Browsing by Author "Gaur, Vineet"

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Branched DNA processing by a thermostable CAS-Cas4 from Thermococcus onnurineus: expanding biochemical landscape of nuclease activity
(Elsevier B.V., 2025) Jain, Muskan; Pattnayak, Asish Kumar; Aggarwal, Sakshi; Rai, Praveen; Kavya, J.; Chandrayan, Sanjeev; Goel, Manisha; Gaur, Vineet
The adaptive immune function of CRISPR-Cas systems in bacteria and archaea is mediated through CRISPR-Associated Proteins (Cas). The adaptation module, typically involving Cas1, Cas2, and Cas4, helps integrate viral “spacer” sequences into the host genome. Cas4 proteins are classified into two types based on neighboring genes: CAS-Cas4, flanked by other cas genes, and Solo-Cas4, which exist independently. While CAS-Cas4 proteins are implicated in adaptation, they remain biochemically uncharacterized in archaea, unlike archaeal Solo-Cas4 proteins. This study biochemically characterizes TON_0321, a CAS-Cas4 protein from the Type IV-C CRISPR cassette of Thermococcus onnurineus. TON_0321 exhibits 5′ to 3′ exonuclease activity and unique structure-dependent endonuclease activity, shedding light on CAS-Cas4 functional diversity. A distinct spatial organization of the catalytic site, angled with the positively charged patch on the protein surface, enables TON_0321 to recognize branching points in DNA substrates. Furthermore, this spatial arrangement facilitates cleavage 2 to 3 nucleotides away from the branch point in the 5′ direction, demonstrating structure-specific endonuclease activity.
Cavity architecture based modulation of ligand binding tunnels in plant START domains
(Elsevier B.V., 2023) Mahtha, Sanjeet Kumar; Kumari, Kamlesh; Gaur, Vineet; Yadav, Gitanjali
The Steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain represents an evolutionarily conserved superfamily of lipid transfer proteins widely distributed across the tree of life. Despite significant expansion in plants, knowledge about this domain remains inadequate in plants. In this work, we explore the role of cavity architectural modulations in START protein evolution and functional diversity. We use deep-learning approaches to generate plant START domain models, followed by surface accessibility studies and a comprehensive structural investigation of the rice START family. We validate 28 rice START domain models, delineate binding cavities, measure pocket volumes, and compare these with mammalian counterparts to understand evolution of binding preferences. Overall, plant START domains retain the ancestral α/β helix-grip signature, but we find subtle variation in cavity architectures, resulting in significantly smaller ligand-binding tunnels in the plant kingdom. We identify cavity lining residues (CLRs) responsible for reduction in ancestral tunnel space, and these appear to be class specific, and unique to plants, providing a mechanism for the observed shift in domain function. For instance, mammalian cavity lining residues A135, G181 and A192 have evolved to larger CLRs across the plant kingdom, contributing to smaller sizes, minimal STARTs being the largest, while members of type-IV HD-Zip family show almost complete obliteration of lipid binding cavities, consistent with their present-day DNA binding functions. In summary, this work quantifies plant START structural & functional divergence, bridging current knowledge gaps.
A combined structural and biochemical approach reveals translocation and stalling of UvrB on the DNA lesion as a mechanism of damage verification in bacterial nucleotide excision repair
(Elsevier B.V., 2020) Jaciuk, Marcin; Swuec, Paolo; Gaur, Vineet; Kasprzak, Joanna M.; Renault, Ludovic; Dobrychłop, Mateusz; Nirwal, Shivlee; Bujnicki, Janusz M.; Costa, Alessandro; Nowotny, Marcin
Nucleotide excision repair (NER) is a DNA repair pathway present in all domains of life. In bacteria, UvrA protein localizes the DNA lesion, followed by verification by UvrB helicase and excision by UvrC double nuclease. UvrA senses deformations and flexibility of the DNA duplex without precisely localizing the lesion in the damaged strand, an element essential for proper NER. Using a combination of techniques, we elucidate the mechanism of the damage verification step in bacterial NER. UvrA dimer recruits two UvrB molecules to its two sides. Each of the two UvrB molecules clamps a different DNA strand using its β-hairpin element. Both UvrB molecules then translocate to the lesion, and UvrA dissociates. The UvrB molecule that clamps the damaged strand gets stalled at the lesion to recruit UvrC. This mechanism allows UvrB to verify the DNA damage and identify its precise location triggering subsequent steps in the NER pathway.
Epitope-directed anti-SARS-CoV-2 scFv engineered against the key spike protein region could block membrane fusion
(John Wiley & Sons, 2023) Jaiswal, Deepika; Kumar, Ujjwal; Gaur, Vineet; Salunke, Dinakar M.
