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Molecular dynamics simulations have been widely used to study solute permeation across biological membranes. The potential of mean force (PMF) for solute permeation is typically computed using enhanced sampling techniques such as umbrella sampling (US). For bulky drug-like permeants, however, obtaining converged PMFs remains challenging and often requires long simulation times, resulting in an unacceptable computational cost. Here, we augmented US with simulated tempering, introducing Simulated Tempering-enhanced US (STeUS), to improve the convergence of PMF calculations for the permeation of methanol and three common drug molecules. Simulate tempering helps to enhance sampling by varying the temperature of the system along a pre-defined temperature ladder. To obtain sufficient sampling of the umbrella histograms, which were computed only from the ground temperature, we modified the simulation time fraction spent at the ground temperature between 1/Kand 50%, whereKis the number of ST temperature states. We found that STeUS accelerates convergence compared to standard US, and the benefit of STeUS is system-dependent. For bulky molecules, for which standard US poorly converged, the application of ST was highly successful, leading to a more than five-fold accelerated convergence of the PMFs. For the small methanol solute, for which conventional US converges moderately, the application of ST is only beneficial if 50% of the STeUS simulation time is spent at the ground temperature. This study establishes STeUS as an efficient and simple method for PMF calculations, thereby strongly reducing the computational cost of routine high-throughput studies of drug permeability.

Funding: This work was supported by the European Research Council (Consolidator Grant 101002326 to J.K.). Ribosomally synthesized and post-translationally modified peptide natural products have provided many highly unusual scaffolds. This includes the intriguing alkaloids crocagins, which possess a tetracyclic core structure and whose biosynthesis has remained enigmatic. Here we use in vitro experiments to demonstrate that three proteins, CgnB, CgnC and CgnE, are sufficient for the production of the hallmark tetracyclic crocagin core from the precursor peptide CgnA. The crystal structures of the homologues CgnB and CgnE reveal them to be the founding members of a peptide-binding protein family and allow us to rationalize their distinct functions. We further show that the hydrolase CgnD liberates the crocagin core scaffold, which is subsequently N-methylated by CgnL. These insights allow us to propose a biosynthetic scheme for crocagins. Bioinformatic analyses based on these data led to the discovery of related biosynthetic pathways that may provide access to a structurally diverse family of peptide-derived pyrroloindoline alkaloids. Publisher PDF

MotivationCompared to eukaryotes, prokaryote genomes are more diverse through different mechanisms, including a higher mutation rate and horizontal gene transfer. Therefore, using a linear representative reference can cause a reference bias. Graph-based pangenome methods have been developed to tackle this problem. However, comparisons in DNA space is still challenging due to this high diversity. In contrast, amino acids have higher similarity due to evolutionary constraints, resulting in conserved amino acids that, however, may be encoded by several synonymous codons. Coding regions cover the majority of the genome in prokaryotes. Thus, building panproteomes leverages the high sequence similarity while not losing much of the genome in non-coding regions.ResultsWe presentPanPA, a method that takes a set of multiple sequence alignments (MSAs) of proteins or protein clusters, indexes them, and builds a graph for each MSA. In the querying step, it can align DNA or amino acid sequences back to these graphs. We first showcase thatPanPAgenerates correct alignments on a panproteome from 1,350E. coli. To demonstrate that panproteomes allow longer phylogenetic distance comparison, we compare DNA and protein alignments from 1,073S. entericaassemblies againstE. colireference genome, pangenome, and panproteome usingBWA,GraphAligner, andPanPArespectively, wherePanPAwas able to produce around 22% more alignments. We also aligned DNA short-reads WGS sample fromS. entericaagainst theE. colireference withBWAand the panproteome withPanPA, wherePanPAwas able to find alignment for 69% of the reads compared to 5% withBWAAvailabilityPanPAis available athttps://github.com/fawaz-dabbaghieh/PanPAContactfawaz@hhu.de,olga.kalinina@helmholtz-hzi.deSupplementary informationSupplementary data are available atBioinformaticsonline.

