Exploiting enzyme evolution for computational protein design.

Recent years have seen an explosion of interest in understanding the physicochemical parameters that shape enzyme evolution, as well as substantial advances in computational enzyme design. This review discusses three areas where evolutionary information can be used as part of the design process: (i) using ancestral sequence reconstruction (ASR) to generate new starting points for enzyme design efforts; (ii) learning from how nature uses conformational dynamics in enzyme evolution to mimic this process in silico; and (iii) modular design of enzymes from smaller fragments, again mimicking the process by which nature appears to create new protein folds. Using showcase examples, we highlight the importance of incorporating evolutionary information to continue to push forward the boundaries of enzyme design studies.

Deep learning in structural bioinformatics: current applications and future perspectives.

In this review article, we explore the transformative impact of deep learning (DL) on structural bioinformatics, emphasizing its pivotal role in a scientific revolution driven by extensive data, accessible toolkits and robust computing resources. As big data continue to advance, DL is poised to become an integral component in healthcare and biology, revolutionizing analytical processes. Our comprehensive review provides detailed insights into DL, featuring specific demonstrations of its notable applications in bioinformatics. We address challenges tailored for DL, spotlight recent successes in structural bioinformatics and present a clear exposition of DL-from basic shallow neural networks to advanced models such as convolution, recurrent, artificial and transformer neural networks. This paper discusses the emerging use of DL for understanding biomolecular structures, anticipating ongoing developments and applications in the realm of structural bioinformatics.

Deep learning for protein structure prediction and design-progress and applications.

Proteins are the key molecular machines that orchestrate all biological processes of the cell. Most proteins fold into three-dimensional shapes that are critical for their function. Studying the 3D shape of proteins can inform us of the mechanisms that underlie biological processes in living cells and can have practical applications in the study of disease mutations or the discovery of novel drug treatments. Here, we review the progress made in sequence-based prediction of protein structures with a focus on applications that go beyond the prediction of single monomer structures. This includes the application of deep learning methods for the prediction of structures of protein complexes, different conformations, the evolution of protein structures and the application of these methods to protein design. These developments create new opportunities for research that will have impact across many areas of biomedical research.

Identification of type VI secretion system effector-immunity pairs using structural bioinformatics.

The type VI secretion system (T6SS) is an important mediator of microbe-microbe and microbe-host interactions. Gram-negative bacteria use the T6SS to inject T6SS effectors (T6Es), which are usually proteins with toxic activity, into neighboring cells. Antibacterial effectors have cognate immunity proteins that neutralize self-intoxication. Here, we applied novel structural bioinformatic tools to perform systematic discovery and functional annotation of T6Es and their cognate immunity proteins from a dataset of 17,920 T6SS-encoding bacterial genomes. Using structural clustering, we identified 517 putative T6E families, outperforming sequence-based clustering. We developed a logistic regression model to reliably quantify protein-protein interaction of new T6E-immunity pairs, yielding candidate immunity proteins for 231 out of the 517 T6E families. We used sensitive structure-based annotation which yielded functional annotations for 51% of the T6E families, again outperforming sequence-based annotation. Next, we validated four novel T6E-immunity pairs using basic experiments in E. coli. In particular, we showed that the Pfam domain DUF3289 is a homolog of Colicin M and that DUF943 acts as its cognate immunity protein. Furthermore, we discovered a novel T6E that is a structural homolog of SleB, a lytic transglycosylase, and identified a specific glutamate that acts as its putative catalytic residue. Overall, this study applies novel structural bioinformatic tools to T6E-immunity pair discovery, and provides an extensive database of annotated T6E-immunity pairs.

Prop3D: A flexible, Python-based platform for machine learning with protein structural properties and biophysical data.

