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Biomedical subjects

M Vijayan

Publications and source records attributed to M Vijayan.

At least 19 recordsLinked to original sources
PubMed2006

Overexpression, purification, crystallization and preliminary X-ray analysis of uracil N-glycosylase from Mycobacterium tuberculosis in complex with a proteinaceous inhibitor.

Uracil N-glycosylase is an enzyme which initiates the pathway of uracil-excision repair of DNA. The enzyme from Mycobacterium tuberculosis was co-expressed with a proteinaceous inhibitor from Bacillus subtilis phage and was crystallized in monoclinic space group C2, with unit-cell parameters a = 201.14, b = 64.27, c = 203.68 A, beta = 109.7 degrees. X-ray data from the crystal have been collected for structure analysis.

Crystallization↗
PubMed2006

Structural studies on peanut lectin complexed with disaccharides involving different linkages: further insights into the structure and interactions of the lectin.

Crystal structures of peanut lectin complexed with Galbeta1-3Gal, methyl-T-antigen, Galbeta1-6GalNAc, Galalpha1-3Gal and Galalpha1-6Glc and that of a crystal grown in the presence of Galalpha1-3Galbeta1-4Gal have been determined using data collected at 100 K. The use of water bridges as a strategy for generating carbohydrate specificity was previously deduced from the complexes of the lectin with lactose (Galbeta1-4Glc) and T-antigen (Galbeta1-3GalNAc). This has been confirmed by the analysis of the complexes with Galbeta1-3Gal and methyl-T-antigen (Galbeta1-3GalNAc-alpha-OMe). A detailed analysis of lectin-sugar interactions in the complexes shows that they are more extensive when the beta-anomer is involved in the linkage. As expected, the second sugar residue is ill-defined when the linkage is 1-->6. There are more than two dozen water molecules which occur in the hydration shells of all structures determined at resolutions better than 2.5 A. Most of them are involved in stabilizing the structure, particularly loops. Water molecules involved in lectin-sugar interactions are also substantially conserved. The lectin molecule is fairly rigid and does not appear to be affected by changes in temperature.

Disaccharides↗
PubMed2006

Cloning, expression, purification, crystallization and preliminary X-ray analysis of peptidyl-tRNA hydrolase from Mycobacterium tuberculosis.

Peptidyl-tRNA hydrolase catalyses the cleavage of the ester link between the peptide and the tRNA in peptidyl-tRNAs that, for various reasons, have dropped off the translating ribosomes. This enzyme from Mycobacterium tuberculosis has been crystallized in three related but distinct forms: P2(1)2(1)2(1), unit-cell parameters a = 36.30, b = 61.85, c = 73.97 A, P2(1), a = 35.83, b = 73.79, c = 59.79 A, beta = 92.3 degrees , and P2(1)2(1)2(1), a = 35.84, b = 57.06, c = 72.59 A. X-ray data have been collected from all three forms.

Bacterial Proteins↗
PubMed2006

Structure of Mycobacterium tuberculosis RuvA, a protein involved in recombination.

The process of recombinational repair is crucial for maintaining genomic integrity and generating biological diversity. In association with RuvB and RuvC, RuvA plays a central role in processing and resolving Holliday junctions, which are a critical intermediate in homologous recombination. Here, the cloning, purification and structure determination of the RuvA protein from Mycobacterium tuberculosis (MtRuvA) are reported. Analysis of the structure and comparison with other known RuvA proteins reveal an octameric state with conserved subunit-subunit interaction surfaces, indicating the requirement of octamer formation for biological activity. A detailed analysis of plasticity in the RuvA molecules has led to insights into the invariant and variable regions, thus providing a framework for understanding regional flexibility in various aspects of RuvA function.

Bacterial Proteins↗
PubMed2006

Invariance and variability in bacterial PanK: a study based on the crystal structure of Mycobacterium tuberculosis PanK.

