismo , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Uracila/análogos & derivadosRESUMO
The last enzyme in the arginine-biosynthesis pathway, argininosuccinate lyase, from Mycobacterium tuberculosis has been cloned, expressed, purified and crystallized, and preliminary X-ray studies have been carried out on the crystals. The His-tagged tetrameric enzyme with a subunit molecular weight of 50.9 kDa crystallized with two tetramers in the asymmetric unit of the orthorhombic unit cell, space group P2(1)2(1)2(1). Molecular-replacement calculations and self-rotation calculations confirmed the space group and the tetrameric nature of the molecule.
Assuntos
Argininossuccinato Liase/química , Proteínas de Bactérias/química , Mycobacterium tuberculosis/química , Subunidades Proteicas/química , Argininossuccinato Liase/genética , Argininossuccinato Liase/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Cristalização , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Histidina/química , Histidina/genética , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Oligopeptídeos/química , Oligopeptídeos/genética , Multimerização Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Previous studies of complexes of Mycobacterium tuberculosis PanK (MtPanK) with nucleotide diphosphates and nonhydrolysable analogues of nucleoside triphosphates in the presence or the absence of pantothenate established that the enzyme has dual specificity for ATP and GTP, revealed the unusual movement of ligands during enzyme action and provided information on the effect of pantothenate on the location and conformation of the nucleotides at the beginning and the end of enzyme action. The X-ray analyses of the binary complexes of MtPanK with pantothenate, pantothenol and N-nonylpantothenamide reported here demonstrate that in the absence of nucleotide these ligands occupy, with a somewhat open conformation, a location similar to that occupied by phosphopantothenate in the `end' complexes, which differs distinctly from the location of pantothenate in the closed conformation in the ternary `initiation' complexes. The conformation and the location of the nucleotide were also different in the initiation and end complexes. An invariant arginine appears to play a critical role in the movement of ligands that takes place during enzyme action. The work presented here completes the description of the locations and conformations of nucleoside diphosphates and triphosphates and pantothenate in different binary and ternary complexes, and suggests a structural rationale for the movement of ligands during enzyme action. The present investigation also suggests that N-alkylpantothenamides could be phosphorylated by the enzyme in the same manner as pantothenate.
Assuntos
Mycobacterium tuberculosis/enzimologia , Ácido Pantotênico/química , Ácido Pantotênico/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Cristalografia por Raios X , Ácido Pantotênico/análogos & derivadosRESUMO
The antimicrobial biocide triclosan [5-chloro-2-(2,4-dichlorophenoxy)phenol] potently inhibits the growth of Plasmodium falciparum in vitro and, in a mouse model, Plasmodium berghei in vivo. Inhibition of [14C]acetate and [14C]malonyl-CoA incorporation into fatty acids in vivo and in vitro, respectively, by triclosan implicate FabI as its target. Here we demonstrate that the enoyl-ACP reductase purified from P. falciparum is triclosan sensitive. Also, we present the evidence for the existence of FabI gene in P. falciparum. We establish the existence of the de novo fatty acid biosynthetic pathway in this parasite, and identify a key enzyme of this pathway for the development of new antimalarials.
Assuntos
Antimaláricos/farmacologia , Malária/parasitologia , Oxirredutases/antagonistas & inibidores , Plasmodium berghei/efeitos dos fármacos , Plasmodium falciparum/efeitos dos fármacos , Triclosan/farmacologia , Sequência de Aminoácidos , Animais , Modelos Animais de Doenças , Enoil-(Proteína de Transporte de Acila) Redutase (NADH) , Ácidos Graxos/biossíntese , Malária/fisiopatologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , NAD/metabolismo , Plasmodium berghei/crescimento & desenvolvimento , Plasmodium falciparum/crescimento & desenvolvimentoRESUMO
The crystal structures of complexes of Mycobacterium tuberculosis pantothenate kinase with the following ligands have been determined: (i) citrate; (ii) the nonhydrolysable ATP analogue AMPPCP and pantothenate (the initiation complex); (iii) ADP and phosphopantothenate resulting from phosphorylation of pantothenate by ATP in the crystal (the end complex); (iv) ATP and ADP, each with half occupancy, resulting from a quick soak of crystals in ATP (the intermediate complex); (v) CoA; (vi) ADP prepared by soaking and cocrystallization, which turned out to have identical structures, and (vii) ADP and pantothenate. Solution studies on CoA binding and catalytic activity have also been carried out. Unlike in the case of the homologous Escherichia coli enzyme, AMPPCP and ADP occupy different, though overlapping, locations in the respective complexes; the same is true of pantothenate in the initiation complex and phosphopantothenate in the end complex. The binding site of MtPanK is substantially preformed, while that of EcPanK exhibits considerable plasticity. The difference in the behaviour of the E. coli and M. tuberculosis enzymes could be explained in terms of changes in local structure resulting from substitutions. It is unusual for two homologous enzymes to exhibit such striking differences in action. Therefore, the results have to be treated with caution. However, the changes in the locations of ligands exhibited by M. tuberculosis pantothenate kinase are remarkable and novel.
