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Structure Analysis Of Plant Lectin Domains
Get PDFLectins are multivalent carbohydrate binding proteins that specifically recognise diverse sugar structures and mediate a variety of biological processes, such as cell-cell and host-pathogen interactions, serum glycoprotein turnover and innate immune responses. Lectins have received considerable attention in recent years on account of their properties leading to wide use in research and biomedical applications. Seeds of leguminous plants are mainly rich sources of lectins, but lectins are also found in all classes and families of organisms. Legume lectins have similar tertiary structures, but exhibit a large variety of quaternary structures. The carbohydrate binding site in them is made up of four loops, the first three of which are highly conserved in all legume lectins. The fourth loop, which is variable, is implicated in conferring specificity. Legume lectins which share the same monosaccharide specificity often exhibit markedly different oligosaccharide specificities. This thesis primarily concerns with structure solution and analysis of lectins from the legume and β-prism II fold families using X-ray crystallography. Apart from having the property of specifically and reversibly binding to carbohydrates, lectins are also interesting models to study sequence-structure relationships, especially of how minor change in the sequence may bring about major changes in oligomerization and binding.
Chapter 1 gives an overview of different structural types of plant lectins and describes in detail, their carbohydrate binding features. The details of the various experimental procedures employed during the course of this research, are explained in Chapter 2.
Chapter 3 describes the crystal structure of a β-prism II fold lectin (RVL), from Remusatia vivipara, an epiphytic plant of traditional medicinal value, and analysis of its binding properties. This lectin was established to have distinct binding properties and has nematicidal activity against a root-knot nematode with the localization site identified as the high-mannose displaying gut-lining in the nematode. The crystal structure of RVL revealed a new quaternary association of this homodimeric lectin, different from those of reported β-prism II lectins. Functional studies on RVL showed that it fails to bind to simple mannose moieties yet showed agglutination with rabbit blood cells (which have mannose moieties on the surface) and some high mannose containing glycoproteins like mucin and asialofetuin. Further, ELISA and glycan array experiments indicated that RVL has high affinity to N-glycans like trimannose pentasaccharide such as in gp120, a capsid glycoprotein of HIV virus, necessary in virus-association with the host cell. The structural basis for this N-glycan binding was revealed through structure analysis and molecular modelling, and it was demonstrated that there are two distinct binding sites per monomer, making RVL a truly multivalent lectin. Evolutionary phylogeny revealed the divergence in the β-prism II fold proteins with regards to the number of sugar-binding regions per domain, oligomerization and specificity.
Chapter 4 deals with the structural studies on a galactose-specific legume lectin (DLL-II) from Dolichos lablab, a leguminous plant. The lectin was found to be a planar tetramer in the crystal structures of the native and ligand bound forms, as expected from our solution studies and phylogenetic analysis. The protein is a heterotetramer with subunits differing only in the presence or absence of a C-terminal helical region at the core of the tetramer. Due to the static disorder in all the crystals, the central helix could be oriented in either direction. Structure analysis of DLL-II proved to be an interesting endeavour as static disorder compounded with twinning in the crystal made the data processing and structure solution a challenging process. Subsequent structure and sequence alignments led to the identification of an adenine-binding pocket in the hydrophobic core of the tetramer. Based on this, DLL-II lectin was co-crystallized with adenine and the structure revealed the presence of adenine at the predicted binding site.
Chapter 5 describes the identification and analysis of potential plant lectins/lectin-like domains in the genome of Oryza sativa, using bioinformatics approaches. This project was initiated to study the occurrence of legume-lectin like domains (a predominant dicot feature) in O. sativa, which is a monocot. Later, a large scale genome analysis for all types of lectin domains was carried out through exhaustive PSI-BLAST, profile matching by HMMer, CDD and MulPSSM. The final validation was carried out by assessing the carbohydrate binding potential of the domain by examining the sugar binding sites. The primary interest in undertaking this work was to find the occurrence of association of these domains with other domains as in protein receptor kinases, where lectin is the receptor domain. Though primarily initiated as a bioinformatics project, further structural characterization was attempted by cloning, expression and purification of some of the annotated lectin proteins using prokaryotic expression systems. The protein expression was attained in reasonable amounts for a few of the annotated legume lectin homologs, however purification is yet to be achieved as the expressed proteins are insoluble.
