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Saha, C. K.

Publications and source records attributed to Saha, C. K..

6 recordsLinked to original sources

FlashFold: a standalone command-line tool for accelerated protein structure prediction

AlphaFold has revolutionized the decades-old issue of precisely predicting protein structures. However, its high accuracy relies on a computationally intensive step that involves searching vast databases for homologous sequences as the query protein of interest. Additionally, predicting the quaternary structure of protein complexes requires prior knowledge of subunit counts, a prerequisite rarely met. To address these limitations, we introduce FlashFold - a fast, user-friendly tool for protein structure prediction. It accelerates homology searches using a compact built-in database, enabling structure predictions up to 3-fold faster than AlphaFold3, with sacrificing little or no accuracy. Unlike others, FlashFold features adaptable built-in databases that allow users to easily incorporate their own privately sequenced data - an option that can positively influence the prediction accuracy. Moreover, it allows users to estimate stoichiometry of protein complexes directly from sequence information. To support high-throughput workflows and streamline downstream decision-making, it generates interactive and filterable summary reports, enabling users to efficiently visualize protein structures, and interpret large volumes of prediction results. FlashFold runs locally on Linux and Mac, eliminating reliance on third-party servers. FlashFold is available at https://github.com/chayan7/flashfold.

bioinformatics↗

Exploring Harmala Alkaloids as Novel Antimalarial Agents against Plasmodium falciparum through Bioinformatics Approaches

Malaria, caused by the Plasmodium falciparum, remains a significant global health challenge, with Plasmodium falciparum accounting for approximately 50% of cases and posing a considerable threat. Despite advances in control measures, malaria continues to cause an estimated one million deaths annually. The complex lifecycle of P. falciparum, involving both vertebrate hosts and Anopheles mosquitoes, complicates eradication efforts. The parasites resistance to existing antimalarial drugs, along with medication toxicity, necessitates innovative therapeutic approaches. Recent research has revealed that harmine, an alkaloid produced by an endophytic gut bacterium of Anopheles mosquitoes, can impede the transmission of the malarial parasite to humans by inhibiting a crucial life stage. This study investigates harmala alkaloids, sourced from plants and bacteria such as Peganum harmala, as potential alternatives to conventional antimalarial drugs. Notably, harmine and harmaline have shown promising antimalarial activity by inhibiting the essential enzyme protein kinase 4 (PK4), which is vital for the parasites survival. These compounds exhibit lower toxicity, effectively inhibiting both the blood stage growth and transmission of the parasite. Using in silico methodologies, including ADME analysis, molecular docking, MD simulation, and toxicity analysis, this study identifies harmala alkaloids as potential inhibitors against crucial P. falciparum proteins. Targeting proteins essential for the parasites survival, similar to established drugs like pfCRT protein, lays the foundation for developing effective antimalarial treatments. The comprehensive screening of harmala alkaloid molecules opens avenues for the pharmaceutical industry to tackle challenges related to drug resistance and toxicity, offering a promising route for the biorational management of malaria.

bioinformatics↗

The structural basis of hyperpromiscuity in a core combinatorial network of Type II toxin-antitoxin and related phage defence systems

Toxin-antitoxin (TA) systems are a large group of small genetic modules found in prokaryotes and their mobile genetic elements. Type II TAs are encoded as bicistronic (two-gene) operons that encode two proteins: a toxin and a neutralising antitoxin. Using our tool NetFlax (standing for Network-FlaGs for toxins and antitoxins) we have performed a large-scale bioinformatic analysis of proteinaceous TAs, revealing interconnected clusters constituting a core network of TA-like gene pairs. To understand the structural basis of toxin neutralisation by antitoxins, we have predicted the structures of 3,419 complexes with AlphaFold2. Together with mutagenesis and functional assays, our structural predictions provide insights into the neutralising mechanism of the hyperpromiscuous Panacea antitoxin domain. In antitoxins composed of standalone Panacea, the domain mediates direct toxin neutralisation, while in multidomain antitoxins the neutralisation is mediated by other domains, such as PAD1, Phd-C and ZFD. We hypothesise that Panacea acts as a sensor that regulates TA activation. We have experimentally validated 16 new NetFlax TA systems. We used functional domain annotations and with metabolic labelling assays to predict their potential mechanisms of toxicity (such as disruption of membrane integrity, inhibition of cell division and abrogation of protein synthesis) as well as biological functions (such as antiphage defence). The interactive version of the NetFlax TA network that includes structural predictions can be accessed at http://netflax.webflags.se/. Significance statementToxin-antitoxin systems are enigmatic components of microbial genomes, with their biological functions being a conundrum of debate for decades. Increasingly, TAs are being found to have a role in defence against bacteriophages. By mapping and experimentally validating a core combinatorial network of TA systems and high-throughput prediction of structural interfaces, we uncover the evolutionary scale of TA partner swapping and discover new toxic effectors. We validate the predicted toxin:antitoxin complex interfaces of four TA systems, uncovering the evolutionary malleable mechanism of toxin neutralisation by Panacea-containing PanA antitoxins. We find TAs are evolutionarily related to several other phage defence systems, cementing their role as important molecular components of the arsenal of microbial warfare.

