Searching for Frataxin Function: Exploring the Analogy with Nqo15, the Frataxin-like Protein of Respiratory Complex I from Thermus thermophilus.

Nqo15 is a subunit of respiratory complex I of the bacterium Thermus thermophilus, with strong structural similarity to human frataxin (FXN), a protein involved in the mitochondrial disease Friedreich's ataxia (FRDA). Recently, we showed that the expression of recombinant Nqo15 can ameliorate the respiratory phenotype of FRDA patients' cells, and this prompted us to further characterize both the Nqo15 solution's behavior and its potential functional overlap with FXN, using a combination of in silico and in vitro techniques. We studied the analogy of Nqo15 and FXN by performing extensive database searches based on sequence and structure. Nqo15's folding and flexibility were investigated by combining nuclear magnetic resonance (NMR), circular dichroism, and coarse-grained molecular dynamics simulations. Nqo15's iron-binding properties were studied using NMR, fluorescence, and specific assays and its desulfurase activation by biochemical assays. We found that the recombinant Nqo15 isolated from complex I is monomeric, stable, folded in solution, and highly dynamic. Nqo15 does not share the iron-binding properties of FXN or its desulfurase activation function.

A STRP-ed definition of Structured Tandem Repeats in Proteins.

Tandem Repeat Proteins (TRPs) are a class of proteins with repetitive amino acid sequences that have been studied extensively for over two decades. Different features at the level of sequence, structure, function and evolution have been attributed to them by various authors. And yet many of its salient features appear only when looking at specific subclasses of protein tandem repeats. Here, we attempt to rationalize the existing knowledge on Tandem Repeat Proteins (TRPs) by pointing out several dichotomies. The emerging picture is more nuanced than generally assumed and allows us to draw some boundaries of what is not a "proper" TRP. We conclude with an operational definition of a specific subset, which we have denominated STRPs (Structural Tandem Repeat Proteins), which separates a subclass of tandem repeats with distinctive features from several other less well-defined types of repeats. We believe that this definition will help researchers in the field to better characterize the biological meaning of this large yet largely understudied group of proteins.

CardIAP: calcium transients confocal image analysis tool.

One of the main topics of cardiovascular research is the study of calcium (Ca2+) handling, as even small changes in Ca2+ concentration can alter cell functionality (Bers, Annu Rev Physiol, 2014, 76, 107-127). Ionic calcium (Ca2+) plays the role of a second messenger in eukaryotic cells, associated with cellular functions such as cell cycle regulation, transport, motility, gene expression, and regulation. The use of fluorometric techniques in isolated cells loaded with Ca2+-sensitive fluorescent probes allows quantitative measurement of dynamic events occurring in living, functioning cells. The Cardiomyocytes Images Analyzer Python (CardIAP) application addresses the need to analyze and retrieve information from confocal microscopy images systematically, accurately, and rapidly. Here we present CardIAP, an open-source tool developed entirely in Python, freely available and useable in an interactive web application. In addition, CardIAP can be used as a standalone Python library and freely installed via PIP, making it easy to integrate into biomedical imaging pipelines. The images that can be generated in the study of the heart have the particularity of requiring both spatial and temporal analysis. CardIAP aims to open the field of cardiomyocytes and intact hearts image processing. The improvement in the extraction of information from the images will allow optimizing the usage of resources and animals. With CardIAP, users can run the analysis to both, the complete image, and portions of it in an easy way, and replicate it on a series of images. This analysis provides users with information on the spatial and temporal changes in calcium releases and characterizes them. The web application also allows users to extract calcium dynamics data in downloadable tables, simplifying the calculation of alternation and discordance indices and their classification. CardIAP aims to provide a tool that could assist biomedical researchers in studying the underlying mechanisms of anomalous calcium release phenomena.

CaviDB: a database of cavities and their features in the structural and conformational space of proteins.

Proteins are the structural, functional and evolutionary units of cells. On their surface, proteins are shaped into numerous depressions and protrusions that provide unique microenvironments for ligand binding and catalysis. The dynamics, size and chemical properties of these cavities are essential for a mechanistic understanding of protein function. Here, we present CaviDB, a novel database of cavities and their features in known protein structures. It integrates the results of commonly used cavity detection software with protein features derived from sequence, structural and functional analyses. Each protein in CaviDB is linked to its corresponding conformers, which also facilitates the study of conformational changes in cavities. Our initial release includes ∼927 773 distinct proteins, as well as the characterization of 36 136 869 cavities, of which 1 147 034 were predicted to be drug targets. The structural focus of CaviDB provides the ability to compare cavities and their properties from different conformational states of the protein. CaviDB not only aims to provide a comprehensive database that can be used for various aspects of drug design and discovery but also contributes to a better understanding of the fundamentals of protein structure-function relationships. With its unique approach, CaviDB represents an indispensable resource for the large community of bioinformaticians in particular and biologists in general. Database URL https://www.cavidb.org.

