316 research outputs found
Structure Analysis Of Plant Lectin Domains
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 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
No full textThe 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
Polyphenol Composition of Nutraceutical Concentrate Obtained from Edible Vegetable Oil Seeds
No full textThis Dissertation / Report is the outcome of investigation carried out by the creator(s) / author(s) at the department/division of Central Food Technological Research Institute (CFTRI), Mysore mentioned below in this page
EXPLORING THE MODERN MYTHOLOGY: ANALYSING KAVITA KANE’S KARNA’S WIFE: THE OUTCAST’S QUEEN AND SITA’S SISTER
No full textIn modern-day literature, the study of mythology has presented authors with a vast and intricate framework to craft elaborate narratives that delve deeply into the intricacies of human behaviour, interpersonal connections, and societal standards. This research paper embarks on a profound and comprehensive analysis of two compelling literary works by the esteemed author Kavita Kane, explicitly focusing on Karna’s Wife: The Outcast’s Queen and Sita’s Sister. Through a meticulous and rigorous examination of the character progression depicted in Karna’s Wife, the primary objective of this study is to uncover and elucidate how Kane adeptly illustrates the transformation of Uruvi’s character, the significant relationships that shape her journey, and the innovative perspective she brings to the enigmatic persona of Karna. Simultaneously, the scrutiny of themes and symbolism in Sita’s Sister delves deeply into the core thematic foundations of the novel, the symbolic elements skilfully utilised by Kane to enhance the storyline, and the crucial influence of familial and societal expectations in moulding the characters within the narrative. By closely analysing these critical components, this research paper strives to illuminate the intricate layers of storytelling and the contemporary reinterpretation of classical mythology in Kane’s literary creations, thereby contributing to the enhanced comprehension of the enduring fascination of mythology within present-day literature
Efficacy of Solvent Extracts of Nelumbo nucifera Gaertn (Nelumbonaceae) and Melia dubia Cav (Meliacae) against Fall Armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae)
No full textSpodoptera frugiperda is a significant pest of economic importance due to its high rate of reproduction, potential for damage and capacity to consume several types of plants. It has become resistant to numerous chemical pesticides. It is challenging to control this pest in field due to the lack of host plant resistance and inadequate management techniques. Bioactive molecules of plant origin hold potential alternative for the chemical pesticides. In the present study, leaves of Nelumbo nucifera and Melia dubia were extracted with acetone, ethyl acetate and benzene. All the solvent extracts of N. nucifera and M. dubia exhibited larval, pupal and adult malformation of S. frugiperda. At higher doses, these plant extract exerted medium antifeedancy. In regarding, insect growth regulatory (IGR) activity, maximum of 33.33% larval malformation at 7% benzene extract of M. dubia, 53.33% pupal malformation at 7% benzene extract of N. nucifera and 33.33% adult malformation at 5% ethyl acetate extract of N. nucifera was recorded. In comparing all the extracts, the benzene extract of N. nucifera showed maximum IGR activity against S. frugiperda at 5%
HR-LCMS Profiling of phytochemical constituents and evaluation of antioxidant, antibacterial, anti-cancerous and anti-inflammatory potentials, plasma biocompatibility and cytotoxicity of Grewia orbiculata Rottler
No full textInfectious diseases are one of the main reasons that are causing a greater number of deaths in the world owing to their strong resistance development and evolution. There is an immediate urgency for the discovery of drugs with a new class or new mode of action to combat these resistant bugs. In the past few decades, we have not been able to find new antibiotics, which are effective on resistant bugs. Instead of searching for synthetic molecules, if we divert our search for alternative sources that are abundant in nature, we can easily find new molecules. Plants are the best as they are known to possess complex molecules that are strong in their potency while being relatively safe for the host and tough on pathogens. With this rationale, the study was conducted to assess the phytochemical constituents of different parts of plant Grewia orbiculataRottler using different solvents and to elucidate the biological activities. From qualitative analysis of all extracts, Methanolic Extract of Bark (MEB) and Ethyl acetate Extract of Leaf (EEL) were found to be rich in total phenolics and total flavonoids. Major phytochemicals found in MEB were Catechin, Epicatechin, and Carnitine and in EEL were Quinin acid, Gallic acid, Catechol, Isoquinoline, Coumaric