Glycans are important polysaccharides on cellular surfaces that are bound to glycoproteins and glycolipids. These are one of the most common post-translational modifications of proteins in eukaryotic cells. They play important roles in protein folding, cell-cell interactions, and other extracellular processes. Changes in glycan structures may influence the course of different diseases, such as infections or cancer. Glycans are commonly represented using the IUPAC-condensed notation. IUPAC-condensed is a textual representation of glycans operating on the same topological level as the Symbol Nomenclature for Glycans (SNFG) that assigns colored, geometrical shapes to the main monomers. These symbols are then connected in tree-like structures, visualizing the glycan structure on a topological level. Yet for a representation on the atomic level, notations such as SMILES should be used. To our knowledge, there is no easy-to-use, general, open-source, and offline tool to convert the IUPAC-condensed notation to SMILES. Here, we present the open-access Python package GlyLES for the generalizable generation of SMILES representations out of IUPAC-condensed representations. GlyLES uses a grammar to read in the monomer tree from the IUPAC-condensed notation. From this tree, the tool can compute the atomic structures of each monomer based on their IUPAC-condensed descriptions. In the last step, it merges all monomers into the atomic structure of a glycan in the SMILES notation. GlyLES is the first package that allows conversion from the IUPAC-condensed notation of glycans to SMILES strings. This may have multiple applications, including straightforward visualization, substructure search, molecular modeling and docking, and a new featurization strategy for machine-learning algorithms. GlyLES is available at https://github.com/kalininalab/GlyLES.

The problem of enteral viral hepatitis is still relevant all over the world. Vietnam is endemic territory of South-East Asia in terms of the prevalence of hepatitis E virus. However, data on the prevalence of HEV infection among the indigenous population of Vietnam are limited, while the data among various minor ethnic groups are not available. Due to the large extent of the country from North to South, the provinces of Vietnam differ in climatic and socio-economic conditions. According to the 2019 population census, there are 54 ethnic groups live in Vietnam with a total population of about 96 million, of which about 63,0% are rural residents. Рopulation of the Northern province Ha Giang is characterized by ethnic diversity and consist of about 22 ethnic groups that have preserved their ethnic identity, which determines the features of their lifestyle and farming. The goal of this study was to conduct a cross-sectional study to assess the prevalence of serological markers of HEV infection among the indigenous population of the Northern province of Vietnam, Ha Giang. Materials and methods . A total of 1127 healthy indigenous residents aged 18 to 83 years (average age 42,8±1.5) who lived in three regions (Yen Minh, Bac Me and Đong Van) of Ha Giang province in 2019 were enrolled in the study. Each region was characterized by the unique ethnic composition of the local population. All blood plasma samples were obtained under the code names according to the Helsinki Declaration. The presence of HEV-specific IgG antibodies (anti-HEV IgG) was determined by the ELISA method using commercial kits "DS-IFA-ANTI-HEV-G" in accordance with the manufacturer's instructions (LLC "NPO "Diagnostic Systems", Nizhny Novgorod, Russia). Results. The occurrence of anti-HEV IgG among the healthy indigenous population in the Northern province Ha Giang was 74,4% (838/1127; 95% CI 71,7-76,8). The highest occurrence of anti-HEV IgG (87,6%) among the observed residents was found in Đong Van region, which was significantly higher compared to those in the Bac Me (χ2=16,37, p=0,000052) and Yen Minh (χ2=214,64, p

Chronic hepatitis B (CHB) is a significant public health problem worldwide. The aim of the present review is to summarize the actual trends in the management of CHB in pregnant women. The prevalence of hepatitis B virus (HBV) infection in pregnant women is usually comparable to that in the general population in the corresponding geographic area. All women have to be screened for hepatitis B surface antigen (HBsAg) during pregnancy. Additional examinations of pregnant women with CHB may include maternal hepatitis B e antigen, HBV viral load, alanine aminotransferase level, and HBsAg level. The management of pregnancy depends on the phase of the HBV infection, which has to be determined before pregnancy. In women of childbearing age with CHB, antiviral therapy can pursue two main goals: Treatment of active CHB, and vertical transmission prevention. During pregnancy, tenofovir is the drug of choice in both cases. A combination of hepatitis B immunoglobulin and vaccine against hepatitis B should be administered within the first 12 h to all infants born to mothers with CHB. In such cases, there are no contraindications to breastfeeding.