Assuntos
Proteínas de Bactérias/metabolismo , Mycobacterium tuberculosis/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Substituição de Aminoácidos , Proteínas de Bactérias/química , Sítios de Ligação , Coenzima A/metabolismo , Cristalização , Cristalografia por Raios X , Proteínas de Escherichia coli/química , Fator VIIa/química , Fator VIIa/metabolismo , Ligantes , Modelos Moleculares , Ácido Pantotênico/análogos & derivados , Ácido Pantotênico/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/química , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidade da EspécieRESUMO
CIA17 is a PP2-like, homodimeric lectin made up of 17â¯kDa subunits present in the phloem exudate of ivy gourd (Coccinia indica). Isothermal titration calorimetric (ITC) studies on the interaction of chitooligosaccharides [(GlcNAc)2-6] showed that the dimeric protein has two sugar binding sites which recognize chitotriose with ~70-fold higher affinity than chitobiose, indicating that the binding site is extended in nature. ITC, atomic force microscopic and non-denaturing gel electrophoresis studies revealed that the high-affinity interaction of CIA17 with chitohexaose (Kaâ¯=â¯1.8â¯×â¯107â¯M-1) promotes the formation of protein oligomers. Computational studies involving homology modeling, molecular docking and molecular dynamics simulations on the binding of chitooligosaccharides to CIA17 showed that the protein binding pocket accommodates up to three GlcNAc residues. Interestingly, docking studies with chitohexaose indicated that its two triose units could interact with binding sites on two protein molecules to yield dimeric complexes of the type CIA17-(GlcNAc)6-CIA17, which can extend in length by the binding of additional chitohexaose and CIA17 molecules. These results suggest that PP2 proteins play a role in plant defense against insect/pathogen attack by directly binding with the higher chain length chitooligosaccharides and forming extended, filamentous structures, which facilitate wound sealing.
Assuntos
Quitosana/química , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Lectinas de Plantas/metabolismo , Acetilação , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Lectinas de Plantas/química , Conformação Proteica , TermodinâmicaRESUMO
The napin from Brassica juncea, oriental mustard, is highly thermostable, proteolysis resistant and allergenic in nature. It consists of two subunits - one small (29 amino acid residues) and one large (86 amino acids residues) - held together by disulfide bonds. The thermal unfolding of napin has been followed by differential scanning calorimetry (DSC) and circular dichroism (CD) measurements. The thermal unfolding is characterized by a three state transition with T(M1) and T(M2) at 323.5 K and 335.8 K, respectively; DeltaC(P1) and DeltaC(P2) are 2.05 kcal mol(-1) K(-1) and 1.40 kcal mol(-1) K(-1), respectively. In the temperature range 310-318 K, the molecule undergoes dimerisation. Isothermal equilibrium unfolding by guanidinium hydrochloride also follows a three state transition, N <_-_-> I <_-_-> U with DeltaG(1H2O) and DeltaG(2H2O) values of 5.2 kcal mol(-1) and 5.1 kcal mol(-1) at 300 K, respectively. Excess heat capacity values obtained, are similar to those obtained from DSC measurements. There is an increase in hydrodynamic radius from 20 A to 35.0 A due to unfolding by guanidinium hydrochloride. In silico alignment of sequences of napin has revealed that the internal repeats (40%) spanning residues 31 to 60 and 73 to 109 are conserved in all Brassica species. The internal repeats may contribute to the greater stability of napin. A thorough understanding of the structure and stability of these proteins is essential before they can be exploited for genetic improvements for nutrition.