Combining Protein Conformational Diversity and Phylogenetic Information Using CoDNaS and CoDNaS-Q.

CoDNaS (http://ufq.unq.edu.ar/codnas/) and CoDNaS-Q (http://ufq.unq.edu.ar/codnasq) are repositories of proteins with different degrees of conformational diversity. Following the ensemble nature of the native state, conformational diversity represents the structural differences between the conformers in the ensemble. Each entry in CoDNaS and CoDNaS-Q contains a redundant collection of experimentally determined conformers obtained under different conditions. These conformers represent snapshots of the protein dynamism. While CoDNaS contains examples of conformational diversity at the tertiary level, a recent development, CoDNaS-Q, contains examples at the quaternary level. In the emerging age of accurate protein structure prediction by machine learning approaches, many questions remain open regarding the characterization of protein dynamism. In this context, most bioinformatics resources take advantage of distinct features derived from protein alignments, however, the complexity and heterogeneity of information makes it difficult to recover reliable biological signatures. Here we present five protocols to explore tertiary and quaternary conformational diversity at the individual protein level as well as for the characterization of the distribution of conformational diversity at the protein family level in a phylogenetic context. These protocols can provide curated protein families with experimentally known conformational diversity, facilitating the exploration of sequence determinants of protein dynamism. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Assessing conformational diversity with CoDNaS Alternate Protocol 1: Assessing conformational diversity at the quaternary level with CoDNaS-Q Basic Protocol 2: Exploring conformational diversity in a protein family Alternate Protocol 2: Exploring quaternary conformational diversity in a protein family Basic Protocol 3: Representing conformational diversity in a phylogenetic context.

CoDNaS-Q: a database of conformational diversity of the native state of proteins with quaternary structure.

SUMMARY: A collection of conformers that exist in a dynamical equilibrium defines the native state of a protein. The structural differences between them describe their conformational diversity, a defining characteristic of the protein with an essential role in multiple cellular processes. Since most proteins carry out their functions by assembling into complexes, we have developed CoDNaS-Q, the first online resource to explore conformational diversity in homooligomeric proteins. It features a curated collection of redundant protein structures with known quaternary structure. CoDNaS-Q integrates relevant annotations that allow researchers to identify and explore the extent and possible reasons of conformational diversity in homooligomeric protein complexes. AVAILABILITY AND IMPLEMENTATION: CoDNaS-Q is freely accessible at http://ufq.unq.edu.ar/codnasq/ or https://codnas-q.bioinformatica.org/home. The data can be retrieved from the website. The source code of the database can be downloaded from https://github.com/SfrRonaldo/codnas-q.

Few Amino Acid Mutations in H6 Influenza A Virus From South American Lineage Increase Viral Replication Efficiency in Poultry.

In chickens, infections due to influenza A virus (IAV) can be mild to severe and lethal. The study of IAV infections in poultry has been mostly limited to strains from the North American and Eurasian lineages, whereas limited information exists on similar studies with strains from the South American lineage (SAm). To better evaluate the risk of introduction of a prototypical SAm IAV strain into poultry, chickens were infected with a wild-type SAm origin strain (WT557/H6N2). The resulting virus progeny was serially passaged in chickens 20 times, and the immunopathological effects of the last passage virus, 20Ch557/H6N2, in chickens were compared to those of the parental strain. A comparison of complete viral genome sequences indicated that the 20Ch557/H6N2 strain contained 13 amino acid differences compared to the wild-type strain. Five of these mutations are in functionally relevant regions of the viral surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). However, despite higher and more prolonged virus shedding in chickens inoculated with the 20Ch557/H6N2 strain compared to those that received the WT557/H6N2 strain, transmission to naïve chickens was not observed for either group. Analyses by flow cytometry of mononuclear cells and lymphocyte subpopulations from the lamina propria and intraepithelial lymphocytic cells (IELs) from the ileum revealed a significant increase in the percentages of CD3+TCRγδ+ IELs in chickens inoculated with the 20Ch557/H6N2 strain compared to those inoculated with the WT557/H6N2 strain.