A part of the results described in this thesis and the other related projects that the author was involved are reported in the following publications.
1) Purification, characterization and molecular cloning of a monocot mannose-binding lectin from Remusatia vivipara with nematicidal activity Bhat GG, Shetty KN, Nagre NN, Neekhra VV, Lingaraju S, Bhat RS, Inamdar SR, Suguna K, Swamy BM. 2010. Glycoconjugate J. 27(3):309-320
2) Modification of the sugar specificity of a plant lectin: structural studies on a point mutant of Erythrina corallodendron lectin Thamotharan S, Karthikeyan T, Kulkarni KA, Shetty KN, Surolia A, Vijayan M & Suguna K. 2011. Acta Crystallographica D 67(3):218-227
3) Crystal structure of a β-prism II lectin from Remusatia vivipara Shetty KN, Bhat GG, Inamdar SR, Swamy BM, Suguna K. 2012. Glycobiology 22(1): 56-69.
4) Structure of a galactose binding lectin from Dolichos lablab
Shetty KN, Lavanyalatha V, Rao RN, SivaKumar N & Suguna K (Under review)
5) Occurrence of lectin-like domains: Oryza sativa genome analysis. Shetty KN & Suguna K. (Manuscript in preparation
Structure Analysis Of FabI And FabZ Enzymes Of The Fatty Acid Biosynthesis Pathway Of Plasmodium Falciparum
Get PDFThe emergence of drug resistant strains of Plasmodium has given a new face to the old disease, malaria. One of the approaches is to block metabolic pathways of the pathogen. The current thesis describes the X-ray crystallographic analysis of two enzymes of the fatty acid biosynthesis pathway of the malaria parasite Plasmodium falciparum. In order to understand the functional mechanism and mode of inhibitor binding, enzyme-inhibitor complexes were characterized, which could help in further improvement of the efficacy of the inhibitors and hence to fight against the disease.
The introductory chapter of the thesis presents a discussion on malaria and different metabolic pathways of the pathogen which could be suitable targets for novel antimalarials. In continuation to that, the pathway of our choice the fatty acid biosynthesis and an overview of the structural features of the enzymes involved in the pathway that have been characterized from different organisms are also described. The second chapter includes the tools of X-ray crystallography that were used for structural studies of the present work. It also discusses the biochemical, biophysical and other computational methods used to further characterize the enzymes under study.
Triclosan, a well known inhibitor of Enoyl Acyl Carrier Protein Reductase (FabI) from several pathogenic organisms, is a promising lead compound to design effective drugs. The X-ray crystal structures of Plasmodium falciparum FabI (PfFabI), in complex with triclosan variants having different substituted and unsubstituted groups at different key functional locations, were determined and compared with triclosan binding which form the basis of chapter 3. The structures revealed that 4 and 2’ substituted compounds have more interactions with the protein, cofactor and solvent molecules as compared to triclosan. New water molecules were found to interact with some of these inhibitors. Substitution at the 2’ position of triclosan caused the relocation of a conserved water molecule, leading to an additional hydrogen bond with the inhibitor. This observation can help in conserved water based inhibitor design. 2’ and 4’ unsubstituted compounds showed a movement away from the hydrophobic pocket to compensate for the interactions made by the halogen groups of triclosan. This compound also makes additional interactions with the protein and cofactor which compensates for the lost interactions due to the unsubstitution at 2’ and 4’. In cell culture, this inhibitor shows less potency, which indicates that the chlorines at 2’ and 4’ positions increase the ability of the inhibitor to cross multilayered membranes. This knowledge helps us to modify the different functional groups of triclosan to get more potent inhibitors.
