Biomedical subjects

Nagasuma R Chandra

Publications and source records attributed to Nagasuma R Chandra.

14 recordsLinked to original sources

PubMed2006

A K52Q substitution in the globular domain of histone H1t modulates its nucleosome binding properties.

A comparison of the globular domain sequences of the somatic H1d and testis-specific H1t revealed a single substitution of lysine 52 in H1d to glutamine 54 in H1t, which is one of the three crucial residues within the second DNA binding site. The globular domains of both histones were modeled using the crystal structure of chicken GH5 as a template and was also docked onto the nucleosome structure. The glutamine residue in histone H1t forms a hydrogen bond with main chain carbonyl of methionine-52 (in H1t) and is spatially oriented away from the nucleosome dyad axis. A consequence of this change was a lower affinity of recombinant histone H1t towards Four-way junction DNA and reconstituted 5S mononucleosomes. When Gln-54 in Histone H1t was mutated to lysine, its binding affinity towards DNA substrates was comparable to that of histone H1d. The differential binding of histones H1d and H1t towards reconstituted mononucleosomes was also reflected in the chromatosome-stop assay.

Amino Acid Sequence↗

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

Lectindb: a plant lectin database.

Lectins, a class of carbohydrate-binding proteins, are now widely recognized to play a range of crucial roles in many cell-cell recognition events triggering several important cellular processes. They encompass different members that are diverse in their sequences, structures, binding site architectures, quaternary structures, carbohydrate affinities, and specificities as well as their larger biological roles and potential applications. It is not surprising, therefore, that the vast amount of experimental data on lectins available in the literature is so diverse, that it becomes difficult and time consuming, if not impossible to comprehend the advances in various areas and obtain the maximum benefit. To achieve an effective use of all the data toward understanding the function and their possible applications, an organization of these seemingly independent data into a common framework is essential. An integrated knowledge base ( Lectindb, http://nscdb.bic.physics.iisc.ernet.in ) together with appropriate analytical tools has therefore been developed initially for plant lectins by collating and integrating diverse data. The database has been implemented using MySQL on a Linux platform and web-enabled using PERL-CGI and Java tools. Data for each lectin pertain to taxonomic, biochemical, domain architecture, molecular sequence, and structural details as well as carbohydrate and hence blood group specificities. Extensive links have also been provided for relevant bioinformatics resources and analytical tools. Availability of diverse data integrated into a common framework is expected to be of high value not only for basic studies in lectin biology but also for basic studies in pursuing several applications in biotechnology, immunology, and clinical practice, using these molecules.

Algorithms↗

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↗

PubMed2005

Defining putative T cell epitopes from PE and PPE families of proteins of Mycobacterium tuberculosis with vaccine potential.

The identification of T cell epitopes from immune relevant antigens of Mycobacterium tuberculosis is a critical step in the development of a vaccine covering diverse populations. Two multigene families, PE-PGRS and PPE make up about 10% of the M. tuberculosis genome. However, the functions of the proteins coded by these large numbers of genes are unknown. All possible nonameric peptide sequences from PE and PPE proteins were analysed in silico for their ability to bind to 33 alleles of class I HLA. These results reveal that of all PE and PPE proteins, a significant number of these peptides are predicted to be high-affinity HLA binders, irrespective of the length of the protein. The pathogen peptides that could behave as self or partially self-peptides in the host were eliminated using a comparative study with human proteome, thus reducing the number of peptides for analysis. The structural basis for recognition of the nonamers by the respective HLA molecules thus predicted was analyzed by molecular modeling. The structural analysis showed good correlation with the binding prediction. The analysis also led to an understanding of the binding profile of the peptides with respect to different alleles of class I HLA. The predicted epitopes can be tested experimentally for their inclusion in a potential vaccine against tuberculosis that is HLA haplotype-specific.

Alleles↗

PubMed2004

A database analysis of jacalin-like lectins: sequence-structure-function relationships.