Impact of protein conformational diversity on AlphaFold predictions.

MOTIVATION: After the outstanding breakthrough of AlphaFold in predicting protein 3D models, new questions appeared and remain unanswered. The ensemble nature of proteins, for example, challenges the structural prediction methods because the models should represent a set of conformers instead of single structures. The evolutionary and structural features captured by effective deep learning techniques may unveil the information to generate several diverse conformations from a single sequence. Here, we address the performance of AlphaFold2 predictions obtained through ColabFold under this ensemble paradigm. RESULTS: Using a curated collection of apo-holo pairs of conformers, we found that AlphaFold2 predicts the holo form of a protein in ∼70% of the cases, being unable to reproduce the observed conformational diversity with the same error for both conformers. More importantly, we found that AlphaFold2's performance worsens with the increasing conformational diversity of the studied protein. This impairment is related to the heterogeneity in the degree of conformational diversity found between different members of the homologous family of the protein under study. Finally, we found that main-chain flexibility associated with apo-holo pairs of conformers negatively correlates with the predicted local model quality score plDDT, indicating that plDDT values in a single 3D model could be used to infer local conformational changes linked to ligand binding transitions. AVAILABILITY AND IMPLEMENTATION: Data and code used in this manuscript are publicly available at https://gitlab.com/sbgunq/publications/af2confdiv-oct2021. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Structural and evolutionary analysis unveil functional adaptations in the promiscuous behavior of serum albumins.

Promiscuous activities have been related to the capacity to catalyze reactions different from those a protein has evolved to sustain. In this work, we rethought the serum albumin's promiscuous behavior using evolutionary and structural analysis. We found that the cross aldol condensation of acetone and p-formylbenzonitrile is a promiscuous reaction conserved in humans serum albumin and in closely related albumins from other mammals. Evolutionary analysis indicates that the residues involved in this promiscuous reaction are evolving under positive selection, an evolutionary pattern indicating a putative functional adaptation. Also, key residues are located in an evolutionary conserved cavity connected with the protein surface with an also conserved tunnel and mutations involving these residues are described in human diseases. Overall, our results suggest that albumin could have evolved to sustain a still unknown biological function among the many others it maintains. Our results could contribute to better characterize the serum albumin family and raise questions about the evolution of protein promiscuity and function.

CoDNaS-RNA: a database of conformational diversity in the native state of RNA.

SUMMARY: Conformational changes in RNA native ensembles are central to fulfill many of their biological roles. Systematic knowledge of the extent and possible modulators of this conformational diversity is desirable to better understand the relationship between RNA dynamics and function. We have developed CoDNaS-RNA as the first database of conformational diversity in RNA molecules. Known RNA structures are retrieved and clustered to identify alternative conformers of each molecule. Pairwise structural comparisons between all conformers within each cluster allows to measure the variability of the molecule. Additional annotations about structural features, molecular interactions and biological function are provided. All data in CoDNaS-RNA is free to download and available as a public website that can be of interest for researchers in computational biology and other life science disciplines. AVAILABILITY AND IMPLEMENTATION: The data underlying this article are available at http://ufq.unq.edu.ar/codnasrna or https://codnas-rna.bioinformatica.org/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Intrinsically Disordered Region Modulates Ligand Binding in Glutaredoxin 1 from Trypanosoma Brucei.

Glutaredoxins are small proteins that share a common well-conserved thioredoxin-fold and participate in a wide variety of biological processes. Among them, class II Grx are redox-inactive proteins involved in iron-sulfur (Fe-S) metabolism. In the present work, we report different structural and dynamics aspects of 1CGrx1 from the pathogenic parasite Trypanosoma brucei that differentiate it from other orthologues by the presence of a parasite-specific unstructured N-terminal extension whose role has not been fully elucidated yet. Previous nuclear magnetic resonance (NMR) studies revealed significant differences with respect to the mutant lacking the disordered tail. Herein, we have performed atomistic molecular dynamics simulations that, complementary to NMR studies, confirm the intrinsically disordered nature of the N-terminal extension. Moreover, we confirm the main role of these residues in modulating the conformational dynamics of the glutathione-binding pocket. We observe that the N-terminal extension modifies the ligand cavity stiffening it by specific interactions that ultimately modulate its intrinsic flexibility, which may modify its role in the storage and/or transfer of preformed iron-sulfur clusters. These unique structural and dynamics aspects of Trypanosoma brucei 1CGrx1 differentiate it from other orthologues and could have functional relevance. In this way, our results encourage the study of other similar protein folding families with intrinsically disordered regions whose functional roles are still unrevealed and the screening of potential 1CGrx1 inhibitors as antitrypanosomal drug candidates.