Background Structural annotation of genetic variants in the context of intermolecular interactions and protein stability can shed light onto mechanisms of disease-related phenotypes. Three-dimensional structures of related proteins in complexes with other proteins, nucleic acids, or ligands enrich such functional interpretation, since intermolecular interactions are well conserved in evolution. Results We present d-StructMAn, a novel computational method that enables structural annotation of local genetic variants, such as single-nucleotide variants and in-frame indels, and implements it in a highly efficient and user-friendly tool provided as a Docker container. Using d-StructMAn, we annotated several very large sets of human genetic variants, including all variants from ClinVar and all amino acid positions in the human proteome. We were able to provide annotation for more than 46% of positions in the human proteome representing over 60% proteins. Conclusions d-StructMAn is the first of its kind and a highly efficient tool for structural annotation of protein-coding genetic variation in the context of observed and potential intermolecular interactions. d-StructMAn is readily applicable to proteome-scale datasets and can be an instrumental building machine-learning tool for predicting genotype-to-phenotype relationships.

In the last 2 years, observational studies have shown that a low 25-hydroxyvitamin D (25(OH)D) level affected the severity of infection with the novel coronavirus (COVID-19). This study aimed to analyze the potential effect of vitamin D supplementation in reducing SARS-CoV-2 infection morbidity and severity in health care workers. Of 128 health care workers, 91 (consisting of 38 medical doctors (42%), 38 nurses (42%), and 15 medical attendants (16%)) were randomized into two groups receiving vitamin D supplementation. Participants of group I (n = 45) received water-soluble cholecalciferol at a dose of 50,000 IU/week for 2 consecutive weeks, followed by 5000 IU/day for the rest of the study. Participants of group II (n = 46) received water-soluble cholecalciferol at a dose of 2000 IU/day. For both groups, treatment lasted 3 months. Baseline serum 25(OH)D level in health care workers varied from 3.0 to 65.1 ng/mL (median, 17.7 (interquartile range, 12.2; 24.7) ng/mL). Vitamin D deficiency, insufficiency, and normal vitamin D status were diagnosed in 60%, 30%, and 10%, respectively. Only 78 subjects completed the study. Vitamin D supplementation was associated with an increase in serum 25(OH)D level, but only intake of 5000 IU/day was accompanied by normalization of serum 25(OH)D level, which occurred in 53% of cases. Neither vitamin D intake nor vitamin D deficiency/insufficiency were associated with a decrease in SARS-CoV-2 morbidity (odds ratio = 2.27; 95% confidence interval, 0.72 to 7.12). However, subjects receiving high-dose vitamin D had only asymptomatic SARS-CoV-2 in 10 (26%) cases; at the same time, participants who received 2000 IU/day showed twice as many SARS-CoV-2 cases, with mild clinical features in half of them.