BACKGROUND: Machine learning (ML) has a rich history in structural bioinformatics, and modern approaches, such as deep learning, are revolutionizing our knowledge of the subtle relationships between biomolecular sequence, structure, function, dynamics and evolution. As with any advance that rests upon statistical learning approaches, the recent progress in biomolecular sciences is enabled by the availability of vast volumes of sufficiently-variable data. To be useful, such data must be well-structured, machine-readable, intelligible and manipulable. These and related requirements pose challenges that become especially acute at the computational scales typical in ML. Furthermore, in structural bioinformatics such data generally relate to protein three-dimensional (3D) structures, which are inherently more complex than sequence-based data. A significant and recurring challenge concerns the creation of large, high-quality, openly-accessible datasets that can be used for specific training and benchmarking tasks in ML pipelines for predictive modeling projects, along with reproducible splits for training and testing. RESULTS: Here, we report 'Prop3D', a platform that allows for the creation, sharing and extensible reuse of libraries of protein domains, featurized with biophysical and evolutionary properties that can range from detailed, atomically-resolved physicochemical quantities (e.g., electrostatics) to coarser, residue-level features (e.g., phylogenetic conservation). As a community resource, we also supply a 'Prop3D-20sf' protein dataset, obtained by applying our approach to CATH . We have developed and deployed the Prop3D framework, both in the cloud and on local HPC resources, to systematically and reproducibly create comprehensive datasets via the Highly Scalable Data Service ( HSDS ). Our datasets are freely accessible via a public HSDS instance, or they can be used with accompanying Python wrappers for popular ML frameworks. CONCLUSION: Prop3D and its associated Prop3D-20sf dataset can be of broad utility in at least three ways. Firstly, the Prop3D workflow code can be customized and deployed on various cloud-based compute platforms, with scalability achieved largely by saving the results to distributed HDF5 files via HSDS . Secondly, the linked Prop3D-20sf dataset provides a hand-crafted, already-featurized dataset of protein domains for 20 highly-populated CATH families; importantly, provision of this pre-computed resource can aid the more efficient development (and reproducible deployment) of ML pipelines. Thirdly, Prop3D-20sf's construction explicitly takes into account (in creating datasets and data-splits) the enigma of 'data leakage', stemming from the evolutionary relationships between proteins.

Protein structure-based gene expression signatures.

Gene expression signatures (GES) connect phenotypes to differential messenger RNA (mRNA) expression of genes, providing a powerful approach to define cellular identity, function, and the effects of perturbations. The use of GES has suffered from vague assessment criteria and limited reproducibility. Because the structure of proteins defines the functional capability of genes, we hypothesized that enrichment of structural features could be a generalizable representation of gene sets. We derive structural gene expression signatures (sGES) using features from multiple levels of protein structure (e.g., domain and fold) encoded by the mRNAs in GES. Comprehensive analyses of data from the Genotype-Tissue Expression Project (GTEx), the all RNA-seq and ChIP-seq sample and signature search (ARCHS4) database, and mRNA expression of drug effects on cardiomyocytes show that sGES are useful for characterizing biological phenomena. sGES enable phenotypic characterization across experimental platforms, facilitates interoperability of expression datasets, and describe drug action on cells.

EGG: Accuracy Estimation of Individual Multimeric Protein Models Using Deep Energy-Based Models and Graph Neural Networks.

Reliable and accurate methods of estimating the accuracy of predicted protein models are vital to understanding their respective utility. Discerning how the quaternary structure conforms can significantly improve our collective understanding of cell biology, systems biology, disease formation, and disease treatment. Accurately determining the quality of multimeric protein models is still computationally challenging, as the space of possible conformations is significantly larger when proteins form in complex with one another. Here, we present EGG (energy and graph-based architectures) to assess the accuracy of predicted multimeric protein models. We implemented message-passing and transformer layers to infer the overall fold and interface accuracy scores of predicted multimeric protein models. When evaluated with CASP15 targets, our methods achieved promising results against single model predictors: fourth and third place for determining the highest-quality model when estimating overall fold accuracy and overall interface accuracy, respectively, and first place for determining the top three highest quality models when estimating both overall fold accuracy and overall interface accuracy.

Molecular Origins of the Mendelian Rare Diseases Reviewed by Orpha.net: A Structural Bioinformatics Investigation.

