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Crystal Structures of Native and AdoMet Bound rRNA Methyltransferase from Sinorhizobium meliloti : Structural Insights into rRNA Recognition. Evolutionary, Structural and Functional Studies on Nucleoid-Associated Proteins HU and IHF
No full textDNA- and RNA-binding proteins play a central role in gene regulation, which includes transcriptional control, alternative splicing, post-translational and transcriptional modifications like methylation and acetylation among other roles. In this way, they control most of the working machinery of the cell in direct or indirect manner. Although more than 60 years ago the structure of DNA was proposed by Watson and Crick, our understanding of how RNA- and DNA-binding proteins interact with the genome and transcriptome remains scarce. One of the most important questions in biology is how a large number of DNA- and RNA-binding proteins find their target, interact and later disassociate. These nucleic acid binding proteins either recognizes the unique structural and chemical signatures of the bases (base readout) which give the specificity or it recognizes a sequence-dependent shape (shape readout).
Methyltransferases are enzymes with diverse folds, which perform methyltransfer to various substrates using mainly S-adenosyl-L-methionine (AdoMet) as a methyl donor. RNA methylation is one of the most crucial post-transcriptional modifications which influences a wide variety of cellular processes like metabolic stabilization of RNA, quality control in protein synthesis, resistance to antibiotics, mRNA reading frame maintenance, splicing, viral nucleoprotein stabilization among others. Specificity in recognition and methylation in ribosomal RNA (rRNA) methyltransferases is very crucial, as rRNA is highly conserved and lack of specificity would influence the stabilization of RNA and thus, will affect the ribosome. In recent years, rRNA modifications which confer resistance to ribosomal antibiotics have also been observed. The mechanism of recognition to their unique rRNA target site with high selectivity and their evolution still remains an enigma. Thus, the evolution of antibiotic resistance-conferring methyltransferases in pathogenic organisms needs to be investigated from the structural and evolutionary perspective.
In the last two decades, many global regulators in both eukaryotes and prokaryotes have been discovered, which promiscuously bind to a large number of DNA sequences. In prokaryotes, they are called as ‘Nucleoid-associated proteins’ (NAPs), which influence the transcriptional process and exhibit multi-specificity or promiscuity. They also take part in the formation of many multi-protein complexes. HU and Integration Host Factor (IHF) are NAPs which belong to prokaryotic DNA-bending protein family (DNABII family). HU and IHF play crucial architectural roles in bacterial DNA condensation and additionally play a regulatory role in many cellular processes. Although sharing structural similarity, the DNA binding and bending features of HU and IHF are strikingly different, allowing them to selectively regulate genes from different genomic locations. HU binds to DNA in a sequence promiscuous manner while IHF is moderately sequence specific. The molecular mechanism of DNA binding multi-specificity (differential specificity with varied binding affinity) of HU/IHF proteins remains unexplored, as little attention has been paid to the determinants at the sequence level.
Now, the fundamental question which the author attempted to understand is the structural and evolutionary determinants of specificity in DNA- and RNA-binding proteins. The candidate has taken nucleoid-associated protein HU and SPOUT superfamily RNA methyltransferase as model systems. As the very limited number of structural folds makes up the DNA- and RNA-binding proteins, it is intriguing to examine closely related nucleic acid binding domains or folds carrying out specific functions. Also, we observed that some proteins having a particular structural fold (or homologous ancestry) bind to DNA or RNA with high specificity, while its other homolog binds promiscuously. These observations tempted us to find the sequence and structural determinants which guide this phenomenon, not just specific to only a single protein family, but, determinants are of more general nature, where results can possibly be applied to other nucleic acid binding proteins too.
The first part of the thesis reports the crystal structures of native and AdoMet bound ribosomal RNA Methyltransferase from Sinorhizobium meliloti (smMtase), by single anomalous dispersion (SAD) phasing on seleno-methionine substituted crystal, which diffracted to 2.28Å and 2.9 Å resolutions respectively in space group P212121. smMtase belong to an rRNA binding SPOUT superfamily protein, which is fused with an RNA binding L30e domain at the N-terminus. We focused our study on these types of proteins among the large superfamily (henceforth termed as SPOUTL30).