Background: The immunological changes associated with COVID-19 are largely unknown. Methods: Patients with COVID-19 showing moderate (n = 18, SpO2 &gt, 93%, respiratory rate &gt, 22 per minute, CRP &gt, 10 mg/L) and severe (n = 23, SpO2 &lt, 30 per minute, PaO2/FiO2 ≤ 300 mmHg, permanent oxygen therapy, qSOFA &gt, 2) infection, and 37 healthy donors (HD) were enrolled. Circulating T- and B-cell subsets were analyzed by flow cytometry. Results: CD4+Th cells were skewed toward Th2-like phenotypes within CD45RA+CD62L− (CM) and CD45RA–CD62L− (EM) cells in patients with severe COVID-19, while CM CCR6+ Th17-like cells were decreased if compared with HD. Within CM Th17-like cells “classical” Th17-like cells were increased and Th17.1-like cells were decreased in severe COVID-19 cases. Circulating CM follicular Th-like (Tfh) cells were decreased in all COVID-19 patients, and Tfh17-like cells represented the most predominant subset in severe COVID-19 cases. Both groups of patients showed increased levels of IgD-CD38++ B cells, while the levels of IgD+CD38− and IgD–CD38− were decreased. The frequency of IgD+CD27+ and IgD–CD27+ B cells was significantly reduced in severe COVID-19 cases. Conclusions: We showed an imbalance within almost all circulating memory Th subsets during acute COVID-19 and showed that altered Tfh polarization led to a dysregulated humoral immune response.

The Q80K polymorphism in the NS3-4A protease of the hepatitis C virus is associated with treatment failure of direct-acting antiviral agents. This polymorphism is highly prevalent in genotype 1a infections and stably transmitted between hosts. Here, we investigated the underlying molecular mechanisms of evolutionarily conserved coevolving amino acids in NS3-Q80K and revealed potential implications of epistatic interactions in immune escape and variants persistence. Using purified protein, we characterized the impact of epistatic amino acid substitutions on the physicochemical properties and peptide cleavage kinetics of the NS3-Q80K protease. We found that Q80K destabilized the protease protein fold (p < 0.0001). Although NS3-Q80K showed reduced peptide substrate turnover (p < 0.0002), replicative fitness in an H77S.3 cell culture model of infection was not significantly inferior to the WT virus. Epistatic substitutions at residues 91 and 174 in NS3-Q80K stabilized the protein fold (p < 0.0001) and leveraged the WT protease stability. However, changes in protease stability inversely correlated with enzymatic activity. In infectious cell culture, these secondary substitutions were not associated with a gain of replicative fitness in NS3-Q80K variants. Using molecular dynamics, we observed that the total number of residue contacts in NS3-Q80K mutants correlated with protein folding stability. Changes in the number of contacts reflected the compensatory effect on protein folding instability by epistatic substitutions. In summary, epistatic substitutions in NS3-Q80K contribute to viral fitness by mechanisms not directly related to RNA replication. By compensating for protein-folding instability, epistatic interactions likely protect NS3-Q80K variants from immune cell recognition.

The article deals with results of research on transmission of religious tradition in local community after the Second World War. The research is based on field materials collected by the author in ethnographic expeditions of 2007-2017 and archive materials. It examines the ways of transmission of religious norms and practices on the example of the population of the Pechersky District of the Pskovskaya Oblast situated outside the sphere of state atheistic ideology until 1940. First it's interpersonal communication within family (seniors-juniors) and in church (priest-parishioners). Second it's implicit influence of environment - acting churches of the Pskovo-Pechersky Monastery and sustainable reproduction of religious tradition by local population. The Soviet educational system implied predominance of social education over domestic one, but it couldn't completely exclude children from the local ecclesiastical culture; they continued to participate in many religious practices even in conditions of absence of systematic religious education. The educational resources of the Pskovo-Pechersky Monastery started to develop immediately after the democratization of society in the 1980s. In the 20th - early 21st centuries monastery functioned as important actor of religion's integration in local educational process both in form of optional catechism lessons and broader context of spiritual-ethical education.