Lectins are known to be important for many biological processes, due to their ability to recognize cell surface carbohydrates with high specificity. Plant lectins have been model systems to study protein-carbohydrate recognition, because individually they exhibit high sensitivity and as a group large diversity in recognizing carbohydrate structures. Although extensive studies have been carried out for legume lectins that have led to interesting insights into the sequence determinants of sugar recognition in them, frameworks with such specific correlations are not available for other plant lectin families. This study reports a large-scale data acquisition and extensive analysis of sequences and structures of beta-prism-I or jacalin-related lectins (JRLs) and shows that hypervariability in the binding site loops generates carbohydrate recognition diversity, a strategy analogous to that in legume lectins. Analyses of the size, conformation, and sequence variability in key regions reveal the existence of a common theme, encoded as a set of structural features over a common scaffold, in defining specificity. This study also points to the remarkable range of domain architectures, often arising out of gene duplication events in lectins of this family. The data analyzed here also indicate a spectacular variety of quaternary associations possible in this family of lectins that have implications for glycan recognition. These results thus provide sequence-structure-function correlations, an understanding of the molecular basis of carbohydrate recognition by beta-prism-I lectins, and also a rationale for engineering specific recognition capabilities in relevant molecules.

Amino Acid Sequence↗

PubMed2003

Delta-aminolevulinic acid dehydratase from Plasmodium falciparum: indigenous versus imported.

The heme biosynthetic pathway of the malaria parasite is a drug target and the import of host delta-aminolevulinate dehydratase (ALAD), the second enzyme of the pathway, from the red cell cytoplasm by the intra erythrocytic malaria parasite has been demonstrated earlier in this laboratory. In this study, ALAD encoded by the Plasmodium falciparum genome (PfALAD) has been cloned, the protein overexpressed in Escherichia coli, and then characterized. The mature recombinant enzyme (rPfALAD) is enzymatically active and behaves as an octamer with a subunit Mr of 46,000. The enzyme has an alkaline pH optimum of 8.0 to 9.0. rPfALAD does not require any metal ion for activity, although it is stimulated by 20-30% upon addition of Mg2+. The enzyme is inhibited by Zn2+ and succinylacetone. The presence of PfALAD in P. falciparum can be demonstrated by Western blot analysis and immunoelectron microscopy. The enzyme has been localized to the apicoplast of the malaria parasite. Homology modeling studies reveal that PfALAD is very similar to the enzyme species from Pseudomonas aeruginosa, but manifests features that are unique and different from plant ALADs as well as from those of the bacterium. It is concluded that PfALAD, while resembling plant ALADs in terms of its alkaline pH optimum and apicoplast localization, differs in its Mg2+ independence for catalytic activity or octamer stabilization. Expression levels of PfALAD in P. falciparum, based on Western blot analysis, immunoelectron microscopy, and EDTA-resistant enzyme activity assay reveals that it may account for about 10% of the total ALAD activity in the parasite, the rest being accounted for by the host enzyme imported by the parasite. It is proposed that the role of PfALAD may be confined to heme synthesis in the apicoplast that may not account for the total de novo heme biosynthesis in the parasite.

Amino Acid Sequence↗

PubMed2003

Molecular modeling of the chromatosome particle.

In an effort to understand the role of the linker histone in chromatin folding, its structure and location in the nucleosome has been studied by molecular modeling methods. The structure of the globular domain of the rat histone H1d, a highly conserved part of the linker histone, built by homology modeling methods, revealed a three-helical bundle fold that could be described as a helix-turn-helix variant with its characteristic properties of binding to DNA at the major groove. Using the information of its preferential binding to four-way Holliday junction (HJ) DNA, a model of the domain complexed to HJ was built, which was subsequently used to position the globular domain onto the nucleosome. The model revealed that the primary binding site of the domain interacts with the extra 20 bp of DNA of the entering duplex at the major groove while the secondary binding site interacts with the minor groove of the central gyre of the DNA superhelix of the nucleosomal core. The positioning of the globular domain served as an anchor to locate the C-terminal domain onto the nucleosome to obtain the structure of the chromatosome particle. The resulting structure had a stem-like appearance, resembling that observed by electron microscopic studies. The C-terminal domain which adopts a high mobility group (HMG)-box-like fold, has the ability to bend DNA, causing DNA condensation or compaction. It was observed that the three S/TPKK motifs in the C-terminal domain interact with the exiting duplex, thus defining the path of linker DNA in the chromatin fiber. This study has provided an insight into the probable individual roles of globular and the C-terminal domains of histone H1 in chromatin organization.