The author also has conducted a phylogenetic study, which revealed 11 major clades, out of which we focused our present study in understanding the sequence conservation and variations of 5 (A-E) clades, for which structural, biochemical and functional data is available. These proteins share homology to antibiotic resistance conferring methyltransferases. The availability of experimentally determined structures of native and AdoMet bound smMtase along with an analysis of other homologous crystal structures has enabled a critical examination of factors influencing RNA binding specificity. Also, the thesis reports for the first time an evolutionary and structural inter-connectivity of the three conserved motifs (I-III) in SPOUT superfamily, which is responsible for AdoMet binding and catalysis. The results highlight that both the location of conserved positive and negatively charged residues influence the RNA binding, specificity, and affinity. The conservation of these residues could be at superfamily, family or at clade level, and the position of these charged residues at specific sites, alters their salt-bridge geometry, which ultimately fixes the conformation of RNA-binding residues, thus defining a particular binding site specific to its cognate RNA. The study conducted by the author reveals that the dynamics of salt-bridge and other directional interactions like hydrogen bonding and aromatic interactions essentially determines the specificity of SPOUTL30.
The second part of the thesis reports evolutionary, structural and functional studies on nucleoid-associated proteins HU and IHF. To understand the sequence determinants, which influence the degree of DNA binding specificity, we undertook a phylogenetic study in conjunction with analysis of three-dimensional structures. The phylogenetic analysis revealed three major clades, belonging to HU, IHFα, and IHFβ like proteins with reference to E. coli. The author observed statistically significant amino acid compositional bias in the DNA binding sites of HU and IHF clade proteins. The author proposes that the molecular mechanisms giving rise to specificity or multi-specificity depend on a combination effect of the amino acid composition of the binding site, its flexibility, ionic and steric constraints. In continuation of this part of the thesis, the candidate examined the role of protein interacting interface of HU-IHF family proteins, understanding its evolutionary history and utilizing it in designing inhibitors for Mycobacterium tuberculosis HU (MtbHU). The present results give a model example of an evolutionary study of a protein interface of nucleoid-associated protein, which is used to understand the interface and computationally design inhibitors targeting it.
The author was a part of the study (Bhowmick et al. 2014, Nature communications) which has determined the crystal structure of Mycobacterium tuberculosis HU, inhibited it using stilbene derivatives (SD1 and SD4) which curtailed the Mtb cell growth. In the present thesis, the candidate observed from microarray analysis that the SD1 stimulon consists of genes involved majorly in lipid biosynthesis pathway, ribosomal genes which affect the overall translation, aerobic respiration pathways, antigenic membrane proteins involved in pathogenicity. Nearly half of the genes in affected by SD1 are essential in nature, thus could explain the curtailing of cellular growth. The whole study provides a system inspired view of probing as well, inhibiting global regulator HU using novel chemical molecules
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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
Structural Characterization of the Chlorophyllide a Oxygenase (CAO) Enzyme Through an In Silico Approach
Get PDFChlorophyllide a oxygenase (CAO) is responsible for converting chlorophyll a to chlorophyll b in a two-step oxygenation reaction. CAO belongs to the family of Rieske-mononuclear iron oxygenases. Although the structure and reaction mechanism of other Rieske monooxygenases have been described, a member of plant Rieske non-heme iron-dependent monooxygenase has not been structurally characterized. The enzymes in this family usually form a trimeric structure and electrons are transferred between the non-heme iron site and the Rieske center of the adjoining subunits. CAO is supposed to form a similar structural arrangement. However, in Mamiellales such as Micromonas and Ostreococcus, CAO is encoded by two genes where non-heme iron site and Rieske cluster localize on the distinct polypeptides. It is not clear if they can form a similar structural organization to achieve the enzymatic activity. In this study, the tertiary structures of CAO from the model plant Arabidopsis thaliana and the Prasinophyte Micromonas pusilla were predicted by deep learning-based methods, followed by energy minimization and subsequent stereochemical quality assessment of the predicted models. Furthermore, the chlorophyll a binding cavity and the interaction of ferredoxin, which is the electron donor, on the surface of Micromonas CAO were predicted. The electron transfer pathway was predicted in Micromonas CAO and the overall structure of the CAO active site was conserved even though it forms a heterodimeric complex. The structures presented in this study will serve as a basis for understanding the reaction mechanism and regulation of the plant monooxygenase family to which CAO belongs