Tandem alternative splice sites (TASS) is a special class of alternative splicing events that are characterized by a close tandem arrangement of splice sites. Most TASS lack functional characterization and are believed to arise from splicing noise. Based on the RNA-seq data from the Genotype Tissue Expression project, we present an extended catalogue of TASS in healthy human tissues and analyze their tissue-specific expression. The expression of TASS is usually dominated by one major splice site (maSS), while the expression of minor splice sites (miSS) is at least an order of magnitude lower. Among 46k miSS with sufficient read support, 9k (20%) are significantly expressed above the expected noise level, and among them 2.5k are expressed tissue-specifically. We found significant correlations between tissue-specific expression of RNA-binding proteins (RBP), tissue-specific expression of miSS, and miSS response to RBP inactivation by shRNA. In combination with RBP profiling by eCLIP, this allowed prediction of novel cases of tissue-specific splicing regulation including a miSS in QKI mRNA that is likely regulated by PTBP1. The analysis of human primary cell transcriptomes suggested that both tissue-specific and cell-type-specific factors contribute to the regulation of miSS expression. More than 20% of tissue-specific miSS affect structured protein regions and may adjust protein-protein interactions or modify the stability of the protein core. The significantly expressed miSS evolve under the same selection pressure as maSS, while other miSS lack signatures of evolutionary selection and conservation. Using mixture models, we estimated that not more than 15% of maSS and not more than 54% of tissue-specific miSS are noisy, while the proportion of noisy splice sites among non-significantly expressed miSS is above 63%., Author summary Pre-mRNA splicing is an important step in the processing of the genomic information during gene expression. During splicing, introns are excised from a gene transcript, and the remaining exons are ligated. Our work concerns one its particular subtype, which involves the so-called tandem alternative splice sites, a group of closely located exon borders that are used alternatively. We analyzed RNA-seq measurements of gene expression provided by the Genotype-Tissue Expression (GTEx) project, the largest to-date collection of such measurements in healthy human tissues, and constructed a detailed catalogue of tandem alternative splice sites. Within this catalogue, we characterized patterns of tissue-specific expression, regulation, impact on protein structure, and evolutionary selection acting on tandem alternative splice sites. In a number of genes, we predicted regulatory mechanisms that could be responsible for choosing one of many tandem alternative splice sites. The results of this study provide an invaluable resource for molecular biologists studying alternative splicing.

Technological advances of next-generation sequencing present new computational challenges to develop methods to store and query these data in time- and memory-efficient ways. We present MetaProFi (https://github.com/kalininalab/metaprofi), a Bloom filter-based tool that, in addition to supporting nucleotide sequences, can for the first time directly store and query amino acid sequences and translated nucleotide sequences, thus bringing sequence comparison to a more biologically relevant protein level. Owing to the properties of Bloom filters, it has a zero false-negative rate, allows for exact and inexact searches, and leverages disk storage and Zstandard compression to achieve high time and space efficiency. We demonstrate the utility of MetaProFi by indexing UniProtKB datasets at organism- and at sequence-level in addition to the indexing of Tara Oceans dataset and the 2585 human RNA-seq experiments, showing that MetaProFi consumes far less disk space than state-of-the-art-tools while also improving performance.

Plastic debris is gradually filling the seas, oceans, and freshwater bodies of the planet. Since the 1950s, a huge amount of plastic has entered into various bodies of water. All these objects decompose at different rates, and aquatic organisms take part in these processes. This book chapter provides an overview of studies carried out in recent decades on the interaction between microorganisms and plastic debris in the aquatic environment. Both prokaryotic communities and algo-bacterial cenoses are considered. A separate section is devoted to the research results of the authors of this book chapter, obtained in the natural environment, contaminated with plastic, and in field experiments in the sea.

Bottromycins are ribosomally synthesized and post-translationally modified peptide natural product antibiotics that are effective against high-priority human pathogens such as methicillin-resistant

Bottromycins are ribosomally synthesized and post-translationally modified peptide natural product antibiotics that are effective against high-priority human pathogens such as methicillin-resistant Staphylococcus aureus. The total synthesis of bottromycins involves at least 17 steps, with a poor overall yield. Here, we report the characterization of the cytochrome P450 enzyme BotCYP from a bottromycin biosynthetic gene cluster. We determined the structure of a close BotCYP homolog and used our data to conduct the first large-scale survey of P450 enzymes associated with RiPP biosynthetic gene clusters. We demonstrate that BotCYP converts a C-terminal thiazoline to a thiazole via an oxidative decarboxylation reaction and provides stereochemical resolution for the pathway. Our data enable the two-pot in vitro production of the bottromycin core scaffold and may allow the rapid generation of bottromycin analogues for compound development.