The study of rare diseases is important not only for the individuals affected but also for the advancement of medical knowledge and a deeper understanding of human biology and genetics. The wide repertoire of structural information now available from reliable and accurate prediction methods provides the opportunity to investigate the molecular origins of most of the rare diseases reviewed in the Orpha.net database. Thus, it has been possible to analyze the topology of the pathogenic missense variants found in the 2515 proteins involved in Mendelian rare diseases (MRDs), which form the database for our structural bioinformatics study. The amino acid substitutions responsible for MRDs showed different mutation site distributions at different three-dimensional protein depths. We then highlighted the depth-dependent effects of pathogenic variants for the 20,061 pathogenic variants that are present in our database. The results of this structural bioinformatics investigation are relevant, as they provide additional clues to mitigate the damage caused by MRD.

Biotite: new tools for a versatile Python bioinformatics library.

BACKGROUND: Biotite is a program library for sequence and structural bioinformatics written for the Python programming language. It implements widely used computational methods into a consistent and accessible package. This allows for easy combination of various data analysis, modeling and simulation methods. RESULTS: This article presents major functionalities introduced into Biotite since its original publication. The fields of application are shown using concrete examples. We show that the computational performance of Biotite for bioinformatics tasks is comparable to individual, special purpose software systems specifically developed for the respective single task. CONCLUSIONS: The results show that Biotite can be used as program library to either answer specific bioinformatics questions and simultaneously allow the user to write entire, self-contained software applications with sufficient performance for general application.

3DVizSNP: a tool for rapidly visualizing missense mutations identified in high throughput experiments in iCn3D.

BACKGROUND: High throughput experiments in cancer and other areas of genomic research identify large numbers of sequence variants that need to be evaluated for phenotypic impact. While many tools exist to score the likely impact of single nucleotide polymorphisms (SNPs) based on sequence alone, the three-dimensional structural environment is essential for understanding the biological impact of a nonsynonymous mutation. RESULTS: We present a program, 3DVizSNP, that enables the rapid visualization of nonsynonymous missense mutations extracted from a variant caller format file using the web-based iCn3D visualization platform. The program, written in Python, leverages REST APIs and can be run locally without installing any other software or databases, or from a webserver hosted by the National Cancer Institute. It automatically selects the appropriate experimental structure from the Protein Data Bank, if available, or the predicted structure from the AlphaFold database, enabling users to rapidly screen SNPs based on their local structural environment. 3DVizSNP leverages iCn3D annotations and its structural analysis functions to assess changes in structural contacts associated with mutations. CONCLUSIONS: This tool enables researchers to efficiently make use of 3D structural information to prioritize mutations for further computational and experimental impact assessment. The program is available as a webserver at https://analysistools.cancer.gov/3dvizsnp or as a standalone python program at https://github.com/CBIIT-CGBB/3DVizSNP .

Surface exposed and charged residues drive thermostability in fungi.

Fungi, though mesophilic, include thermophilic and thermostable species, as well. The thermostability of proteins observed in these fungi is most likely to be attributed to several molecular factors, such as the presence of salt bridges and hydrogen bond interactions between side chains. These factors cannot be generalized for all fungi. Factors impacting thermostability can guide how fungal thermophilic proteins gain thermostability. We curated a dataset of proteins for 14 thermophilic fungi and their evolutionarily closer mesophiles. Additionally, the proteome of Chaetomium thermophilum and its evolutionarily related mesophile Chaetomium globosum was analyzed. Using eggNOG, we categorized the proteomes into clusters of orthologous groups (COGs). While the individual count of proteins is over-represented in mesophiles (for COGs S, G, L, and Q), there are certain features that are significantly enriched in thermophiles (such as charged residues, exposed residues, polar residues, etc.). Since fungi are known to be cellulolytic and chitinolytic by nature, we selected 37 existing carbohydrate-active enzymes (CAZyme) families in Eurotiales, Mucorales, and Sordariales. We looked at closely similar sequences and their modeled structures for further comparison. Comparing solvent accessibilities of thermophilic and mesophilic proteins, exposed and intermediate residues are observed higher in thermophiles whereas buried residues are observed higher in mesophiles. For specific five CAZYme families (GH7, GH11, GH18, GH45, and CBM1) we looked at position-specific substitutions between thermophiles and mesophiles. We also found that there are relatively more intramolecular interactions in thermophiles compared to mesophiles. Thus, we found factors such as surface exposed residues and charged residues that are highly likely to impart thermostability in fungi, and this study sets the stage for further studies in the area of fungal thermostability.