Assuntos
Mostardeira/metabolismo , Proteínas de Plantas/química , Albuminas 2S de Plantas , Sequência de Aminoácidos , Varredura Diferencial de Calorimetria , Cromatografia , Cromatografia em Gel , Dicroísmo Circular , Guanidina/química , Dados de Sequência Molecular , Desnaturação Proteica , Dobramento de Proteína , Homologia de Sequência de Aminoácidos , Temperatura , Termodinâmica , Fatores de TempoRESUMO
RecA protein is a crucial and central component of the homologous recombination and DNA repair machinery. Despite numerous studies on the protein, several issues concerning its action, including the allosteric regulation mechanism have remained unclear. Here we report, for the first time, a crystal structure of a complex of Mycobacterium smegmatis RecA (MsRecA) with dATP, which exhibits a fully ordered C-terminal domain, with a second dATP molecule bound to it. ATP binding is an essential step for all activities of RecA, since it triggers the formation of active nucleoprotein filaments. In the crystal filament, dATP at the first site communicates with a dATP of the second site of an adjacent subunit, through conserved residues, suggesting a new route for allosteric regulation. In addition, subtle but definite changes observed in the orientation of the nucleotide at the first site and in the positions of the segment preceding loop L2 as well as in the segment 102-105 situated between the 2 nt, all appear to be concerted and suggestive of a biological role for the second bound nucleotide.
Assuntos
Proteínas de Bactérias/química , Nucleotídeos de Desoxiadenina/química , Mycobacterium smegmatis , Recombinases Rec A/química , Regulação Alostérica , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Modelos Moleculares , Ligação Proteica , Estrutura Terciária de Proteína , Recombinases Rec A/metabolismo , TermodinâmicaRESUMO
Two point mutants and the corresponding double mutant of Mycobacterium tuberculosis pantothenate kinase have been prepared and biochemically and structurally characterized. The mutants were designed to weaken the affinity of the enzyme for the feedback inhibitor CoA. The mutants exhibit reduced activity, which can be explained in terms of their structures. The crystals of the mutants are not isomorphous to any of the previously analysed crystals of the wild-type enzyme or its complexes. The mycobacterial enzyme and its homologous Escherichia coli enzyme exhibit structural differences in their nucleotide complexes in the dimer interface and the ligand-binding region. In three of the four crystallographically independent mutant molecules the structure is similar to that in the E. coli enzyme. Although the mutants involve changes in the CoA-binding region, the dimer interface and the ligand-binding region move in a concerted manner, an observation which might be important in enzyme action. This work demonstrates that the structure of the mycobacterial enzyme can be transformed into a structure similar to that of the E. coli enzyme through minor perturbations without external influences such as those involving ligand binding.
Assuntos
Coenzima A/metabolismo , Mutação , Mycobacterium tuberculosis/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Sítios de Ligação , Cristalização , Cristalografia por Raios X , Escherichia coli/enzimologia , Ligantes , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Ligação Proteica , Conformação ProteicaRESUMO
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.
Assuntos
Adjuvantes Imunológicos , Carboidratos , Lectinas de Plantas , Conformação Proteica , Adjuvantes Imunológicos/química , Adjuvantes Imunológicos/metabolismo , Artocarpus/química , Calorimetria , Metabolismo dos Carboidratos , Carboidratos/química , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Lectinas de Plantas/química , Lectinas de Plantas/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/metabolismoRESUMO
Monosialoganglioside liposomes are rapidly taken up by the liver as compared to dicetylphosphate, phosphatidic acid or neutral liposomes. Asialoganglioside GM 1 liposomes are taken up with the same avidity as ganglioside GM 1 liposomes. Competition experiments with asialofetuin suggest that this uptake is mediated by specific recognition of the terminal galactose residues of the glyco-lipid liposomes by the receptor present on the plasma membrane of the parenchymal cells of liver. Thus liposomes containing glycolipids with terminal beta-galactosyl residues should provide an approach for specifically directing biologically active molecules to liver parenchymal cells.
Assuntos
Gangliosídeos/metabolismo , Lipossomos/metabolismo , Fígado/metabolismo , Sacarase/metabolismo , Animais , Técnicas In Vitro , Cinética , Masculino , Ligação Proteica , Ratos , Ácidos Siálicos , alfa-Fetoproteínas/metabolismoRESUMO
The galactose binding toxin (RCAII) from Ricinus communis was affinity-immobilised at varying densities on a polysaccharide matrix and reacted with glutaraldehyde. The critical density below which inter-molecular cross-links were not formed was determined. At this density RCAII was monoconjugated to lysozyme. This approach could serve as a prototype for enzyme-lectin and enzyme-antipolysaccharide antibody monoconjugation.
