| dc.description.abstract | DNA- 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. | en_US |