The newly emerged SARS-CoV-2 causing coronavirus disease (COVID-19) resulted in >500 million infections. A great deal about the molecular processes of virus infection in the host is getting uncovered. Two sequential proteolytic cleavages of viral spike protein by host proteases are prerequisites for the entry of the virus into the host cell. The first cleavage occurs at S1/S2 site by the furin protease, and the second cleavage at a fusion activation site, the S2' site, by the TMPRSS2 protease. S2' cleavage site is present in the S2 domain of spike protein followed by a fusion peptide. Given the S2' site to be conserved among all the SARS-CoV-2 variants, we chose an S2' epitope encompassing the S2' cleavage site and generated single-chain antibodies (scFvs) through an exhaustive phage display library screening. Crystal structure of a scFv in complex with S2' epitope was determined. Incidentally, S2' epitope in the scFv bound structure adopts an alpha-helical conformation equivalent to the conformation of the epitope in the spike protein. Furthermore, these scFvs can bind to the spike protein expressed either in vitro or on the mammalian cell surface. We illustrate a molecular model based on structural and biochemical insights into the antibody-S2' epitope interaction emphasizing scFvs mediated blocking of virus entry into the host cell by restricting the access of TMPRSS2 protease and consequently inhibiting the S2' cleavage competitively.
Erasing methylation marks on DNA by plant-specific DEMETER family DNA glycosylases
(Springer Nature Publishing AG, 2025) Rai, Praveen; Kumari, Poonam; Gaur, Vineet
Plants, being sessile and incapable of evading the continually fluctuating environment, display enormous plasticity in responding to constantly changing environmental challenges through assimilating changes in the genome (resulting in sequence polymorphism) and epigenome (resulting in gene expression alterations). DNA methylation is one of the reversible epigenetic modifications imparting a swift tunability to the genome through a complex interplay of methylation and demethylation processes. Plants have diligently repurposed the base excision repair machinery to actively erase methylation marks on DNA by evolving a specific family of HhH DNA glycosylases collectively called the DEMETER family. The DML family comprises four types of HhH DNA glycosylases: DEMETER, REPRESSOR OF SILENCING 1, and the paralogs DML2 and DML3 (DEMETER-LIKE proteins 2 and 3). The DML family enzymes are bifunctional glycosylases with a bipartite glycosylase domain and a C-terminal domain crucial for the catalytic activity. Removal of methylation proceeds through a classical mechanism of DNA bending and base flipping. DML family DNA glycosylases play roles in maintaining genome stability, plant development, and various biotic and abiotic stresses. This review aims to provide a comprehensive summary of the current understanding of the molecular mechanisms and biological functions of the DEMETER family DNA glycosylases in plants.
Holliday junction resolution by At-HIGLE: an SLX1 lineage endonuclease from Arabidopsis thaliana with a novel in-built regulatory mechanism
(Oxford University Press, 2022) Verma, Prabha; Kumari, Poonam; Negi, Shreya; Yadav, Gitanjali; Gaur, Vineet
Holliday junction is the key homologous recombination intermediate, resolved by structure-selective endonucleases (SSEs). SLX1 is the most promiscuous SSE of the GIY-YIG nuclease superfamily. In fungi and animals, SLX1 nuclease activity relies on a non-enzymatic partner, SLX4, but no SLX1-SLX4 like complex has ever been characterized in plants. Plants exhibit specialized DNA repair and recombination machinery. Based on sequence similarity with the GIY-YIG nuclease domain of SLX1 proteins from fungi and animals, At-HIGLE was identified to be a possible SLX1 like nuclease from plants. Here, we elucidated the crystal structure of the At-HIGLE nuclease domain from Arabidopsis thaliana, establishing it as a member of the SLX1-lineage of the GIY-YIG superfamily with structural changes in DNA interacting regions. We show that At-HIGLE can process branched-DNA molecules without an SLX4 like protein. Unlike fungal SLX1, At-HIGLE exists as a catalytically active homodimer capable of generating two coordinated nicks during HJ resolution. Truncating the extended C-terminal region of At-HIGLE increases its catalytic activity, changes the nicking pattern, and monomerizes At-HIGLE. Overall, we elucidated the first structure of a plant SLX1-lineage protein, showed its HJ resolving activity independent of any regulatory protein, and identified an in-built novel regulatory mechanism engaging its C-terminal region.