Recent studies identified signal peptidase complex subunit 1 (SPCS1) as a proviral host factor for Flaviviridae viruses, including HCV. One of the SPCS1’s roles in flavivirus propagation was attributed to its regulation of signal peptidase complex (SPC)-mediated processing of flavivirus polyprotein, especially C-prM junction. However, whether SPCS1 also regulates any SPC-mediated processing sites within HCV polyprotein remains unclear. In this study, we determined that loss of SPCS1 specifically impairs the HCV E2-p7 processing by the SPC. We also determined that efficient separation of E2 and p7, regardless of its dependence on SPC-mediated processing, leads to SPCS1 dispensable for HCV assembly These results suggest that SPCS1 regulates HCV assembly by facilitating the SPC-mediated processing of E2-p7 precursor. Structural modeling suggests that intrinsically delayed processing of the E2-p7 is likely caused by the structural rigidity of p7 N-terminal transmembrane helix-1 (p7/TM1/helix-1), which has mostly maintained membrane-embedded conformations during molecular dynamics (MD) simulations. E2-p7-processing-impairing p7 mutations narrowed the p7/TM1/helix-1 bending angle against the membrane, resulting in closer membrane embedment of the p7/TM1/helix-1 and less access of E2-p7 junction substrate to the catalytic site of the SPC, located well above the membrane in the ER lumen. Based on these results we propose that the key mechanism of action of SPCS1 in HCV assembly is to facilitate the E2-p7 processing by enhancing the E2-p7 junction site presentation to the SPC active site. By providing evidence that SPCS1 facilitates HCV assembly by regulating SPC-mediated cleavage of E2-p7 junction, equivalent to the previously established role of this protein in C-prM junction processing in flavivirus, this study establishes the common role of SPCS1 in Flaviviridae family virus propagation as to exquisitely regulate the SPC-mediated processing of specific, suboptimal target sites.

Alternative splicing plays a major role in regulating the functional repertoire of the proteome. However, isoform-specific effects to protein-protein interactions (PPIs) are usually overlooked, making it impossible to judge the functional role of individual exons on a systems biology level. We overcome this barrier by integrating protein-protein interactions, domain-domain interactions and residue-level interactions information to lift exon expression analysis to a network level. Our user-friendly database DIGGER is available at https://exbio.wzw.tum.de/digger and allows users to seamlessly switch between isoform and exon-centric views of the interactome and to extract sub-networks of relevant isoforms, making it an essential resource for studying mechanistic consequences of alternative splicing.

The pipeline of antibiotics has been for decades on an alarmingly low level. Considering the steadily emerging antibiotic resistance, novel tools are needed for early and easy identification of effective anti-infective compounds. In Gram-negative bacteria, the uptake of anti-infectives is especially limited. We here present a surprisingly simple in vitro model of the Gram-negative bacterial envelope, based on 20% (w/v) potato starch gel, printed on polycarbonate 96-well filter membranes. Rapid permeability measurements across this polysaccharide hydrogel allowed to correctly predict either high or low accumulation for all 16 tested anti-infectives in living Escherichia coli. Freeze-fracture TEM supports that the macromolecular network structure of the starch hydrogel may represent a useful surrogate of the Gram-negative bacterial envelope. A random forest analysis of in vitro data revealed molecular mass, minimum projection area, and rigidity as the most critical physicochemical parameters for hydrogel permeability, in agreement with reported structural features needed for uptake into Gram-negative bacteria. Correlating our dataset of 27 antibiotics from different structural classes to reported MIC values of nine clinically relevant pathogens allowed to distinguish active from nonactive compounds based on their low in vitro permeability specifically for Gram-negatives. The model may help to identify poorly permeable antimicrobial candidates before testing them on living bacteria., Graphical abstract Image 1