Pantothenate kinase (PanK) is a ubiquitous and essential enzyme that catalyzes the first step of the universal coenzyme A biosynthetic pathway. In this step, pantothenate (vitamin B(5)) is converted to 4'-phosphopantothenate, which subsequently forms coenzyme A in four enzymatic steps. The complex of this enzyme from Mycobacterium tuberculosis (MtPanK) with a derivative of the feedback inhibitor coenzyme A has been crystallized in two forms and its structure solved. The structure was refined in both forms using room-temperature and low-temperature X-ray data. In both forms, the MtPanK subunit has a mononucleotide-binding fold with a seven-stranded central beta-sheet and helices on either side. However, there is a small though significant difference in subunit association between the two forms. The structure is also grossly similar to the enzyme from Escherichia coli. The active-site pocket and the dimeric interface are on two opposite sides of the PanK subunit. The enzymes from M. tuberculosis and E. coli exhibit several differences, particularly at the dimeric interface. On the other hand, the coenzyme A-binding region is almost entirely conserved. A delineation of the invariant and variable features of the PanK structure further indicates that the dimeric interface is very variable, while the coenzyme A-binding site is substantially invariant. A sequence alignment involving various bacterial PanKs is in agreement with this conclusion. The strong correlation between structural plasticity, evolutionary conservation and variability and function exhibited by the molecule could be important in the design of species-specific inhibitors of the enzyme.

Amino Acid Sequence↗
PubMed2006

Crystallographic identification of an ordered C-terminal domain and a second nucleotide-binding site in RecA: new insights into allostery.

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.

Allosteric Regulation↗
PubMed2006

X-ray studies of crystalline complexes involving amino acids and peptides. XLIII. Adipic acid complexes of L- and DL-lysine.

The asymmetric unit of the DL-lysine complex of adipic acid [bis(DL-lysinium) adipate], 2C6H15N2O2+.C6H8O(4)2-, contains a zwitterionic singly charged lysinium cation and half a doubly charged adipate anion (the complete anion has inversion symmetry). That of the L-lysine complex (lysinium hydrogen adipate), C6H15N2O2+.C6H9O4-, consists of a lysinium cation and a singly charged hydrogen adipate anion. In both structures, the lysinium cations organize into layers interconnected by adipate or hydrogen adipate anions. However, the arrangement of the molecular ions in the layer is profoundly different in the DL- and L-lysine complexes. The hydrogen adipate anions in the L-lysine complex form linear arrays in which adjacent ions are interconnected by a symmetric O...H...O hydrogen bond.

Adipates↗
PubMed2005

Structural biology of mycobacterial proteins: the Bangalore effort.

As part of an international effort and a national programme, structural analysis of mycobacterial proteins involved in recombination and repair, stringent response and protein synthesis has been undertaken, and work on proteins in a couple of metabolic pathways has been initiated. Already X-ray analysed are Mycobacterium tuberculosis and Mycobacterium smegmatis RecA and their nucleotide complexes, and different crystal forms of M. tuberculosis single-stranded DNA binding protein, M. smegmatis DNA binding protein from stationary phase cells and M. tuberculosis ribosome recycling factor. A comparative study involving these structures and those of similar proteins from other sources brings out the special features of the mycobacterial proteins, which are likely to be useful in selective inhibitor design. The structures provide insights into the plasticity of the molecules and its biological implications, and yield valuable information on their assembly and quaternary structure. They also provide leads for further structural investigations.

Bacterial Proteins↗
PubMed2005

A new relaxed state in horse methemoglobin characterized by crystallographic studies.

A new relaxed state has been characterized in the crystals of horse methemoglobin grown at neutral pH at low ionic concentration and their low humidity variants. The crystals provide an example for improvement in X-ray diffraction quality with reduced solvent content. Only the classical R state has been so far observed in liganded horse hemoglobin. The state characterized in the present study lies in between the R state and the R2 state characterized earlier in liganded human hemoglobin. The results presented here, along with those of earlier studies, suggest that relaxed and tense hemoglobin can access ensembles of states.

Animals↗
PubMed2005

Structure of Mycobacterium smegmatis single-stranded DNA-binding protein and a comparative study involving homologus SSBs: biological implications of structural plasticity and variability in quaternary association.