Animals↗

PubMed2003

Computational analysis of multivalency in lectins: structures of garlic lectin-oligosaccharide complexes and their aggregates.

Multivalency in lectins is a phenomenon that has been discussed at considerable length. The structural basis for the role of multivalency in garlic lectin has been investigated here through computational studies. Biochemical studies have shown that the binding affinity of garlic lectin for high mannose oligosaccharides is orders of magnitude greater than that for mannose. Modeling and energy calculations clearly indicate that such increase in affinity cannot be accounted for by binding of these oligosaccharides at any of the six sites of a garlic lectin dimer. These studies also indicate that a given oligosaccharide cannot bind simultaneously to more than one binding site on a lectin dimer. The possibility of a given oligosaccharide simultaneously binding to and hence linking two or more lectin molecules was therefore explored. This study showed that trimannosides and higher oligomers can cross-link lectin dimers, amplifying the protein-oligosaccharide interactions severalfold, thus explaining the role of multivalency in enhancing affinity. A comprehensive exploration of all possible cross-links posed a formidable computational problem. Even a partial exploration involving a carefully chosen region of the conformational space clearly showed that a given dimer pair can be cross-linked not only by a single oligosaccharide molecule but also simultaneously by two oligosaccharides. The number of such possible double cross-links, including those forming interesting tetrameric structures, generally increases with the size of the oligosaccharide, correlating with the biochemical data. In addition to their immediate relevance to garlic lectin, these studies are of general interest in relation to lectin-oligosaccharide interactions.

Computational Biology↗

PubMed2003

Structural studies on MtRecA-nucleotide complexes: insights into DNA and nucleotide binding and the structural signature of NTP recognition.

RecA protein plays a crucial role in homologous recombination and repair of DNA. Central to all activities of RecA is its binding to Mg(+2)-ATP. The active form of the protein is a helical nucleoprotein filament containing the nucleotide cofactor and single-stranded DNA. The stability and structure of the helical nucleoprotein filament formed by RecA are modulated by nucleotide cofactors. Here we report crystal structures of a MtRecA-ADP complex, complexes with ATPgammaS in the presence and absence of magnesium as well as a complex with dATP and Mg+2. Comparison with the recently solved crystal structures of the apo form as well as a complex with ADP-AlF4 confirms an expansion of the P-loop region in MtRecA, compared to its homologue in Escherichia coli, correlating with the reduced affinity of MtRecA for ATP. The ligand bound structures reveal subtle variations in nucleotide conformations among different nucleotides that serve in maintaining the network of interactions crucial for nucleotide binding. The nucleotide binding site itself, however, remains relatively unchanged. The analysis also reveals that ATPgammaS rather than ADP-AlF4 is structurally a better mimic of ATP. From among the complexed structures, a definition for the two DNA-binding loops L1 and L2 has clearly emerged for the first time and provides a basis to understand DNA binding by RecA. The structural information obtained from these complexes correlates well with the extensive biochemical data on mutants available in the literature, contributing to an understanding of the role of individual residues in the nucleotide binding pocket, at the molecular level. Modeling studies on the mutants again point to the relative rigidity of the nucleotide binding site. Comparison with other NTP binding proteins reveals many commonalties in modes of binding by diverse members in the structural family, contributing to our understanding of the structural signature of NTP recognition.

Adenosine Diphosphate↗

PubMed2003

Crystal structures of Mycobacterium smegmatis RecA and its nucleotide complexes.

The crystal structures of Mycobacterium smegmatis RecA (RecA(Ms)) and its complexes with ADP, ATPgammaS, and dATP show that RecA(Ms) has an expanded binding site like that in Mycobacterium tuberculosis RecA, although there are small differences between the proteins in their modes of nucleotide binding. Nucleotide binding is invariably accompanied by the movement of Gln 196, which appears to provide the trigger for transmitting the effect of nucleotide binding to the DNA-binding loops. These observations provide a framework for exploring the known properties of the RecA proteins.

Adenosine Diphosphate↗

PubMed2002

Prediction of an HMG-box fold in the C-terminal domain of histone H1: insights into its role in DNA condensation.