Certain residues in the substrate binding tunnel of PfFabI were mutated to identify the role of these residues in substrate binding and protein stability, which forms the 4th chapter of the thesis. The substrate binding site residue Ala372 of PfFabI has been mutated to Methionine and Valine which increased the affinity of the enzyme towards triclosan to almost double, close to that of Escherichia coli FabI (EcFabI) which has a Methionine at the structurally similar position of Ala372 of PfFabI. Kinetic studies of the mutants of PfFabI and the crystal structure analysis of the A372M mutant revealed that a more hydrophobic environment enhances the affinity of the enzyme for the inhibitor. A triclosan derivative showed a 3-fold increase in the affinity towards the mutants compared to the wild type, due to additional interactions with the A372M mutant as revealed by the crystal structure. The enzyme has a conserved salt bridge which stabilizes the substrate binding loop and appears to be important for the active conformation of the enzyme. A second set of mutants generated to check this hypothesis exhibited loss of function, except in one case where, the crystal structure showed that the substrate binding loop is stabilized by a water bridge network.
The main focus of chapter 5 is β-Hydroxyacyl-acyl carrier protein dehydratase of Plasmoduim falciparum (PfFabZ) which catalyzes the third and important reaction of the fatty acid elongation cycle. The crystal structure of PfFabZ was available in its hexameric (active) and dimeric (inactive) forms. However, until now PfFabZ has not been crystallized with any bound inhibitors. We have designed a new condition to crystallize PfFabZ with its inhibitors bound in the active site, and determined the crystal structures of three of these complexes. This is the first report of the crystal structures of PfFabZ with competitive inhibitor complexes and the first such study on any FabZ enzyme with active site inhibitors. These inhibitors in the active site stabilize the substrate binding loop, revealing the substrate binding tunnel with an overall shape of “U”. In the crystal structure, the residue Phe169 located in the middle of the tunnel was found to be in two different conformations, open and closed, implying that it controls the length of the tunnel and makes it suitable for accommodating longer substrates merely by changing its side chain conformation. The hydrophobic nature of the substrate binding channel signifies the specificity for the hydrophobic tail of fatty acid substrates. The volume of the active site tunnel is determined by the sequence as well as by the conformation of the substrate binding site loop region and varies between organisms for accommodating fatty acids of different chain lengths. All PfFabZ inhibitors reported here bind to the active site through specific contacts like hydrogen bonds with catalytic residues and hydrophobic interactions. This report on the crystal structures of the complexes of PfFabZ provides the structural basis of the inhibitory mechanism of the enzyme, that could be used to improve the potency of inhibitors against an important component of fatty acid synthesis common to many infectious organisms.
The hot dog fold has been found in more than sixty proteins since the first report of its existence about a decade ago. The fold appears to have a strong association with fatty acid biosynthesis, its regulation and metabolism, as the proteins with this fold are predominantly coenzyme A-binding enzymes with a variety of substrates located at their active sites. We have analyzed the structural features and sequences of proteins having the hot dog fold. This study reveals that though the basic architecture of the fold is well conserved in these proteins, significant differences exist in their sequence, nature of substrate and oligomerization. Segments with certain conserved sequence motifs seem to play crucial structural and functional roles in various classes of these proteins. The analysis discussed in chapter 6, led to predictions regarding the functional classification and identification of possible catalytic residues of a number of hot dog fold-containing hypothetical proteins whose structures were determined in high throughput structural genomics projects.
Rv0098, predicted to be the FabZ of Mycobacterium tuberculosis, was cloned, expressed, purified, crystallized, and X-ray diffraction data were collected. Molecular replacement trials with all “hot dog” fold proteins failed to yield any significant solution due to the low sequence similarity (<20%) of Rv0098 compared to other FabZs. During the trials of structure solution by multiple isomorphous replacement method, structure of Rv0098 was published and it was shown to be a long-chain fatty acyl-CoA thioesterase (FcoT). The crystal structure of Rv0098 did not explain the molecular basis of substrate specificity of varying chain lengths. Molecular dynamics studies were carried out, which revealed that certain residues of the substrate binding tunnel are flexible and thus modulates the length of the tunnel. Flexibility of the loop at the base of the tunnel was also found to be important for determining the length of the tunnel for accommodating appropriate substrates. The structural basis of accommodating long chain substrates by Rv0098 is discussed in chapter 7, by combining the crystallographic and molecular dynamics studies.