The structure of Mycobacterium smegmatis single-stranded DNA-binding protein (SSB) has been determined using three data sets collected from related crystals. The structure is similar to that of its homologue from Mycobacterium tuberculosis, indicating that the clamp arrangement that stabilizes the dimer and the ellipsoidal shape of the tetramer are characteristic features of mycobacterial SSBs. The central OB fold is conserved in mycobacterial SSBs as well as those from Escherichia coli, Deinococcus radiodurans and human mitochondria. However, the quaternary structure exhibits considerable variability. The observed plasticity of the subunit is related to this variability. The crystal structures and modelling provide a rationale for the variability. The strand involved in the clamp mechanism, which leads to higher stability of the tetramer, appears to occur in all high-G+C Gram-positive bacteria. The higher stability is perhaps required by these organisms. The mode of DNA binding of mycobacterial SSBs is different from that of E. coli SSB partly on account of the difference in the shape of the tetramers. Another difference between the two modes is that the former contains additional ionic interactions and is more susceptible to salt concentration.

Crystallography, X-Ray↗
PubMed2005

Unusual sugar specificity of banana lectin from Musa paradisiaca and its probable evolutionary origin. Crystallographic and modelling studies.

The crystal structure of a complex of methyl-alpha-D-mannoside with banana lectin from Musa paradisiaca reveals two primary binding sites in the lectin, unlike in other lectins with beta-prism I fold which essentially consists of three Greek key motifs. It has been suggested that the fold evolved through successive gene duplication and fusion of an ancestral Greek key motif. In other lectins, all from dicots, the primary binding site exists on one of the three motifs in the three-fold symmetric molecule. Banana is a monocot, and the three motifs have not diverged enough to obliterate sequence similarity among them. Two Greek key motifs in it carry one primary binding site each. A common secondary binding site exists on the third Greek key. Modelling shows that both the primary sites can support 1-2, 1-3, and 1-6 linked mannosides with the second residue interacting in each case primarily with the secondary binding site. Modelling also readily leads to a bound branched mannopentose with the nonreducing ends of the two branches anchored at the two primary binding sites, providing a structural explanation for the lectin's specificity for branched alpha-mannans. A comparison of the dimeric banana lectin with other beta-prism I fold lectins, provides interesting insights into the variability in their quaternary structure.

Amino Acid Motifs↗
PubMed2005

X-ray studies on crystalline complexes involving amino acids and peptides. XLII. Adipic acid complexes of L- and DL-arginine and supramolecular association in arginine-dicarboxylic acid complexes.

The adipic acid complexes of DL-arginine and L-arginine are made up of zwitterionic, singularly positively charged arginium ions and doubly negatively charged adipate ions, with a 2:1 stoichiometry. One of the two crystallographically independent arginium ions in the L-arginine complex has a conformation hitherto unobserved in crystal structures containing the amino acid. In the present study the structural data on arginine complexes of saturated dicarboxylic acids with 0-5 C atoms separating the two carboxyl functions are given. In terms of molecular aggregation, formic and acetic acid complexes behave in a similar way to those involving fairly long carboxylic acids such as adipic acid. By and large, the supramolecular assembly in complexes involving dicarboxylic acids with 3 or more C atoms separating the carboxyl groups (glutaric, adipic and pimelic acids), and those involving formic and acetic acids, have common features. The aggregation patterns in complexes involving oxalic, malonic and maleic acids do not share striking features among themselves (except for the mode of hydrogen-bonded dimerization of arginium ions) or with those involving larger dicarboxylic acids. Complexes of succinic acid, the shortest linear dicarboxylic acid, share features with those involving shorter as well as longer dicarboxylic acids. The difference in the behaviour of long and short dicarboxylic acids and the ambiguous behaviour of succinic acid can be broadly related to their lengths.

Adipates↗
PubMed2005

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

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

Adjuvants, Immunologic↗
PubMed2005

X-ray structural studies of Mycobacterium tuberculosis RRF and a comparative study of RRFs of known structure. Molecular plasticity and biological implications.