Assuntos
Polissacarídeos , Proteínas , Reagentes de Ligações Cruzadas , Densitometria , Galactose , Glutaral , Lectinas , Muramidase , Polímeros/síntese química , Ligação Proteica , Toxinas BiológicasRESUMO
A galactose-specific lectin isolated from Ricinus communis beans has been covalently coupled to Sepharose 4B activated with cyanogen bromide. The immobilized lectin retains its polysaccharide-binding property. The Sepharose-lectin can be used for the purification of polysaccharides containing terminal nonreducing galactose. Only a small fraction of 'native fetuin' and 'native ceruloplasmin' are retarded on Sepharose-lectin. On analysis it was observed that they had a lower content of sialic acids as compared to the native and unbound glycoproteins (sialated fractions). However, on desialation, fetuin and ceruloplasmin were completely adsorbed to Sepharose-lectin. The asialoglycoproteins interact strongly with Sepharose-lectin as compared to 'partially sialated glycoproteins'. This has been attributed to the exposure of galactose residues of these glycoproteins on enzymatic desialation. These experiments demonstrated that Sepharose-lectin interacts with glycoproteins through their terminal, non-reducing galactose. On the basis of these experiments it is suggested that Sepharose-lectin can be used as an analytical tool for separation of 'fully sialated glycoproteins' from the 'partially sialated glycoproteins'.
Assuntos
Galactose/análise , Glicoproteínas/isolamento & purificação , Lectinas , Ceruloplasmina/isolamento & purificação , Cromatografia de Afinidade , Glicoproteínas/análise , Lectinas de Plantas , Plantas Tóxicas , Ricinus , Sefarose , Ácidos Siálicos/análise , alfa-Fetoproteínas/isolamento & purificaçãoRESUMO
Legume lectins family of proteins, despite having the same 'jelly roll' tertiary structural fold at monomeric level, exhibit considerable variation in their quaternary structure arising out of small changes in their sequence. Nevertheless, their folding behavior and stability correlates very well with their patterns of assembly into dimers and tetramers. A conservation of their fold during evolution, its wide distribution in many protein families together with the availability of structural information on them make them interesting as proteins to explore the effect of inter- versus intra-subunit interactions in the stability of multimeric proteins. Additionally, as 'natural mutants' of quaternary association, proteins of legume lectin family provide interesting paradigms for studies addressing the effect of subunit oligomerization on the stability, folding and function as well as the evolution of multimeric structures.
Assuntos
Fabaceae/química , Lectinas/química , Plantas Medicinais , Sequência de Aminoácidos , Varredura Diferencial de Calorimetria , Dimerização , Modelos Moleculares , Dados de Sequência Molecular , Lectinas de Plantas , Desnaturação Proteica , Dobramento de Proteína , Alinhamento de SequênciaRESUMO
The process cascade leading to the final accommodation of the carbohydrate ligand in the lectin's binding site comprises enthalpic and entropic contributions of the binding partners and solvent molecules. With emphasis on lactose, N-acetyllactosamine, and thiodigalactoside as potent inhibitors of binding of galactoside-specific lectins, the question was addressed to what extent these parameters are affected as a function of the protein. The microcalorimetric study of carbohydrate association to the galectin from chicken liver (CG-16) and the agglutinin from Viscum album (VAA) revealed enthalpy-entropy compensation with evident protein type-dependent changes for N-acetyllactosamine. Reduction of the entropic penalty by differential flexibility of loops or side chains and/or solvation properties of the protein will have to be reckoned with to assign a molecular cause to protein type-dependent changes in thermodynamic parameters for lectins sharing the same monosaccharide specificity.