RNA tertiary structure prediction in CASP15 by the GeneSilico group: Folding simulations based on statistical potentials and spatial restraints.

Ribonucleic acid (RNA) molecules serve as master regulators of cells by encoding their biological function in the ribonucleotide sequence, particularly their ability to interact with other molecules. To understand how RNA molecules perform their biological tasks and to design new sequences with specific functions, it is of great benefit to be able to computationally predict how RNA folds and interacts in the cellular environment. Our workflow for computational modeling of the 3D structures of RNA and its interactions with other molecules uses a set of methods developed in our laboratory, including MeSSPredRNA for predicting canonical and non-canonical base pairs, PARNASSUS for detecting remote homology based on comparisons of sequences and secondary structures, ModeRNA for comparative modeling, the SimRNA family of programs for modeling RNA 3D structure and its complexes with other molecules, and QRNAS for model refinement. In this study, we present the results of testing this workflow in predicting RNA 3D structures in the CASP15 experiment. The overall high score of the computational models predicted by our group demonstrates the robustness of our workflow and its individual components in terms of predicting RNA 3D structures of acceptable quality that are close to the target structures. However, the variance in prediction quality is still quite high, and the results are still too far from the level of protein 3D structure predictions. This exercise led us to consider several improvements, especially to better predict and enforce stacking interactions and non-canonical base pairs.

Challenges in bridging the gap between protein structure prediction and functional interpretation.

The rapid evolution of protein structure prediction tools has significantly broadened access to protein structural data. Although predicted structure models have the potential to accelerate and impact fundamental and translational research significantly, it is essential to note that they are not validated and cannot be considered the ground truth. Thus, challenges persist, particularly in capturing protein dynamics, predicting multi-chain structures, interpreting protein function, and assessing model quality. Interdisciplinary collaborations are crucial to overcoming these obstacles. Databases like the AlphaFold Protein Structure Database, the ESM Metagenomic Atlas, and initiatives like the 3D-Beacons Network provide FAIR access to these data, enabling their interpretation and application across a broader scientific community. Whilst substantial advancements have been made in protein structure prediction, further progress is required to address the remaining challenges. Developing training materials, nurturing collaborations, and ensuring open data sharing will be paramount in this pursuit. The continued evolution of these tools and methodologies will deepen our understanding of protein function and accelerate disease pathogenesis and drug development discoveries.

An efficient computational protocol for template-based design of peptides that inhibit interactions involving SARS-CoV-2 proteins.

The RNA-dependent RNA polymerase (RdRp) complex of SARS-CoV-2 lies at the core of its replication and transcription processes. The interfaces between holo-RdRp subunits are highly conserved, facilitating the design of inhibitors with high affinity for the interaction interface hotspots. We, therefore, take this as a model protein complex for the application of a structural bioinformatics protocol to design peptides that inhibit RdRp complexation by preferential binding at the interface of its core subunit nonstructural protein, nsp12, with accessory factor nsp7. Here, the interaction hotspots of the nsp7-nsp12 subunit of RdRp, determined from a long molecular dynamics trajectory, are used as a template. A large library of peptide sequences constructed from multiple hotspot motifs of nsp12 is screened in-silico to determine sequences with high geometric complementarity and interaction specificity for the binding interface of nsp7 (target) in the complex. Two lead designed peptides are extensively characterized using orthogonal bioanalytical methods to determine their suitability for inhibition of RdRp complexation. Binding affinity of these peptides to accessory factor nsp7, determined using a surface plasmon resonance (SPR) assay, is slightly better than that of nsp12: dissociation constant of 133nM and 167nM, respectively, compared to 473nM for nsp12. A competitive ELISA is used to quantify inhibition of nsp7-nsp12 complexation, with one of the lead peptides giving an IC50 of 25µM . Cell penetrability and cytotoxicity are characterized using a cargo delivery assay and MTT cytotoxicity assay, respectively. Overall, this work presents a proof-of-concept of an approach for rational discovery of peptide inhibitors of SARS-CoV-2 protein-protein interactions.