Part of the work presented in the thesis has been reported in the following publications.
Karmodiya, K., Sajad, S., Sinha, S., Maity, K., Suguna, K. and Surolia, N. (2007) Conformational stability and thermodynamic characterization of homotetrameric Plasmodium falciparum beta-ketoacyl-ACP reductase. IUBMB Life 59, 441-9.
Pidugu, L. S., Maity, K., Ramaswamy, K., Surolia, N. and Suguna, K. (2009) Analysis of proteins with the 'hot dog' fold: prediction of function and identification of catalytic residues of hypothetical proteins. BMC Struct Biol 9, 37.
Kapoor, N., Banerjee, T., Babu, P., Maity, K., Surolia, N. and Surolia, A. (2009) Design, development, synthesis, and docking analysis of 2'-substituted triclosan analogs as inhibitors for Plasmodium falciparum enoyl-ACP reductase. IUBMB Life 61, 1083-91.
Maity, K., Bhargav, S. P., Sankaran, B., Surolia, N., Surolia, A. and Suguna, K. (2010) X-ray crystallographic analysis of the complexes of enoyl acyl carrier protein reductase of Plasmodium falciparum with triclosan variants to elucidate the importance of different functional groups in enzyme inhibition. IUBMB Life 62, 467-76.
Maity, K., Banerjee, T., Narayanappa, P., Surolia, N., Surolia, A. and Suguna, K. (2010) Effect of substrate binding loop mutations on the structure, kinetics and inhibition of Enoyl Acyl Carrier Protein Reductase from Plasmodium falciparum. (Communicated)
Maity, K., Bharat, S. V., Kapoor, N., Surolia, N., Surolia, A. and Suguna, K. (2010) Insights into the functional and inhibitory mechanism of the β-Hydroxyacyl-Acyl Carrier Protein Dehydratase of Plasmodium falciparum from the crystal structures of its complexes with active site inhibitors. (Communicated
A Multi-Language Comparison of Influences on Author Verification using Character N-Grams
No full textWe create a new multi-language corpus for author verification based on Wikipedia talkpages, and evaluate the influence that differences in topic and time have on character n-gram author profiles. Topic alignment between two texts is found to increase author verification precision, and an authors writing style is found to change over time, but not more significantly after 3 years than after 1 year.Information ArchitectureWISElectrical Engineering, Mathematics and Computer Scienc
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
The vanishing author in computer-generated works: a critical analysis of recent Australian case law
Get PDFAbstract
The use of software is ubiquitous in the creation of many copyright works, yet the requirement in copyright law that every work have a human author who engages in independent intellectual effort means that its use may prevent copyright subsistence. Several recent Australian cases have refocused attention on authorship as an essential criterion of copyright subsistence, and these cases suggest that much computer-produced output may be authorless and thus lack copyright protection. This article, the first in a two-part series, analyses how each case deals with the question of authorship of computer-produced works and why the use of software diminishes copyright protection for a significant number of computer-generated works. The article critiques the application of conventional notions of human authorship developed in the pre-computer age to modern productions and suggests alternative approaches to authorship that satisfy both the major objectives of copyright policy and the need to adapt to the computer age. The article argues that, without a broader judicial approach to authorship of computer-generated works, Parliament must remedy the lacuna in protection for these ‘authorless’ works. Possible solutions for reform are suggested. In a forthcoming article, the author comprehensively examines those reform proposals
Diffusive author(s), cohesive author: Analysis of S/N (1994)
No full textThis study indicates the ways in which various aspects of the author(s) are brought forth in Dumb type’s performance art, the S/N production. Previous research has suggested a non-hierarchical organization of Dumb type and the absence of a “privileged author” in Dumb type’s collaborative work, S/N. However, the results that I have investigated from member’s interviews on the creative process of S/N along with my analysis of the recorded images of S/N, indicate a different aspect of the author(s). First, S/N was created through, so to speak, the collective ideas of the members of Dumb type. Further, S/N has at least nine quotations from previous performances, installations, and printed writings, besides the work-in-progress technique. Explicating one of the “author functions” as given by Michel Foucault, each text has plural subjects of the author. However, it has been revealed from members’ interviews that Teiji Furuhashi had a decision-making role in selecting the members’ ideas within the performance. Since then, S/N has had plural subjects of creation; however, Furuhashi is one of the subjects of creation along with the “privileged author.” S/N has plural authors (diffusive authors) yet at the same time, it has a “privileged author,” Teiji Furuhashi (cohesive author)