"Protein" no longer means what it used to.

Every biologist knows that the word protein describes a group of macromolecules essential to sustain life on Earth. As biologists, we are invariably trained under a protein paradigm established since the early twentieth century. However, in recent years, the term protein unveiled itself as an euphemism to describe the overwhelming heterogeneity of these compounds. Most of our current studies are targeted on carefully selected subsets of proteins, but we tend to think and write about these as representative of the whole population. Here we discuss how seeking for universal definitions and general rules in any arbitrarily segmented study would be misleading about the conclusions. Of course, it is not our purpose to discourage the use of the word protein. Instead, we suggest to embrace the extended universe of proteins to reach a deeper understanding of their full potential, realizing that the term encompasses a group of molecules very heterogeneous in terms of size, shape, chemistry and functions, i.e. the term protein no longer means what it used to.

PED in 2021: a major update of the protein ensemble database for intrinsically disordered proteins.

The Protein Ensemble Database (PED) (https://proteinensemble.org), which holds structural ensembles of intrinsically disordered proteins (IDPs), has been significantly updated and upgraded since its last release in 2016. The new version, PED 4.0, has been completely redesigned and reimplemented with cutting-edge technology and now holds about six times more data (162 versus 24 entries and 242 versus 60 structural ensembles) and a broader representation of state of the art ensemble generation methods than the previous version. The database has a completely renewed graphical interface with an interactive feature viewer for region-based annotations, and provides a series of descriptors of the qualitative and quantitative properties of the ensembles. High quality of the data is guaranteed by a new submission process, which combines both automatic and manual evaluation steps. A team of biocurators integrate structured metadata describing the ensemble generation methodology, experimental constraints and conditions. A new search engine allows the user to build advanced queries and search all entry fields including cross-references to IDP-related resources such as DisProt, MobiDB, BMRB and SASBDB. We expect that the renewed PED will be useful for researchers interested in the atomic-level understanding of IDP function, and promote the rational, structure-based design of IDP-targeting drugs.

Intrinsically Disordered Protein Ensembles Shape Evolutionary Rates Revealing Conformational Patterns.

Intrinsically disordered proteins (IDPs) lack stable tertiary structure under physiological conditions. The unique composition and complex dynamical behaviour of IDPs make them a challenge for structural biology and molecular evolution studies. Using NMR ensembles, we found that IDPs evolve under a strong site-specific evolutionary rate heterogeneity, mainly originated by different constraints derived from their inter-residue contacts. Evolutionary rate profiles correlate with the experimentally observed conformational diversity of the protein, allowing the description of different conformational patterns possibly related to their structure-function relationships. The correlation between evolutionary rates and contact information improves when structural information is taken not from any individual conformer or the whole ensemble, but from combining a limited number of conformers. Our results suggest that residue contacts in disordered regions constrain evolutionary rates to conserve the dynamic behaviour of the ensemble and that evolutionary rates can be used as a proxy for the conformational diversity of IDPs.

RepeatsDB in 2021: improved data and extended classification for protein tandem repeat structures.

The RepeatsDB database (URL: https://repeatsdb.org/) provides annotations and classification for protein tandem repeat structures from the Protein Data Bank (PDB). Protein tandem repeats are ubiquitous in all branches of the tree of life. The accumulation of solved repeat structures provides new possibilities for classification and detection, but also increasing the need for annotation. Here we present RepeatsDB 3.0, which addresses these challenges and presents an extended classification scheme. The major conceptual change compared to the previous version is the hierarchical classification combining top levels based solely on structural similarity (Class > Topology > Fold) with two new levels (Clan > Family) requiring sequence similarity and describing repeat motifs in collaboration with Pfam. Data growth has been addressed with improved mechanisms for browsing the classification hierarchy. A new UniProt-centric view unifies the increasingly frequent annotation of structures from identical or similar sequences. This update of RepeatsDB aligns with our commitment to develop a resource that extracts, organizes and distributes specialized information on tandem repeat protein structures.

MobiDB: intrinsically disordered proteins in 2021.