Assuntos
Galactosídeos/metabolismo , Hemaglutininas/metabolismo , Plantas/metabolismo , Animais , Aves , Calorimetria/métodos , Galectinas , Ligantes , Ligação Proteica , TermodinâmicaRESUMO
Recognition of cell-surface carbohydrates by lectins has wide implications in important biological processes. The ability of plant lectins to detect subtle variations in carbohydrate structures found on molecules, cells and organisms have made them a paradigm for protein-carbohydrate recognition. Legume lectins, one of the most well studied family of plant proteins, display a considerable repertoire of carbohydrate specificities owing perhaps to the sequence hypervariability in the loops constituting their combining site. However, lack of a rigorous framework to explain their carbohydrate binding specificities has precluded a rational approach to alter their ligand binding activity in a meaningful manner. This study reports an extensive analysis of sequences and structures of several legume lectins and shows that despite the hypervariability of their combining regions they exhibit within a significant pattern of uniformity. The results show that the size of the binding site loop D is invariant in the Man/Glc specific lectins and is possibly a primary determinant of the monosaccharide specificities of the legume lectins. Analyses of size and sequence variability of loops reveal the existence of a common theme that subserves to define their binding specificities. These results thus provide not only a framework for understanding the molecular basis of carbohydrate recognition by legume lectins but also a rationale for redesign of their ligand binding propensities.
Assuntos
Metabolismo dos Carboidratos , Fabaceae/química , Lectinas/química , Plantas Medicinais , Acetilgalactosamina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Dissacarídeos/metabolismo , Fucose/metabolismo , Ligação de Hidrogênio , Lectinas/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Monossacarídeos/metabolismo , Lectinas de Plantas , Proteínas de Plantas/metabolismo , Conformação Proteica , Estrutura Secundária de Proteína , Alinhamento de Sequência , SoftwareRESUMO
Structures of two crystal forms of the dimeric acidic winged bean agglutinin (WBAII) complexed with methyl-alpha-D-galactose have been determined at 3.0 A and 3.3 A resolution. The subunit structure and dimerisation of the lectin are similar to those of the basic lectin from winged bean (WBAI) and the lectin from Erythrina corallodendron (EcorL). The conformation of a loop and its orientation with respect to the rest of the molecule in WBAII are, however, different from those in all the other legume lectins of known structure. This difference appears to have been caused by the formation of two strategically placed salt bridges in the former. Modelling based on the crystal structures provides a rationale for the specificity of the lectin, which is very different from that of WBAI, for the H-antigenic determinant responsible for O blood group reactivity. It also leads to a qualitative explanation for the thermodynamic data on sugar-binding to the lectin, with special emphasis on the role of a tyrosyl residue in the variable loop in the sugar-binding region in generating the carbohydrate specificity of WBAII.
Assuntos
Fabaceae/química , Lectinas/química , Lectinas/metabolismo , Metilgalactosídeos/metabolismo , Plantas Medicinais , Sais/metabolismo , Sistema ABO de Grupos Sanguíneos/imunologia , Sítios de Ligação , Cristalografia por Raios X , Dimerização , Glicosilação , Lectinas/genética , Lectinas/imunologia , Ligantes , Magnoliopsida/química , Metilgalactosídeos/química , Metilgalactosídeos/imunologia , Modelos Moleculares , Oligossacarídeos/química , Oligossacarídeos/imunologia , Oligossacarídeos/metabolismo , Lectinas de Plantas , Ligação Proteica , Conformação Proteica , Subunidades Proteicas , Sais/química , Eletricidade Estática , Especificidade por Substrato , Tirosina/genética , Tirosina/metabolismoRESUMO
Four new crystal forms of the anti-T lectin from jackfruit (Artocarpus integrifolia) have been prepared and characterized. Three of them, two monoclinic (P21, a = 59.4 A, b = 83.3 A, c = 63.5 A, beta = 107.7 degrees; C2, a = 106.1 A, b = 53.9 A, c = 128.0 A, beta = 95.0 A) and one orthorhombic (C222(1), a = 98.1 A, b = 67.3 A, c = 95.1 A) were grown with 2-methylpentan-2,4-diol (MPD) as the precipitant while the fourth, an hexagonal form (P6(1)22, a = b = 129.6 A, c = 157.9 A), was obtained in the presence of methyl-alpha-D-galactopyranoside with polyethylene glycol 4000 as the precipitant. The reported relative molecular mass (Mr) of the lectin was found to be inconsistent with the solvent content of the crystals estimated using measured densities. The Mr was redetermined using size-exclusion chromatography in the presence of methyl-alpha-D-galactopyranoside and Ferguson-plot analysis of mobilities in polyacrylamide gel electrophoresis. The redetermined Mr (66,000) is consistent with the measured crystal densities. The orthorhombic and the hexagonal forms, which have one half molecule and one molecule, respectively, in the asymmetric unit, are suitable for high-resolution X-ray analysis.