The crystal structure of Mycobacterium tuberculosis ribosome recycling factor has been determined and refined against three X-ray diffraction data sets, two collected at room temperature and the other at 100K. The two room-temperature data sets differ in the radiation damage suffered by the crystals before the data used for processing were collected. A comparison between the structures refined against the two data sets indicates the possibility of radiation-induced conformational change. The L-shaped molecule is composed of a long three-helix bundle domain (domain I) and a globular domain (domain II) connected by a linker region. The main difference between the room-temperature structure and the low temperature structure is in the rotation of domain II about an axis close to its libration axis. This observation and a detailed comparative study of ribosome recycling factors (RRFs) of known structures led to an elaboration of the present understanding of the structural variability of RRF. The variability involves a change in the angle between the two arms of the molecule, a rotation of domain II in a plane nearly perpendicular to the axis of the helix bundle and an internal rotation of domain II. Furthermore, the domains and the linker could be delineated into fixed and variable regions in a physically meaningful manner. The relative mobility of the domains of the molecule in the crystal structure appears to be similar to that in the ribosome--RRF complex. That permits a meaningful discussion of the structural features of RRF in terms of ribosome--RRF interactions. The structure also provides insights into the results of inter-species complementation studies.

Bacterial Proteins↗
PubMed2004

Expression, purification, crystallization and preliminary X-ray crystallographic analysis of pantothenate kinase from Mycobacterium tuberculosis.

Pantothenate kinase is an essential enzyme in the bacterial life cycle. It catalyzes the phosphorylation of pantothenate (vitamin B5) to 4'-phosphopantothenate, the first step in the coenzyme A biosynthetic pathway. The enzyme from Mycobacterium tuberculosis, MW 35.7 kDa, has been cloned, expressed, purified and crystallized in two different trigonal crystal forms, both belonging to space group P3(1)21. Two complete data sets of resolution 2.5 A (form I) and 2.9 A (form II) from crystals with unit-cell parameters a = b = 78.3, c = 115.45 A and a = b = 107.63, c = 89.85 A, respectively, were collected at room temperature on a home X-ray source. Structures of both crystal forms were solved for one subunit in the asymmetric unit by molecular replacement.

Bacterial Proteins↗
PubMed2004

Effect of glycosylation on the structure of Erythrina corallodendron lectin.

The three-dimensional structure of the recombinant form of Erythrina corallodendron lectin, complexed with lactose, has been elucidated by X-ray crystallography at 2.55 A resolution. Comparison of this non-glycosylated structure with that of the native glycosylated lectin reveals that the tertiary and quaternary structures are identical in the two forms, with local changes observed at one of the glycosylation sites (Asn17). These changes take place in such a way that hydrogen bonds with the neighboring protein molecules in rECorL compensate those made by the glycan with the protein in ECorL. Contrary to an earlier report, this study demonstrates that the glycan attached to the lectin does not influence the oligomeric state of the lectin. Identical interactions between the lectin and the non-covalently bound lactose in the two forms indicate, in line with earlier reports, that glycosylation does not affect the carbohydrate specificity of the lectin. The present study, the first of its kind involving a glycosylated protein with a well-defined glycan and the corresponding deglycosylated form, provides insights into the structural aspects of protein glycosylation.

Binding Sites↗
PubMed2004

X-ray analysis of Mycobacterium smegmatis Dps and a comparative study involving other Dps and Dps-like molecules.

The structure of the DNA binding protein from starved cells from Mycobacterium smegmatis has been determined in three crystal forms and has been compared with those of similar proteins from other sources. The dodecameric molecule can be described as a distorted icosahedron. The interfaces among subunits are such that the dodecameric molecule appears to have been made up of stable trimers. The situation is similar in the proteins from Escherichia coli and Agrobacterium tumefaciens, which are closer to the M.smegmatis protein in sequence and structure than those from other sources, which appear to form a dimer first. Trimerisation is aided in the three proteins by the additional N-terminal stretches that they possess. The M.smegmatis protein has an additional C-terminal stretch compared to other related proteins. The stretch, known to be involved in DNA binding, is situated on the surface of the molecule. A comparison of the available structures permits a delineation of the rigid and flexible regions in the molecule. The subunit interfaces around the molecular dyads, where the ferroxidation centres are located, are relatively rigid. Regions in the vicinity of the acidic holes centred around molecular 3-fold axes, are relatively flexible. So are the DNA binding regions. The crystal structures of the protein from M.smegmatis confirm that DNA molecules can occupy spaces within the crystal without disturbing the arrangement of the protein molecules. However, contrary to earlier suggestions, the spaces do not need to be between layers of protein molecules. The cubic form provides an arrangement in which grooves, which could hold DNA molecules, criss-cross the crystal.

Amino Acid Sequence↗