microbiology↗

Panacea: a hyperpromiscuous antitoxin protein domain for the neutralisation of diverse toxin domains

Toxin-Antitoxin (TA) gene pairs are ubiquitous in microbial chromosomal genomes and plasmids, as well as bacteriophages. They act as regulatory switches, with the toxin limiting the growth of bacteria and archaea by compromising diverse essential cellular targets, and the antitoxin counteracting the toxic effect. To uncover previously uncharted TA diversity across microbes and bacteriophages, we analysed the conservation of genomic neighbourhoods using our computational tool FlaGs (for Flanking Genes), which allows high-throughput detection of TA-like operons. Focussing on the widespread but poorly experimentally characterised antitoxin domain DUF4065, our in silico analyses indicated that DUF4065-containing proteins serve as broadly distributed antitoxin components in putative TA-like operons with dozens of different toxic domains with multiple different folds. Given the versatility of DUF4065, we have renamed the domain to Panacea (and proteins containing the domain, PanA) after the Greek goddess of universal remedy. We have experimentally validated nine PanA-neutralised TA pairs. While the majority of validated PanA-neutralised toxins act as translation inhibitors or membrane disruptors, a putative nucleotide cyclase toxin from a Burkholderia prophage compromises replication and translation, as well as inducing RelA-dependent accumulation of the nucleotide alarmone (p)ppGpp. We find that Panacea-containing antitoxins form a complex with their diverse cognate toxins, characteristic of the direct neutralisation mechanisms employed by Type II TA systems. Finally, through directed evolution we have selected PanA variants that can neutralise non-cognate TA toxins, thus experimentally demonstrating the evolutionary plasticity of this hyperpromiscuous antitoxin domain. SignificanceToxin-antitoxin systems are enigmatic and diverse elements of bacterial and bacteriophage genomes. We have uncovered remarkable versatility of an antitoxin protein domain, that has evolved to neutralise dozens of different toxin domains. We find that antitoxins carrying this domain - Panacea - form complexes with their cognate toxins, indicating a direct neutralisation mechanism, and that Panacea can be evolved to neutralise a non-cognate and non-homologous toxin with just two amino acid substitutions. This raises the possibility that this domain could be an adaptable universal, or semi-universal protein neutraliser with significant biotechnological and medical potential.

microbiology↗

Discovery of small alarmone synthetases and their inhibitors as toxin-antitoxin loci

Under stressful conditions, bacterial RelA-SpoT Homologue (RSH) enzymes synthesise the alarmone (p)ppGpp, a nucleotide messenger. (p)ppGpp rewires bacterial transcription and metabolism to cope with stress, and at high concentrations inhibits the process of protein synthesis and bacterial growth to save and redirect resources until conditions improve. Single domain Small Alarmone Synthetases (SASs) are RSH family members that contain the (p)ppGpp synthesis (SYNTH) domain, but lack the hydrolysis (HD) domain and regulatory C-terminal domains of the long RSHs such as Rel, RelA and SpoT. We have discovered that multiple SAS subfamilies can be encoded in broadly distributed conserved bicistronic operon architectures in bacteria and bacteriophages that are reminiscent of those typically seen in toxin-antitoxin (TA) operons. We have validated five of these SASs as being toxic (toxSASs), with neutralisation by the protein products of six neighbouring antitoxin genes. The toxicity of Cellulomonas marina ToxSAS FaRel is mediated by alarmone accumulation combined with depletion of cellular ATP and GTP pools, and this is counteracted by its HD domain-containing antitoxin. Thus, the ToxSAS-antiToxSAS system is a novel TA paradigm comprising multiple different antitoxins that exemplifies how ancient nucleotide-based signalling mechanisms can be repurposed as TA modules during evolution, potentially multiple times independently.

microbiology↗

Predicting Functional Associations using Flanking Genes (FlaGs)

Analysis of conservation of gene neighbourhoods over different evolutionary levels is important for understanding operon and gene cluster evolution, and predicting functional associations. Our tool FlaGs (Flanking Genes) takes a list of NCBI protein accessions as in input, clusters neighbourhood-encoded proteins into homologous groups using sensitive sequence searching, and outputs a graphical visualization of the gene neighbourhood and its conservation, along with a phylogenetic tree annotated with flanking gene conservation. FlaGs has demonstrated utility for molecular evolutionary analysis, having uncovered a new toxin-antitoxin system in prokaryotes and bacteriophages. FlaGs can be downloaded from https://github.com/GCA-VH-lab/FlaGs or run at www.webflags.se.

bioinformatics↗