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
クロロフィル代謝系に含まれるマグネシウム脱離酵素とクロロフィリドaオキシゲナーゼの構造解析
Get PDFIn land plants, green algae and some cyanobacteria, chlorophyll a and chlorophyll b form the principal components of the photosynthetic machinery that play crucial role in absorption, transmission, and transformation of light energy. The difference between the two chlorophyll species is the presence of a formyl group at the C7 position in chlorophyll b while a methyl group occurs at the same position in chlorophyll a. Both chlorophylls possess distinct absorption spectra in the blue and red regions, which allows this combination of pigments to utilize a wide range of light spectra for photosynthesis. The light harvesting complexes (LHCs) of photosynthetic organisms are composed of core and peripheral antenna complexes. While chlorophyll a is present in the core antenna of photosystems I and II as chlorophyll-protein complexes, chlorophyll b mainly resides in the peripheral antenna complexes along with other pigments like fucoxanthin. Moreover, chlorophyll a is vital for photochemistry in oxygenic photosynthe ic organisms whereas chlorophyll b is necessary for stabilizing the major light-harvesting chlorophyll-binding proteins and also in regulating the photosynthetic antenna size by altering the chlorophyll a/b ratio. Chlorophyll biosynthesis must be finely regulated for efficient photosynthetic performance during the formation of photosystems at the greening stage and also during adaptation to various environmental conditions. Not only chlorophyll biosynthesis but also chlorophyll degradation needs to be regulated because the latter plays a crucial role in mobilizing resources from chloroplast to developing organs. In addition, chlorophyll breakdown forms a key part of nitrogen recycling and is important in avoiding cellular photodamage. Before degradation, chlorophyll b must be converted to chlorophyll a because chlorophyll b derivatives are not catalyzed in the later steps of the chlorophyll degradation pathway. The interconversion pathway between chlorophyll a and chlorophyll b is referred to as the chloroph ll cycle. Chlorophyll a is converted to chlorophyll b in two successive steps by chlorophyll(ide) a oxygenase (CAO). In the first step of chlorophyll b conversion, the enzyme chlorophyll b reductase (CBR) reduces the formyl group of chlorophyll b to produce 7-hydroxymethyl chlorophyll a. In the final step, chlorophyll a is formed by the enzyme 7-hydroxymethyl chlorophyll a reductase (HCAR), the structure of which resembles an archaeal F420-reducing [NiFe] hydrogenase. Chlorophyll a is then converted to a primary fluorescent Chl catabolite by four continuous steps. First, central magnesium (Mg) ion in chlorophyll a is extracted by a Mg-dechelatase enzyme encoded by the Stay-Green (SGR) gene to form pheophytin a, which is then hydrolyzed to become pheophorbide a and phytol by pheophytinase (PPH). As the porphyrin of pheophorbide a is cleaved by pheophorbide a oxygenase (PAO), the green color completely fades in chlorophyll catabolite, leading to the formation of red chlorophyll catabolite. Subsequently, it is turned to the primary fluorescent chlorophyll catabolite by red chlorophyll catabolite reductase (RCCR) which is transferred out of the chloroplasts and isomerized to non-fluorescent products by acidic pH in the vacuole. My PhD study provides insights into the structural characteristics of two chlorophyll metabolic pathway enzymes – SGR and CAO. Chapter 1 deals with the Mg-dechelatase enzyme which catalyzes Mg2+ dechelation from chlorophyll a. This reaction is the first committed step of chlorophyll degradation pathway in plants and is thus indispensable for the process of leaf senescence. There is no structural information available for this or its related enzymes. This chapter provides insight into the structure and reaction mechanism of the enzyme through biochemical and computational analysis of an SGR homolog from the Chloroflexi Anaerolineae (AbSGR-h). Recombinant AbSGR-h with its intact sequence and those with mutations were overexpressed in Escherichia coli and their M -dechelatase activity was compared. Two aspartates – D34 and D62 were found to be essential for catalysis, while R26, Y28, T29, and D114 were responsible for structural maintenance. Gel filtration analysis of the recombinant AbSGR-h revealed the formation of a homo-oligomer. The three-dimensional structure of AbSGR-h was