Department of Biological Sciences
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Item Molecular mechanism of the enhanced viral fitness contributed by secondary mutations in the hemagglutinin protein of oseltamivir resistant H1N1 influenza viruses: Modeling studies of antibody and receptor binding(Elsevier, 2015-02) Basu, SushmitaThe envelope protein hemagglutinin (HA) of influenza viruses is primarily associated with host antibody and receptor interactions. The HA protein is known to maintain a functional balance with neuraminidase (NA), the other major envelope protein. Prior to 2007–2008, human seasonal H1N1 viruses possessing the NA H274Y mutation, which confers oseltamivir resistance, generally had low growth capability. Subsequently, secondary mutations that compensate for the deleterious effect of the NA H274Y mutation have been identified. The molecular mechanism of how the defect could be counteracted by these secondary mutations is not fully understood. We studied here the effect of three such mutations (T86K, K144E and R192K) in the HA protein, which are located at either the HA receptor binding site or in the H1N1 antigenic sites. Molecular docking and dynamics studies showed that, of the three mutations, the R192K mutation could have mediated neutralizing antibody escape and decreased receptor binding affinity, either or both of which may have contributed to increased viral fitness. The study suggests the molecular basis of enhanced viral fitness induced by secondary mutations in the evolution of oseltamivir-resistant influenza strains.Item A structural perspective of RNA recognition by intrinsically disordered proteins(Springer, 2016-05) Basu, SushmitaProtein-RNA recognition is essential for gene expression and its regulation, which is indispensable for the survival of the living organism at one hand, on the other hand, misregulation of this recognition may lead to their extinction. Polymorphic conformation of both the interacting partners is a characteristic feature of such molecular recognition that promotes the assembly. Many RNA binding proteins (RBP) or regions in them are found to be intrinsically disordered, and this property helps them to play a central role in the regulatory processes. Sequence composition and the length of the flexible linkers between RNA binding domains in RBPs are crucial in making significant contacts with its partner RNA. Polymorphic conformations of RBPs can provide thermodynamic advantage to its binding partner while acting as a chaperone. Prolonged extensions of the disordered regions in RBPs also contribute to the stability of the large cellular machines including ribosome and viral assemblies. The involvement of these disordered regions in most of the significant cellular processes makes RBPs highly associated with various human diseases that arise due to their misregulation.Item Effect of neighbouring residues in conformational plasticity of intrinsically disordered proteins(Elsevier, 2018-02) Basu, SushmitaEffect of neighbouring residues in conformational plasticity of intrinsically disordered regions. The concept of unstructured proteins has opened new avenues in the field of structural biology. Intrinsically disordered proteins (IDPs) are the new class of proteins which have been found to be a major player in many significant cellular functions. IDPs have been characterised by its physicochemical properties as well as its molecular interaction behaviour. Detailed study of IDPs can lead to a better understanding of protein folding and its functioning. To understand the source of disorderedness in the disordered regions (IDRs) in IDPs, we studied how the sequence environment of a disordered region correlates to its randomness. Here, we analysed the physicochemical and structural features like amino acid propensities, net charge, hydropathy index, secondary structure propensity, relative surface accessibility, interaction density and H-bonds to characterise the neighbours of the IDRs. Five residues, each towards N and C terminal of the disordered region are considered as the neighbours of IDRs. These neighbouring residues are found to be enriched in disorder promoting amino acids and have higher propensity to form loops than other secondary structures. Solvent accessibility of neighbouring residues also showed increasing trend as we move towards the IDRs. The variation of other parameters along with the above observation indicates that the neighbouring residues of IDRs induce a degree of flexibility to the adjoining IDRs. Based on our findings, we are designing an algorithm using random forest, which shall predict the disordered region based on its neighbouring sequences. The information on IDRs and its neighbours can be useful for proteins to be expressed or characterised for the first time. It can also provide a lead in understanding the molecular mechanism behind the