acid, Kaempferol, and Quercetin of G. orbiculata. Upon testing the biopotentials of these extracts, it was found that among the different solvent extracts of leaves, twigs, buds, and bark, MEB showed the highest biological potential and therapeutic value. The antioxidant property of MEB assessed through DPPH and ABTS assays resulted in an IC50 value of 50 µg/mL and 36 µg/mL, respectively. The metal chelating property of MEB gave a FRAP value of 24 ± 0.093 mmol/g equivalent to that of Tannic acid. Further, MEB was found to possess very good antibacterial activity against human pathogens such as Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterococcus faecalis, Enterococcus faecium, Streptococcus epidermidis, and Mycobacterium smegmatis. In addition, MEB also showed good anti-cancerous property against A549 cells, having IC50 value of 98.73 µg/mL. The anti-inflammatory assay with MEB showed protection of BSA denaturation up to a concentration of 1000 µg/mL. Finally, the biocompatibility assay with blood showed no significant agglutination of RBCs up to a concentration of 200 µg/mL and cytotoxicity of MEB resulted in less than 50 inhibition of HTE cell proliferation at the highest concentration of 320 µg/mL, proving its non-toxic nature towards normal cells. Our study is the first to report and evaluate the therapeutic value of the plant G. orbiculata. MEB was found to possess very good therapeutic potential and can be used as potent antimicrobial agent to treat deadly human infections. © 2022, The Author(s) under exclusive licence to Society for Plant Research
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
Structural and functional studies on DNA damage inducible protein 1 (Ddi1) from protozoa
No full textStructural and functional investigations on DNA-damage inducible protein1 (Ddi1) from Entamoeba histolytica, Trypanosoma cruzi and Toxoplasma gondii have been carried out. Ddi1 belongs to the ubiquitin receptor family of proteins. One of its domains is similar to the retroviral aspartic proteinases. It has been shown that this domain is the target of HIV-protease inhibitors that were being used in the treatment of AIDS and it was observed that these drugs reduced the infection caused by many parasitic protozoa such as Trypanosoma and Leishmania species that are responsible for prevalent opportunistic diseases in AIDS patients. The retroviral protease-like domain (RVP) present in Ddi1 proteins of these organisms was identified as the target of these drugs. The binding of the RVP domain of Ddi1 from E. histolytica, T. cruzi and T. gondii with HIV protease inhibitors; and the binding of ubiquitin and K48-linked diubiquitin with the ubiquitin associated domain (UBA) have been established by Biolayer Interferometry (BLI). The crystal structure of the RVP domain of Ddi1 from T. gondii (ToxoDdi1-RVP) shows that it forms a homodimer similar to that observed in HIV protease and the reported structures of the same domain from S. cerevisiae, L. major and human. The ‘flap’ regions in ToxoDdi1-RVP are similar to the flaps of HIV protease which close-in upon substrate/inhibitor binding. Both the ‘flap’ regions are clearly visible in the electron density maps. Though the native form of the domain shows an open dimeric structure, normal mode analysis reveals that it can take up a closed conformation resulting from relative
movements of the subunits. Comparison of the structure of ToxoDdi1-RVP with the available crystal structures of Ddi1-RVP from other organisms revealed that the active site architecture is conserved in all the proteins with differences in the back β-sheet topology and the size of the binding cavity
Structural Studies on DNA Damage Inducible Protein 1 (Ddi1) of Leishmania and the Rotavirus Nonstructural Protein NSP4
No full textStructuraj investigations on the Ddi1 (DNA-damage inducible protein 1) of Leishmania major and on the rotavirus nonstructural protein NSP4 were carried out. Ddi1 belongs to the ubiquitin receptor family of proteins. One of its domains is similar to the retroviral aspartic proteinases. It has been shown that this domain is the target of HIV-protease inhibitors that were being used in the treatment of AIDS and it was observed that these drugs effectively controlled opportunistic diseases caused by many parasitic protozoa such as Leishmania and Plasmodium species. The retroviral protease-like domains present in Ddi1 proteins of these organisms were identified as the targets of these drugs. Structural studies on Ddi1 from L. major have been carried out, in an attempt to provide a platform for the design of anti-protozoal compounds. Rotavirus NSP4, the first viral enterotoxin to be identified, is a multifunctional glycoprotein that plays critical roles in viral pathogenesis and morphogenesis. As part of an ongoing project on the structural characterization of NSP4, we determined the structure of the diarrhea-inducing region of this protein from the rotavirus strain MF66.
