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.

Bioinformatics calls the school: Use of smartphones to introduce Python for bioinformatics in high schools.

The dynamic nature of technological developments invites us to rethink the learning spaces. In this context, science education can be enriched by the contribution of new computational resources, making the educational process more up-to-date, challenging, and attractive. Bioinformatics is a key interdisciplinary field, contributing to the understanding of biological processes that is often underrated in secondary schools. As a useful resource in learning activities, bioinformatics could help in engaging students to integrate multiple fields of knowledge (logical-mathematical, biological, computational, etc.) and generate an enriched and long-lasting learning environment. Here, we report our recent project in which high school students learned basic concepts of programming applied to solving biological problems. The students were taught the Python syntax, and they coded simple tools to answer biological questions using resources at hand. Notably, these were built mostly on the students' own smartphones, which proved to be capable, readily available, and relevant complementary tools for teaching. This project resulted in an empowering and inclusive experience that challenged differences in social background and technological accessibility.

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.

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.

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.

ProtMiscuity: a database of promiscuous proteins.

Promiscuous behaviour in proteins and enzymes remains a challenging feature to understand the structure-function relationship. Here we present ProtMiscuity, a manually curated online database of proteins showing catalytic promiscuity. ProtMiscuity contains information about canonical and promiscuous activities comprising 88 different reactions in 57 proteins from 40 different organisms. It can be searched or browsed by protein names, organisms and descriptions of canonical and promiscuous reactions. Entries provide information on reaction substrates, products and kinetic parameters, mapping of active sites to sequence and structure and links to external resources with biological and functional annotations. ProtMiscuity could assist in studying the underlying mechanisms of promiscuous reactions by offering a unique and curated collection of experimentally derived data that is otherwise hard to find, retrieve and validate from literature.

Identification of inhibitors of the RGS homology domain of GRK2 by docking-based virtual screening.

AIMS: G protein-coupled receptor (GPCR) kinases (GRKs) are mainly involved in the desensitization of GPCRs. Among them, GRK2 has been described to be upregulated in many pathological conditions and its crucial role in cardiac hypertrophy, hypertension, and heart failure promoted the search for pharmacological inhibitors of its activity. There have been several reports of potent and selective inhibitors of GRK2, most of them directed to the kinase domain of the protein. However, the homologous to the regulator of G protein signaling (RH) domain of GRK2 has also been shown to regulate GPCRs signaling. Herein, we searched for potential inhibitors of receptor desensitization mediated by RH domain of GRK2. MATERIALS AND METHODS: We performed a docking-based virtual screening utilizing the crystal structure of GRK2 to search for potential inhibitors of the interaction between GRK2 and Gαq protein. To evaluate the biological activity of compounds we measured, calcium response of histamine H1 receptor (H1R) using Fura-2AM dye and H1R internalization by saturation binding experiments in A549 cells. GRK2(45-178)GFP translocation was determined in HeLa cells through confocal fluorescence imaging. KEY FINDINGS: We identified inhibitors of GRK2 able to reduce the RH mediated desensitization of the histamine H1 receptor and GRK2 translocation to plasma membrane. Also candidates presented adequate lipophilia and cytotoxicity profile. SIGNIFICANCE: We obtained compounds with the ability of reducing RH mediated actions of GRK2 that can be useful as a starting point in the development of novel drug candidates aimed to treat pathologies were GRK2 plays a key role.

Phylogenetic and phylodynamic analyses of human metapneumovirus in Buenos Aires (Argentina) for a three-year period (2009-2011).

Human metapneumovirus, which belongs to the Paramyxoviridae family and has been classified as a member of the Pneumovirus genus, is genetically and clinically similar to other family members such as human respiratory syncytial virus. A total of 1146 nasopharyngeal aspirates from pediatric patients with moderate and severe acute lower respiratory tract infections, hospitalized at the Ricardo Gutierrez Childrens Hospital (Buenos Aires, Argentina), were tested by real time RT-PCR for human metapneumovirus. Results showed that 168 (14.65%) were positive. Thirty-six of these 168 samples were randomly selected to characterize positive cases molecularly. The phylogenetic analysis of the sequences of the G and F genes showed that genotypes A2 and B2 cocirculated during 2009 and 2010 and that only genotype A2 circulated in 2011 in Argentina. Genotype A2 prevailed during the study period, a fact supported by a higher effective population size (Neτ) and higher diversity as compared to that of genotype B2 (10.9% (SE 1.3%) vs. 1.7% (SE 0.4%), respectively). The phylogeographic analysis of the G protein gene sequences showed that this virus has no geographical restrictions and can travel globally harbored in hosts. The selection pressure analysis of the F protein showed that although this protein has regions with polymorphisms, it has vast structural and functional constraints. In addition, the predicted B-linear epitopes and the sites recognized by previously described monoclonal antibodies were conserved in all Argentine sequences. This points out this protein as a potential candidate to be the target of future humanized antibodies or vaccines.