Tertiary structure assessment at CASP15.

The results of tertiary structure assessment at CASP15 are reported. For the first time, recognizing the outstanding performance of AlphaFold 2 (AF2) at CASP14, all single-chain predictions were assessed together, irrespective of whether a template was available. At CASP15, there was no single stand-out group, with most of the best-scoring groups-led by PEZYFoldings, UM-TBM, and Yang Server-employing AF2 in one way or another. Many top groups paid special attention to generating deep Multiple Sequence Alignments (MSAs) and testing variant MSAs, thereby allowing them to successfully address some of the hardest targets. Such difficult targets, as well as lacking templates, were typically proteins with few homologues. Local divergence between prediction and target correlated with localization at crystal lattice or chain interfaces, and with regions exhibiting high B-factor factors in crystal structure targets, and should not necessarily be considered as representing error in the prediction. However, analysis of exposed and buried side chain accuracy showed room for improvement even in the latter. Nevertheless, a majority of groups produced high-quality predictions for most targets, which are valuable for experimental structure determination, functional analysis, and many other tasks across biology. These include those applying methods similar to those used to generate major resources such as the AlphaFold Protein Structure Database and the ESM Metagenomic atlas: the confidence estimates of the former were also notably accurate.

Sine-skewed toroidal distributions and their application in protein bioinformatics.

In the bioinformatics field, there has been a growing interest in modeling dihedral angles of amino acids by viewing them as data on the torus. This has motivated, over the past years, new proposals of distributions on the torus. The main drawback of most of these models is that the related densities are (pointwise) symmetric, despite the fact that the data usually present asymmetric patterns. This motivates the need to find a new way of constructing asymmetric toroidal distributions starting from a symmetric distribution. We tackle this problem in this article by introducing the sine-skewed toroidal distributions. The general properties of the new models are derived. Based on the initial symmetric model, explicit expressions for the shape and dependence measures are obtained, a simple algorithm for generating random numbers is provided, and asymptotic results for the maximum likelihood estimators are established. An important feature of our construction is that no extra normalizing constant needs to be calculated, leading to more flexible distributions without increasing the complexity of the models. The benefit of employing these new sine-skewed toroidal distributions is shown on the basis of protein data, where, in general, the new models outperform their symmetric antecedents.

Target shortage and less explored multiple targeting: hurdles in the development of novel antifungals but overcome/addressed effectively through structural bioinformatics.

Billions of people are affected by fungal infection worldwide, which is a major cause of morbidity and mortality in humans. Regardless of development in the field of antifungal therapeutics over the last three decades, multidrug resistance and limited efficacy of available antifungal drugs are very prominent and still a great hurdle in the patient treatment. The current antifungal pipeline is dry, which is needed to be strengthened. Although several strategies have been implemented over time to discover novel promising antifungal leads, but very little emphasis has been given to address the gap of fungal target identification. Undeniably, the need for identifying novel cellular fungal targets is as vital as discovering novel antifungal leads and a structural bioinformatics approach could be an effective strategy in this regard. To address the issue, we have performed in silico screening to identify a few potent multiple targeting ligands and their respective antifungal targets. Thus, we offer a perspective on the phenomena of 'target shortage' and least explored 'multiple targeting' being the most underrated challenges in antifungal drug discovery. 'Structural bioinformatics' could be an effective approach in the recognition of new/innovative antifungal target and identification/development of novel antifungal lead molecule aiming multiple molecular targets of the fungal pathogen.

Structural dynamics and allostery of Rab proteins: strategies for drug discovery and design.