Chapter 1 presents an overview of Ddi1 and NSP4 of the rotavirus with an emphasis on their structural features. The methods employed during the course of the present work are described in Chapter 2.
Structural studies on the retroviral protease-like domain of Ddi1 (Ddi1-RVP) of L. major is presented in Chapter 3. Apart from this domain, Ddi1 of L. major also has a ubiquitin-associated and ubiquitin-like domains whereas P. falciparum has only the ubiquitin-associated domain. Activity of the full length Ddi1 of L. major and the retroviral protease domain of P. falciparum using an HIV protease substrate was shown to be inhibited by an HIV protease inhibitor, saquinavir. Binding of saquinavir to the proteins was also confirmed by Biolayer Interferometry studies. The crystal structure of the retroviral protease domain of L. major Ddi1 has been determined. It forms a homodimeric structure similar to that of HIV protease and the reported structure of the same domain from Saccharomyces cerevisiae. The loops in Ddi1-RVP are similar to the 'flap' regions of the HIV protease which close-in upon substrate/inhibitor binding; they are visible in the electron density maps, unlike the case of the S. cerevisiae protein. Though the native form of the domain shows an open dimeric structure, normal mode analysis reveals that it can take up a closed conformation resulting from relative movements of the subunits. The present structure of Ddi1-RVP of L. major with the defined 'flap'-like loops will be helpful in the design of effective drugs against protozoal diseases, starting with HIV protease inhibitors as the lead compounds.
Chapter 4 describes the structural investigations carried out on the diarrhea-inducing region of the nonstructural protein NSP4 of the rotavirus strain MF66 which forms an α-helical coiled-coil structure. Crystal structures of a synthetic peptide and of two recombinant proteins spanning this region showed parallel tetrameric organization of this domain with a bound Ca2+ ion at the core. Subsequently, we determined the structure of NSP4 from a different strain as a pentamer without the bound Ca2+ ion. This new structure provides more insights into understanding some of the functions of NSP4 such as the release of ions into the cytoplasm and binding to the double-layered particle (DLP). We also established conditions responsible for these structural transitions. The crystal structure of the coiled-coil domain of NSP4 presented in this chapter shows an entirely different structure which is an antiparallel tetramer. This explains our failure to determine the structure by the molecular replacement method using known oligomers. The structure was solved by the Sulphur-SAD method using diffraction data collected with Cr Ka radiation. The study reveals that the structural diversity of NSP4 is not limited. We could relate sequence variations and pH conditions to the differences in oligomeric assemblies. Surface properties of the domain suggest that the new form is likely to interact with different sets of proteins compared to those that interact with the parallel tetramers or pentamers. Further investigations are needed to establish this property
The Effects Of Shifting Temperature On The Growth Of Listeria Monocytogenes And Salmonella Typhimurium In Goat Milk Samples Collected From Local Dairy Farms
No full textThe main objective of this study was to determine the adaptation and survival curves
of Listeria monocytogenes and Salmonella Typhimurium in goat milk samples
collected in local dairy farm in Penang Island. This research was conducted to
understand the effects of shifting L. monocytogenes and S. Typhimurium from 37°C
to various selected stress temperature in goat milk samples as growth media. The
study was divided into 3 main parts. First part of the study was to determine the
prevalence microorganisms and pathogens especially L. monocytogenes, S.
Typhimurium, total plate count, yeast and mould count and coliform count in goat
milk samples from 2 farms. The second part was to establish the growth profile for
these two pathogens at 37°C in Tryone Soy Broth media. From the plotted growth
curve, stationary phase at 10-7 CFU/ml at 18 and 10 hours of growth was used as the
starting inoculum for the stress conditions for L. monocytogenes and S.
Typhimurium, respectively. In the third part of study, these pathogens were grown at
37°C in TSB medium before shifting to goat milk at different temperature ranging
from 0°C to 90°C. Results of shifting L. monocytogenes and S. Typhimurium from
37°C to lower temperatures from 0 up to 15°C showed bacteriostatic effects, while
shifting from 37°C to 25°C showed the ability of L. monocytogenes and S.
Typhimurium to grow gradually in goat milk. In contrast, shifting of L.
monocytogenes and S. Typhimurium to higher temperatures (from 50°C to 70°C)
exhibited growth of both pathogens after few hours of stress and demonstrated
straight-line death kinetics
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