polymorphic interactions that are involved with IDPs.Item Two B-box proteins regulate photomorphogenesis by oppositely modulating HY5 through their diverse C-terminal domains(OUP, 2018-04) Yadukrishnan, PremachandranThe Arabidopsis (Arabidopsis thaliana) BBX family comprises several positive and negative regulators of photomorphogenesis. BBX24, a member of BBX structural group IV, acts as a negative regulator of photomorphogenesis, whereas another member from the same group, BBX21, is a positive regulator. The molecular basis for the functional diversity shown by these related BBX family members is unknown. Using domain-swap lines, we show that the C-terminal regions of BBX24 and BBX21 specify their function. Because both BBX21 and BBX24 work in close association with HY5, we hypothesized that these proteins differentially regulate the levels or activity of HY5 to fulfill their opposite roles. We show that BBX21 can regulate HY5 post-transcriptionally and the two proteins can coordinate to promote photomorphogenesis. By contrast, BBX24 interferes with the binding of HY5 to the promoter of an anthocyanin biosynthetic gene, possibly by heterodimerizing with HY5 and preventing it from binding DNA. Our finding that both BBX21 and BBX24 regulate HY5 activity post-transcriptionally, in opposite ways, suggests that closely related B-box proteins execute contrasting functions through differential regulation of HY5.Item Opposite roles of group IV BBX proteins: Exploring missing links between structural and functional diversity(Taylor & Francis, 2018-08) Yadukrishnan, PremachandranBBX proteins are a family of zinc finger transcription factors that are versatile regulators of plant development. The 32 BBX proteins in Arabidopsis are subdivided into five structural groups based on their domain structure. Members of group IV play important and diverse roles in light-regulated development. The N-terminal B-box domains mediate DNA binding and transcriptional regulation. The C-terminal region determines the functional diversity of the structurally similar group IV members as reported in our recent study investigating the basis of functional diversification between BBX21 and BBX24. We also found that multi-layered regulation of HY5 by the BBX proteins leads to a diverse repertoire of developmental effects. Here we provide a comprehensive structure-function analysis of the group IV BBX proteins.Item The B-box bridge between light and hormones in plants(Elsevier, 2019-02) Yadukrishnan, PremachandranPlant development is meticulously modulated by interactions between the surrounding environment and the endogenous phytohormones. Light, as an external signal coordinates with the extensive networks of hormones inside the plant to execute its effects on growth and development. Several proteins in plants have been identified for their crucial roles in mediating light regulated development. Among these are the B-box (BBX) family of transcription factors characterized by the presence of zinc-finger B-box domain in their N-terminal region. In Arabidopsis there are 32 BBX proteins that are divided into five structural groups on the basis of the domains present. Several BBX proteins play important roles in seedling photomorphogenesis, neighbourhood detection and photoperiodic regulation of flowering. There is increasing evidence that besides light signaling BBX proteins also play integral roles in several hormone signaling pathways in plants. Here we attempt to comprehensively integrate the roles of multiple BBX proteins in various light and hormone signaling pathways. We further discuss the role of the BBX proteins in mediating crosstalk between the two signaling pathways to harmonize plant growth and development. Finally, we try to analyse the conservation of BBX genes across species and discuss the role of BBX proteins in regulating economically important traits in crop plants.Item The B-box-containing microprotein miP1a/BBX31 regulates photomorphogenesis and UV-B protection(OUP, 2019-04) Yadukrishnan, PremachandranThe bZIP transcription factor ELONGATED HYPOCOTYL5 (HY5) represents a major hub in the light-signaling cascade both under visible and UV-B light. The mode of transcriptional regulation of HY5, especially under UV-B light, is not well characterized. B-BOX (BBX) transcription factors regulate HY5 transcription and also posttranscriptionally modulate HY5 to control photomorphogenesis under white light. Here, we identify BBX31 as a key signaling intermediate in visible and UV-B light signal transduction in Arabidopsis (Arabidopsis thaliana). BBX31 expression is induced by UV-B radiation in a fluence-dependent manner. HY5 directly binds to the promoter of BBX31 and regulates its transcript levels. Loss- and gain-of-function mutants of BBX31 indicate that it acts as a negative regulator of photomorphogenesis under white light but is a positive regulator