The MobiDB database (URL: https://mobidb.org/) provides predictions and annotations for intrinsically disordered proteins. Here, we report recent developments implemented in MobiDB version 4, regarding the database format, with novel types of annotations and an improved update process. The new website includes a re-designed user interface, a more effective search engine and advanced API for programmatic access. The new database schema gives more flexibility for the users, as well as simplifying the maintenance and updates. In addition, the new entry page provides more visualisation tools including customizable feature viewer and graphs of the residue contact maps. MobiDB v4 annotates the binding modes of disordered proteins, whether they undergo disorder-to-order transitions or remain disordered in the bound state. In addition, disordered regions undergoing liquid-liquid phase separation or post-translational modifications are defined. The integrated information is presented in a simplified interface, which enables faster searches and allows large customized datasets to be downloaded in TSV, Fasta or JSON formats. An alternative advanced interface allows users to drill deeper into features of interest. A new statistics page provides information at database and proteome levels. The new MobiDB version presents state-of-the-art knowledge on disordered proteins and improves data accessibility for both computational and experimental users.

Revenant: a database of resurrected proteins.

Revenant is a database of resurrected proteins coming from extinct organisms. Currently, it contains a manually curated collection of 84 resurrected proteins derived from bibliographic data. Each protein is extensively annotated, including structural, biochemical and biophysical information. Revenant contains a browse capability designed as a timeline from where the different proteins can be accessed. The oldest Revenant entries are between 4200 and 3500 million years ago, while the younger entries are between 8.8 and 6.3 million years ago. These proteins have been resurrected using computational tools called ancestral sequence reconstruction techniques combined with wet-laboratory synthesis and expression. Resurrected proteins are commonly used, with a noticeable increase during the past years, to explore and test different evolutionary hypotheses such as protein stability, to explore the origin of new functions, to get biochemical insights into past metabolisms and to explore specificity and promiscuous behaviour of ancient proteins.

Exploring Conformational Space with Thermal Fluctuations Obtained by Normal-Mode Analysis.

Proteins in their native states can be represented as ensembles of conformers in dynamical equilibrium. Thermal fluctuations are responsible for transitions between these conformers. Normal-modes analysis (NMA) using elastic network models (ENMs) provides an efficient procedure to explore global dynamics of proteins commonly associated with conformational transitions. In the present work, we present an iterative approach to explore protein conformational spaces by introducing structural distortions according to their equilibrium dynamics at room temperature. The approach can be used either to perform unbiased explorations of conformational space or to explore guided pathways connecting two different conformations, e.g., apo and holo forms. In order to test its performance, four proteins with different magnitudes of structural distortions upon ligand binding have been tested. In all cases, the conformational selection model has been confirmed and the conformational space between apo and holo forms has been encompassed. Different strategies have been tested that impact on the efficiency either to achieve a desired conformational change or to achieve a balanced exploration of the protein conformational multiplicity.

Curating the gnomAD database: Report of novel variants in the globin-coding genes and bioinformatics analysis.

Massive parallel sequencing technologies are facilitating the faster identification of sequence variants with the consequent capability of untangling the molecular bases of many human genetic syndromes. However, it is not always easy to understand the impact of novel variants, especially for missense changes, which can lead to a spectrum of phenotypes. This study presents a custom-designed multistep methodology to evaluate the impact of novel variants aggregated in the genome aggregation database for the HBB, HBA2, and HBA1 genes, by testing and improving its performance with a dataset of previously described alterations affecting those same genes. This approach scored high sensitivity and specificity values and showed an overall better performance than sequence-derived predictors, highlighting the importance of protein conformation and interaction specific analyses in curating variant databases. This study also describes the strengths and limitations of these structural studies and allows identifying residues in the globin chains more prone to tolerate substitutions.

DisProt: intrinsic protein disorder annotation in 2020.

The Database of Protein Disorder (DisProt, URL: https://disprot.org) provides manually curated annotations of intrinsically disordered proteins from the literature. Here we report recent developments with DisProt (version 8), including the doubling of protein entries, a new disorder ontology, improvements of the annotation format and a completely new website. The website includes a redesigned graphical interface, a better search engine, a clearer API for programmatic access and a new annotation interface that integrates text mining technologies. The new entry format provides a greater flexibility, simplifies maintenance and allows the capture of more information from the literature. The new disorder ontology has been formalized and made interoperable by adopting the OWL format, as well as its structure and term definitions have been improved. The new annotation interface has made the curation process faster and more effective. We recently showed that new DisProt annotations can be effectively used to train and validate disorder predictors. We believe the growth of DisProt will accelerate, contributing to the improvement of function and disorder predictors and therefore to illuminate the 'dark' proteome.