Structural Studies On Winged Bean Agglutinins
No full textLectins are multivalent carbohydrate binding proteins that specifically recognise diverse sugar structures and mediate a variety of biological processes, such as cell-cell and host-pathogen interactions, serum glycoprotein turnover and innate immune responses. Lectins have received considerable attention in recent years on account of their properties which have led to their wide use in research and biomedical applications. Seeds of leguminous plants are rich sources of lectins, but they are also found in all classes and families of organisms. Legume lectins have similar tertiary structures, but exhibit a large variety of quaternary structures. The carbohydrate binding site in them is made up of four loops, the first three of which are highly conserved in all legume lectins. The fourth loop, which is variable, is implicated in conferring specificity. Legume lectins which share the same monosaccharide specificity often exhibit markedly different oligosaccharide specificities. The introductory chapter gives a broad overview of lectins from a structural point of view.
The rest of the thesis is primarily concerned with structural studies on lectins from seeds of the winged bean (Psophocarpus tetragonolobus). Winged bean seeds contain a basic lectin (WBAI) (pi > 9.5) and an acidic lectin (WBAII) (pi -5.5). Both these lectins are N-glycosylated homodimers with about 240 amino acid residues per monomer. They show a high affinity for methyl-a-D-galactose at the monosaccharide level but have entirely different affinities for oligosaccharides. WBAI agglutinates human type A and B erythrocytes but not O type, while WBAII binds specifically to the terminally monofucosylated H-antigenic (responsible for O blood group reactivity) determinants on the cell surface. In this context, the current study seeks to characterise the carbohydrate binding site of a saccharide-free form of WBAI and determine the structural basis of carbohydrate recognition in WBAII. The study also aims to identify the factors responsible for the differences in carbohydrate specificities between WBAI and WBAII.
Diffraction data from a saccharide-free crystal form of WBAI and two crystal forms (Form I and II) of WBAII complexed with methyl-a-D-galactose were collected on a MAR imaging plate system mounted on a Rigaku RU200 rotating anode X-ray generator. The data were processed using the MAR-XDS and DENZO/SCALEPACK suites of programs. The structures were solved by the molecular replacement method using AMoRe. The model used in the case of WBAI and Form I of WBAII was the structure of WBAI in complex with methyl-a-D-galactose (PDB coderlWBL), while the structure of Form II of WBAH was solved using a partially refined model of Form I. The refinements and model building were performed using the programs X-PLOR/CNS and O respectively.
A comparison of the structures of the saccharide-free and bound forms of WBAI revealed three water molecules occupying the carbohydrate binding site, which mimic the hydrogen bonded interactions made by the saccharide in the structure of the complex. Also a shift of -0.6 A in the variable loop, towards the saccharide in the structure of the complex was observed. Significant differences in the conformation of a loop involved in crystal packing interactions were also observed. An analysis of protein hydration demonstrates, among other things, the role of water molecules in stabilising the structure of the loops around the carbohydrate binding site.
The crystal structures of the two forms of WBAH were solved at 3.0 A and 3.3. A resolution. The structure of the complex revealed the role of the length of the variable loop in generating the difference in oligosaccharide specificity between WBAI and WB All. The difference in the pi values between the two lectins is caused by substitutions occurring in loops and edges of sheets. A distinct structural difference between WBAH and all the other legume lectins of known structure is in the new disposition of the 34-45 loop with an r.m.s deviation of -6.0A in Coc positions compared to its position in other lectins. This change in conformation is caused by the formation of salt bridges by amino acid residues unique to WB All in the 34-45 loop and its neighbourhood. Thermodynamic studies on the binding of H-antigenic determinant to WBAII showed a predominance of entropic contribution suggesting a hydrophobically driven binding, not yet observed in lectin-sugar interactions. An analysis involving the docking of H-type II trisaccharide (Fuca(l-2)Galf}(l-4)GlcNAc) into the carbohydrate binding site and a comparison with the binding sites of other legume lectins revealed the role of a Tyr in the variable loop and an Asn in the second loop that are unique to WBAII in generating this unique binding property.