Rab proteins represent the largest family of the Rab superfamily guanosine triphosphatase (GTPase). Aberrant human Rab proteins are associated with multiple diseases, including cancers and neurological disorders. Rab subfamily members display subtle conformational variations that render specificity in their physiological functions and can be targeted for subfamily-specific drug design. However, drug discovery efforts have not focused much on targeting Rab allosteric non-nucleotide binding sites which are subjected to less evolutionary pressures to be conserved, hence are likely to offer subfamily specificity and may be less prone to undesirable off-target interactions and side effects. To discover druggable allosteric binding sites, Rab structural dynamics need to be first incorporated using multiple experimentally and computationally obtained structures. The high-dimensional structural data may necessitate feature extraction methods to identify manageable representative structures for subsequent analyses. We have detailed state-of-the-art computational methods to (i) identify binding sites using data on sequence, shape, energy, etc., (ii) determine the allosteric nature of these binding sites based on structural ensembles, residue networks and correlated motions and (iii) identify small molecule binders through structure- and ligand-based virtual screening. To benefit future studies for targeting Rab allosteric sites, we herein detail a refined workflow comprising multiple available computational methods, which have been successfully used alone or in combinations. This workflow is also applicable for drug discovery efforts targeting other medically important proteins. Depending on the structural dynamics of proteins of interest, researchers can select suitable strategies for allosteric drug discovery and design, from the resources of computational methods and tools enlisted in the workflow.

The impact of structural bioinformatics tools and resources on SARS-CoV-2 research and therapeutic strategies.

SARS-CoV-2 is the causative agent of COVID-19, the ongoing global pandemic. It has posed a worldwide challenge to human health as no effective treatment is currently available to combat the disease. Its severity has led to unprecedented collaborative initiatives for therapeutic solutions against COVID-19. Studies resorting to structure-based drug design for COVID-19 are plethoric and show good promise. Structural biology provides key insights into 3D structures, critical residues/mutations in SARS-CoV-2 proteins, implicated in infectivity, molecular recognition and susceptibility to a broad range of host species. The detailed understanding of viral proteins and their complexes with host receptors and candidate epitope/lead compounds is the key to developing a structure-guided therapeutic design. Since the discovery of SARS-CoV-2, several structures of its proteins have been determined experimentally at an unprecedented speed and deposited in the Protein Data Bank. Further, specialized structural bioinformatics tools and resources have been developed for theoretical models, data on protein dynamics from computer simulations, impact of variants/mutations and molecular therapeutics. Here, we provide an overview of ongoing efforts on developing structural bioinformatics tools and resources for COVID-19 research. We also discuss the impact of these resources and structure-based studies, to understand various aspects of SARS-CoV-2 infection and therapeutic development. These include (i) understanding differences between SARS-CoV-2 and SARS-CoV, leading to increased infectivity of SARS-CoV-2, (ii) deciphering key residues in the SARS-CoV-2 involved in receptor-antibody recognition, (iii) analysis of variants in host proteins that affect host susceptibility to infection and (iv) analyses facilitating structure-based drug and vaccine design against SARS-CoV-2.

Identifying Potential Molecular Targets in Fungi Based on (Dis)Similarities in Binding Site Architecture with Proteins of the Human Pharmacolome.

Invasive fungal infections represent a public health problem that worsens over the years with the increasing resistance to current antimycotic agents. Therefore, there is a compelling medical need of widening the antifungal drug repertoire, following different methods such as drug repositioning, identification and validation of new molecular targets and developing new inhibitors against these targets. In this work we developed a structure-based strategy for drug repositioning and new drug design, which can be applied to infectious fungi and other pathogens. Instead of applying the commonly accepted off-target criterion to discard fungal proteins with close homologues in humans, the core of our approach consists in identifying fungal proteins with active sites that are structurally similar, but preferably not identical to binding sites of proteins from the so-called "human pharmacolome". Using structural information from thousands of human protein target-inhibitor complexes, we identified dozens of proteins in fungal species of the genera Histoplasma, Candida, Cryptococcus, Aspergillus and Fusarium, which might be exploited for drug repositioning and, more importantly, also for the design of new fungus-specific inhibitors. As a case study, we present the in vitro experiments performed with a set of selected inhibitors of the human mitogen-activated protein kinases 1/2 (MEK1/2), several of which showed a marked cytotoxic activity in different fungal species.