Biology subjects

Mets, T.

Publications and source records attributed to Mets, T..

3 recordsLinked to original sources

bioRxiv2023

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↗

bioRxiv2023

What is hidden in the darkness? Deep-learning assisted large-scale protein family curation uncovers novel protein families and folds

Driven by the development and upscaling of fast genome sequencing and assembly pipelines, the number of protein-coding sequences deposited in public protein sequence databases is increasing exponentially. Recently, the dramatic success of deep learning-based approaches applied to protein structure prediction has done the same for protein structures. We are now entering a new era in protein sequence and structure annotation, with hundreds of millions of predicted protein structures made available through the AlphaFold database. These models cover most of the catalogued natural proteins, including those difficult to annotate for function or putative biological role based on standard, homology-based approaches. In this work, we quantified how much of such "dark matter" of the natural protein universe was structurally illuminated by AlphaFold2 and modelled this diversity as an interactive sequence similarity network that can be navigated at https://uniprot3d.org/atlas/AFDB90v4. In the process, we discovered multiple novel protein families by searching for novelties from sequence, structure, and semantic perspectives. We added a number of them to Pfam, and experimentally demonstrate that one of these belongs to a novel superfamily of translation-targeting toxin-antitoxin systems, TumE-TumA. This work highlights the role of large-scale, evolution-driven protein comparison efforts in combination with structural similarities, genomic context conservation, and deep-learning based function prediction tools for the identification of novel protein families, aiding not only annotation and classification efforts but also the curation and prioritisation of target proteins for experimental characterisation.

bioinformatics↗

bioRxiv2021

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↗