predicted by a deep learning-based method, which was eva uated by protein structure quality evaluation programs while structural stability of wild-type and mutant forms were investigated through molecular dynamics simulations. Furthermore, in concordance with the results of the enzyme assay, molecular docking concluded the significance of D34 in ligand interaction. By combining biochemical analysis and computational prediction, the study unveils the detailed structural characteristics of the enzyme, including the probable pocket of interaction and the residues of structural and functional importance. Chapter 2 also deals with the in-depth analysis of the structure of Mg-dechelatase enzyme. The crystal structure of a highly active SGR homolog from Anaerolineae (AbSGR-h) bacterium at 1.75 Å resolution has been reported. A previous study revealed the catalytic significance of D34 residue in AbSGR-h protein for interaction with the central Mg of chlorophyll a. Therefore, recombinant WT AbSGR-h and three mutants (D34E, D34N, and D34Q) were overexpressed in E. coli and urified by nickel column and size exclusion chromatography. Gel filtration profiles of the WT and three mutant proteins were found to be similar thus confirming the role of D34 to be solely catalytic rather than maintaining the multimeric conformation of the protein. Activity analysis revealed substantial decrease of Mg-dechelation level for the D34E mutant and loss of activity for the D34N and D34Q mutants. The kinetic parameters of WT and D34E mutant AbSGR-h were elucidated by Michaelis-Menten analysis. Furthermore, molecular docking analysis showed stable interaction of the central Mg ion of chlorophyll a with the carbonyl oxygen atom of D34 residue in the crystal structure of AbSGR-h monomer within a distance of 4.4 Å. Besides, the catalytic triad found in AbSGR-h was found to show high resemblance with those observed in hydrolases. This study enhances the existing knowledge about the reaction mechanism of Mg-dechelatase and also provides the first crystal structure of a homolog from the SGR family. Chapter 3 highlights the structural characteristics of the CAO enzyme, that is responsible for converting chlorophyll a to chlorophyll b. CAO belongs to the family of Rieske mononuclear iron oxygenases. Here, the tertiary structures of CAO from the Prasinophyte Micromonas pusilla (MpCAO) and model plant Arabidopsis thaliana (AtCAO) were predicted by deep learning-based methods, followed by energy minimization and subsequent stereochemical quality assessment of the predicted models. Although plant CAO structure exhibits the three-fold symmetric homotrimer form, like most other Rieske non-heme iron oxygenases, Micromonas CAO exist as two distinct polypeptides (MpCAO1 and MpCAO2). Thus, its heterodimeric association was computationally investigated. Furthermore, the chlorophyll a binding cavity on the surface of MpCAO2 was predicted and molecular docking analysis revealed presence of the substrate at the vicinity of the mononuclear iron center. This study enables the structural visualization of the electron trasfer pathway between the two distinct subunits of MpCAO. Mg-dechelatase or SGR plays an indispensable role in chlorophyll metabolism because it catalyzes the committed step of the chlorophyll degradation pathway where it removes Mg2+ from chlorophyll a to produce pheophytin a. Despite such importance, neither the three-dimensional nor the reaction mechanism has been elucidated until now. Combining the information from the tertiary protein structure, obtained by computational prediction as well as X-ray crystallography, and biochemical analysis, the reaction mechanism of the enzyme was proposed. There are two classes of metal dechelatase known to date – heme oxygenase and Mg-dechelatase. The former enzyme cleaves a porphyrin ring to extract Fe2+ in a totally different mechanism from that of Mg-dechelatase. This study proposes a novel reaction mechanism for a metal dechelatase enzyme based on structural analysis. Unexpectedly, my structural model suggests that deprotonated side chain of D34 may coordinate stably with Mg of chlorophyll. This coordination can be suppo ed to destabilize Mg-tetrapyrrole ring interaction, resulting in extraction of Mg from chlorophyll. This study will become a basis for further studies on this enzyme, such as those for substrate specificity, screening for inhibitors and evolutionary analysis. Furthermore, the tertiary and quaternary structure of CAO was also predicted computationally with special emphasis on the heterodimeric association between the two polypeptides of Micromonas CAO, leading to the prediction of a reaction mechanism for the enzyme. These structure-based enzyme studies will provide the clue to understand enzyme properties such as substrate specificity or regulatory mechanism of its activity, which facilitates understanding of plant life
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