Microscopic and transcriptomic analyses of Dalbergoid legume peanut reveal a divergent evolution leading to Nod Factor dependent epidermal crack-entry and terminal bacteroid differentiation
(American Phytopathological Society, 2022) Raul, Bikash; Bhattacharjee, Oindrila; Ghosh, Amit; Upadhyay, Priya; Tembhare, Kunal; Singh, Ajeet; Shaheen, Tarannum; Ghosh, Asim Kumar; Torres-Jerez, Ivone; Krom, Nick; Clevenger, Josh; Udvardi, Michael; Scheffler, Brian E.; Ozias-Akins, Peggy; Sharma, Ravi Datta; Bandyopadhyay, Kaustav; Gaur, Vineet; Kumar, Shailesh; Sinharoy, Senjuti
Root nodule symbiosis (RNS) is the pillar behind sustainable agriculture and plays a pivotal role in the environmental nitrogen cycle. Most of the genetic, molecular, and cell-biological knowledge on RNS come from model legumes that exhibit a root-hair mode of bacterial infection in contrast to the Dalbergoid legumes exhibiting crack-entry of rhizobia. As a step towards understanding this important group of legumes, we have combined microscopic analysis and temporal transcriptome to obtain a dynamic view of plant gene expression during Arachis hypogaea (peanut) nodule development. We generated a comprehensive transcriptome data by mapping the reads to A. hypogaea, and two diploid progenitor genomes. Additionally, we performed BLAST searches to identify nodule-induced yet-to-be annotated peanut genes. Comparison between peanut, Medicago truncatula, Lotus japonicus, and Glycine max showed upregulation of 61 peanut orthologs among 111 tested known RNS-related genes, indicating conservation in mechanisms of nodule development among members of the Papilionoid family. Unlike model legumes, recruitment of class 1 phytoglobin derived symbiotic hemoglobin (SymH) in peanut indicates diversification of oxygen scavenging mechanisms in the Papilionoid family. Finally, absence of cysteine-rich motif-1 containing-NCRs, but the recruitment of defensin like NCRs suggest a diverse molecular mechanism of terminal bacteroid differentiation. In summary, our work describes genetic conservation and diversification in legume-rhizobial symbiosis in the Papilionoid family, as well as among members of the Dalbergoid legumes.
Structural aspects of DNA repair and recombination in crop improvement
(Frontiers Media S.A., 2020) Verma, Prabha; Tandon, Reetika; Yadav, Gitanjali; Gaur, Vineet
The adverse effects of global climate change combined with an exponentially increasing human population have put substantial constraints on agriculture, accelerating efforts towards ensuring food security for a sustainable future. Conventional plant breeding and modern technologies have led to the creation of plants with better traits and higher productivity. Most crop improvement approaches (conventional breeding, genome modification, and gene editing) primarily rely on DNA repair and recombination (DRR). Studying plant DRR can provide insights into designing new strategies or improvising the present techniques for crop improvement. Even though plants have evolved specialized DRR mechanisms compared to other eukaryotes, most of our insights about plant-DRRs remain rooted in studies conducted in animals. DRR mechanisms in plants include direct repair, nucleotide excision repair (NER), base excision repair (BER), mismatch repair (MMR), non-homologous end joining (NHEJ) and homologous recombination (HR). Although each DRR pathway acts on specific DNA damage, there is crosstalk between these. Considering the importance of DRR pathways as a tool in crop improvement, this review focuses on a general description of each DRR pathway, emphasizing on the structural aspects of key DRR proteins. The review highlights the gaps in our understanding and the importance of studying plant DRR in the context of crop improvement.
A structure and knowledge-based combinatorial approach to engineering universal scFv antibodies against influenza M2 protein
(BioMed Central Ltd, 2023) Kumar, Ujjwal; Goyal, Priya; Madni, Zaid K.; Kamble, Kajal; Gaur, Vineet; Rajala, Maitreyi S.; Salunke, Dinakar M.
Background: The influenza virus enters the host via hemagglutinin protein binding to cell surface sialic acid. Receptor-mediated endocytosis is followed by viral nucleocapsid uncoating for replication aided by the transmembrane viral M2 proton ion channel. M2 ectodomain (M2e) is a potential universal candidate for monoclonal antibody therapy owing to its conserved nature across influenza virus subtypes and its importance in viral propagation. Methods: The phage-displayed naive human antibody libraries were screened against the short stretch of the N-terminal 10-mer peptide (SLLTEVETPI) of the M2e. ELISA, BLI, and flow cytometry assays were used to examine scFv binding to M2e epitopes. The scFv crystal structures were determined to examine the nature of the interactions. The potencies of the scFvs against the influenza virus were demonstrated by real-time PCR and confocal microscopy imaging. Results: The four unique scFv clones were obtained from the scFv phage-display antibody libraries and shown to exhibit binding with the 10-mer conserved part of the M2e and with full-length M2 protein expressed on the HEK293T cells. The crystal structure of scFv AU1 with M2e peptide showed the peptide as a dimer in the parallel beta-sheet conformation bound at the interface of two scFv CDRs. The scFv AU1 significantly restricted the release of H1N1 virus progeny from the infected A549 cells. Conclusion: This structural and biochemical study showcased the binding of antibody scFv molecules with M2e peptide dimer, providing the structural insights for the function effect in terms of recognizing and restricting the release of new viral particles from an infected host cell.