Identification of biologically relevant motifs in proteins is a long-standing problem in bioinformatics, especially when considering distantly related proteins where sequence analysis alone becomes increasingly difficult. Here we present a novel approach to identify such motifs in protein three-dimensional structures without depending on sequence alignment by representing structures as graphs in the form of residue interaction networks and employing a modified frequent subgraph mining algorithm. These networks represent residues as vertices while contacts between residues are denoted by edges labeled with Euclidean distances. We use frequent subgraph mining to determine all subgraphs that are subgraph isomorphic to, i.e. are contained in, at least a given number of such networks generated from structures in the same protein family. For this we introduce two extensions of the classical frequent subgraph mining: approximate matching of distance-based labels to account for small variations between protein structures and scoring as well as score-based filtering of subgraphs in order to identify structurally conserved motifs and to counteract the expanding size of the search space. This approach was then validated by demonstrating that it can rediscover previously characterized functionally important structural motifs in selected protein families. For further validation we show that it is also able to identify motifs that correspond to patterns in the PROSITE database. We then applied our approach to all superfamilies in the SCOP database and found an enrichment of residues in the ligand binding site in the discovered motifs evidencing their functional importance. Finally we use the approach to discover a novel structural motif in jelly-roll capsid proteins found in members of the picornavirus-like superfamily. This is presented together with an efficient open source implementation of the algorithm called RINminer.Author summaryAs the evolutionary distance between proteins increases, their sequence identity drops rapidly, whereas functionally important sequence motifs and three-dimensional (3D) structural scaffold, in which they are embedded, are more conserved. We developed an approach that automatically identifies such motifs by converting protein 3D structures into a set of graphs and then employing the frequent subgraph mining framework. In these graphs, residues are represented as vertices, and if two residues interact in the corresponding protein 3D structure, they are connected by an edge labeled with the Euclidean distance between the residues. In the classical setting of frequent subgraph mining, all subgraphs from a database of graphs are enumerated and the ones that are exactly found, i.e. are subgraph isomorphic, in more than a certain number of graphs are listed as supported. Our approach introduces two new concepts: approximately isomorphic subgraphs and an efficient scoring scheme that allows to retain only biologically relevant subgraph in the enumeration step. Approximate isomorphism allows edge labels not to match exactly, and thus account for natural deviations between 3D structures of related proteins. With our approach, we were able to automatically rediscover known motifs from PROSITE, as well as in three well-studied extremely diverse protein families. We predicted functionally important residues in SCOP superfamilies and demonstrated that they tend to lie in structurally meaningful regions: ligand-binding sites and protein core. Additionally, we present a previously unreported structural motif in jelly-roll viral capsids.

Tyrosine phosphorylation, a highly regulated post-translational modification, is carried out by the enzyme tyrosine kinase (TK). TKs are important mediators in signaling cascades, facilitating diverse biological processes in response to stimuli. TKs may acquire mutations leading to malignancy and are viable targets for anti-cancer drugs. Mast/stem cell growth factor receptor KIT is a TK involved in cell differentiation, whose dysregulation leads to various types of cancer, including gastrointestinal stromal tumors, leukemia, and melanoma. KIT can be targeted by a range of inhibitors that predominantly bind to the inactive state of the enzyme. A mutation Y823D in the activation loop of KIT is known to be responsible for the loss of sensitivity to some drugs in metastatic tumors. We used all-atom molecular dynamics simulations to study the impact of Y823D on the KIT conformation and dynamics and compared it to the effect of phosphorylation of Y823. We simulated in total 6.4 μs of wild-type, mutant and phosphorylated KIT in the active- and inactive-state conformations. We found that Y823D affects the protein dynamics differently: in the active state, the mutation increases the protein stability, whereas in the inactive state it induces local destabilization, thus shifting the dynamic equilibrium towards the active state, altering the communication between distant regulatory regions. The observed dynamics of the Y823D mutant is similar to the dynamics of KIT phosphorylated at position Y823, thus we hypothesize that this mutation mimics a constitutively active kinase, which is not responsive to inhibitors that bind its inactive conformation.