Earlier work on peanut lectin and WBAI demonstrated that the modes of dimerisation of legume lectins are governed by features intrinsic to the protein. A phylogenetic analysis of the sequences of all legume lectins whose structures are available has been performed to examine the relationship among the various classes of oligomers and classes of sugar specificity. The information thus obtained showed that groups of legume lectins that share a common mode of dimerisation cluster together. A sequence alignment based on structures revealed amino acid residues unique to each of these clusters that may be important in determining the modes of observed dimerisation.
While pursuing structural studies on WBAI and WBAII, the author has also been involved in an ongoing small molecule project in the laboratory, which involves preparation and X-ray structure determination of the complexes of carboxylic acids with amino acids and peptides. The work carried out in the project is described in the appendix
Dissipative Range Scaling of Higher Order Structure Functions for Velocity and Passive Scalars
No full textDifferently to Kolmogorov's second similarity hypothesis, we find that the 2n-th order velocity and scalar structure functions scale with n-th order moment of the energy dissipation and the scalar dissipation, respectively. The origins of this scaling are analyzed by the transport equations of the fourth order velocity and scalar increment moments and by direct numerical simulations
Fast implementation of iterative adaptive approach for wideband unambiguous radar detection
No full textAccepted author manuscriptMicrowave Sensing, Signals & System
Ratio of n-6/n-3 in the diets of beef cattle
No full textEffects of feeding heat-treated canola (C), soybean (S) and flax (F) or mixtures on growth and slaughter characteristics, taste and fatty acid (FA) composition of beef tissue were investigated using 128 crossbred steers to determine the potential of improving the nutritional quality of beef for humans. For Trial 1 (48 steers), dietary treatments were: roasted C, extruded C, roasted S, extruded S, roasted F and extruded F. For Trial 2 (80 steers), the dietary treatments were: S:F (1:1), S:C (1:1), C:F (1:1) and S:F:C (1:1:1), and the oilseeds were processed either by roasting or extruding before mixing. Soybean meal and soybean oil were used to give equivalent lipid and protein contents to each experimental diet. The basal diet consisted of grass silage, barley grain, vitamins and minerals. Steers were fed for a minimum of 100d then slaughtered at a uniform degree of finish. Growth and slaughter characteristics of the steers were only slightly affected by dietary treatment in that the soybean-fed steers consumed more feed and had a higher average daily gain than the canola or flax-fed animals in Trial 1. There was no difference in taste panel parameters for any of the treatments. Inclusion of flax in the diet increased the total n-3 content of meat. Similar results were found for canola and C18:1n-9 although this was not the case for soybean and the n-6 FA. For the n-6 FA in the PL and neutral lipid fractions of the meat samples, levels were correlated with high dietary levels of n-6 or n-9 with low levels of n-3 while for the n-3 FA, levels were correlated with high dietary n-3 levels and low n-6 levels. Oilseed processing method did not have an effect on any fatty acid levels. It is possible to modify the FA composition of beef meat toward a healthier profile by including heat-treated oilseeds in the diet to influence the degree of lipid metabolism in the rumen.ID: S0377840111004007; M3: Article; Accession Number: S0377840111004007; Author: M.A. McNiven (a, ⁎); Author: J.L. Duynisveld (b); Author: T. Turner (a); Author: A.W. Mitchell (a); Affiliation: Department of Health Management, Atlantic Veterinary College, University of PEI, Charlottetown, PEI, Canada C1A 4P3; Affiliation: Agriculture and Agri-Food Canada, Nappan, NS, Canada B0L 1C0; Keyword: Oilseeds; Keyword: Roasted; Keyword: Extruded; Keyword: Fatty acids; Keyword: Healthy fat; Number of Pages: 11; Language: English
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