In eukaryotes, histone H1 promotes the organization of polynucleosome filaments into chromatin fibers, thus contributing to the formation of an important structural framework responsible for various DNA transaction processes. The H1 protein consists of a short N-terminal "nose," a central globular domain, and a highly basic C-terminal domain. Structure prediction of the C-terminal domain using fold recognition methods reveals the presence of an HMG-box-like fold. We recently showed by extensive site-directed and deletion mutagenesis studies that a 34 amino acid segment encompassing the three S/TPKK motifs, within the C-terminal domain, is responsible for DNA condensing properties of H1. The position of these motifs in the predicted structure corresponds exactly to the DNA-binding segments of HMG-box-containing proteins such as Lef-1 and SRY. Previous analyses have suggested that histone H1 is likely to bend DNA bound to the C-terminal domain, directing the path of linker DNA in chromatin. Prediction of the structure of this domain provides a framework for understanding the higher order of chromatin organization.

Amino Acid Sequence↗

PubMed2002

Identification of a 34 amino acid stretch within the C-terminus of histone H1 as the DNA-condensing domain by site-directed mutagenesis.

The C-terminus of histone H1 is necessary for the folding of polynucleosomal arrays into higher-order structure(s) and contains octapeptide repeats each having DNA binding S/TPKK motifs. These repeat motifs were earlier shown to mimic the DNA/chromatin-condensing properties of the C-terminus of histone H1 (Khadake, J. R., and Rao, M. R. S. (1995) Biochemistry 36, 1041-1051). In the present study, we have generated a series of C-terminal mutants of rat histone H1d and studied their DNA-condensation properties. The single proline to alanine mutation in the S/TPKK motifs either singly or in combination resulted in only a 20% decrease in the DNA-condensation property of histone H1. Deletion of all the three S/TPKK motifs resulted in a 45% decrease in DNA condensation. When the three octapeptide repeats encompassing the S/TPKK motifs were deleted, there was again a 45% decrease in DNA condensation. On the other hand, when the entire 34 amino acid stretch (residue 145-178) was deleted, there was nearly a 90% decrease in DNA condensation brought about by histone H1d. Interestingly, deletion of the 10 amino acid spacer between the octapeptide repeats (residues 161-170) also reduced the DNA condensation by 70%. Deletion of the region (residues 115-141) immediately before the 34 amino acid stretch and after the globular domain and the region (residues 184-218) immediately after the 34 amino acid stretch had only a marginal effect on DNA condensation. The importance of the 34 amino acid stretch, including the 10 amino acid spacer, was also demonstrated with the recombinant histone H1d C-terminus. We have also determined the induced alpha-helicity of histone H1 and its various mutants in the presence of 60% trifluoroethanol, and the experimentally determined induced helical contents agree with the theoretical predictions of secondary structural elements in the C-terminus of histone H1d. Thus, we have identified a 34 amino acid stretch in the C-terminus of histone H1d as the DNA-condensing domain.

Amino Acid Sequence↗

PubMed2002

Re-refinement using reprocessed data to improve the quality of the structure: a case study involving garlic lectin.

The structure of dimeric garlic lectin was previously determined to an effective resolution of 2.8A using X-ray intensity data processed by the XDS package and refined using X-PLOR [Chandra et al. (1999), J. Mol. Biol. 285, 1157--1168]. Repeated attempts to grow better crystals with a view to improving the definition of the structure did not succeed. The available raw data were then reprocessed using DENZO. The structure was re-refined with both X-PLOR and CNS separately using the reprocessed data, which extended to a resolution of 2.2A. These two sets of refinements and the two sets using the XDS-processed data afforded an opportunity to compare the performance of different data-processing and refinement packages when dealing with data from weakly diffracting crystals. The best results were obtained when CNS was employed for refinement using data processed by DENZO. The quality and the resolution of the map and the definition of the structure improved substantially. In particular, the amino-acid residues at the variable locations in the sequence, and hence the isolectins, could be identified with a high degree of confidence. It could be established that the crystal asymmetric unit contains two identical heterodimers. The new refined structure also provided a better definition of other finer structural details.

Amino Acid Sequence↗