of UV-B signaling. Genetic interaction studies suggest that BBX31 regulates photomorphogenesis independent of HY5. We found no evidence for a direct BBX31-HY5 interaction, and they primarily regulate different sets of genes in white light. Under high doses of UV-B radiation, BBX31 promotes the accumulation of UV-protective flavonoids and phenolic compounds. It enhances tolerance to UV-B radiation by regulating genes involved in photoprotection and DNA repair in a HY5-dependent manner. Under UV-B radiation, overexpression of BBX31 enhances HY5 transcriptional levels in a UV RESISTANCE LOCUS8-dependent manner, suggesting that BBX31 might regulate HY5 transcription.Item BBX31 promotes hypocotyl growth, primary root elongation and UV-B tolerance in Arabidopsis(Taylor & Francis, 2019-03) Yadukrishnan, PremachandranPhotomorphogenesis is an important developmental process that helps the seedlings adapt to external light conditions. B-Box proteins are a family of transcription factors that regulate photomorphogenic responses. BBX31 negatively regulates photomorphogenesis under visible light. In contrast, it promotes photomorphogenesis under UV-B and enhances tolerance to high doses of UV-B radiation. BBX31 and HY5 independently and oppositely regulate the ability of seedlings to adapt to varying light intensities. BBX31 also regulates primary root elongation under low intensities of white light. GC-MS and HPLC-based metabolite profiling identified differential accumulation of multiple primary and secondary metabolites in 35S:BBX31 that might enhance tolerance to UV-B.Item Cyclodextrin interaction with specific channel CymA from K. Oxytoca(Cell Press, 2015-01) Prajapati, Jigneshkumar DahyabhaiThe outer membrane acts as a selective uptake barrier in Gram negative bacteria. It contains protein channels (porins) which provide an entry pathway for hydrophilic molecules like small nutrient molecules and β-lactam antibiotics. However the CymA channel is known to take up cyclodextrin molecules giving bacteria the ability to survive on cyclodextrins. Hence understanding uptake of these molecules via porins is vital to comprehend the transport mechanism across the cell membrane. Electrophysiology forms a promising approach to study the permeation of molecules across outer membrane and thereby understanding molecular interactions with the channel. Here we present cyclodextrin interaction studies of CymA from K. oxytoca using single channel electrophysiology. Detailed single channel analysis revealed inherent asymmetric gating characteristics of the channel. Analysis of the ion current reduction through CymA in presence of cyclodextrin led revealed kinetic parameters of substrate binding. To further elucidate the affinity sites of substrate to the channel, mutation of certain channel residues has been performed. An altered channel gating behaviour is observed. To obtain an atomistic view we complement our studies with all-atom molecular dynamics simulation to study the various conductance states of the channel in the absence of cyclodextrin and to get molecular insight into the uptake of cyclodextrins as well.Item Understanding the translocation of fluoroquinolones through OmpC using the metadynamics(Cell Press, 2015-01) Prajapati, Jigneshkumar D.The outer membrane of Gram-negative bacteria such as Escherichia coli acts as a selective permeable barrier between cell and external environment. Water filled outer membrane proteins called as porins were identified for exchange of hydrophilic solutes and hydrophilic antibiotics. One of the most abundant outer membrane porins in E. coli is OmpC and many studies revealed that down-regulation or mutation of this porin shows reduced accumulation of antibacterials in bacterial cells [1]. Fluoroquinolones, used since 1980, are the most common treatment for urinary tract infection caused by E. coli and today this treatment is ineffective in more than half of the patients globally due to widespread resistance. So far the influx kinetics of fluoroquinolones with OmpC has been characterized on free standing lipid bilayers formed on a glass substrate [2]. In particular, detailed analysis of antibiotic interaction with a single OmpC channel using electrophysiology can provide a kinetic description. Here we have investigated two fluoroquinolones, Ciprofloxacin and Enrofloxacin, using an advanced molecular dynamics technique, i.e., metadynamics [3,4]. These free energy calculations help to identify the most favorable paths and activation energies required for molecules to translocate through the OmpC channel. Furthermore, we have also investigated the translocation of the same molecules in the presence of different salts to understand the altered translocation kinetics [5]. Moreover, the identification of favorable interactions networks is important to determine the most prominent residues required for translocation.