PPI3D: a web server for searching, analyzing and modeling protein-protein, protein-peptide and protein-nucleic acid interactions.

Structure-resolved protein interactions with other proteins, peptides and nucleic acids are key for understanding molecular mechanisms. The PPI3D web server enables researchers to query preprocessed and clustered structural data, analyze the results and make homology-based inferences for protein interactions. PPI3D offers three interaction exploration modes: (i) all interactions for proteins homologous to the query, (ii) interactions between two proteins or their homologs and (iii) interactions within a specific PDB entry. The server allows interactive analysis of the identified interactions in both summarized and detailed manner. This includes protein annotations, structures, the interface residues and the corresponding contact surface areas. In addition, users can make inferences about residues at the interaction interface for the query protein(s) from the sequence alignments and homology models. The weekly updated PPI3D database includes all the interaction interfaces and binding sites from PDB, clustered based on both protein sequence and structural similarity, yielding non-redundant datasets without loss of alternative interaction modes. Consequently, the PPI3D users avoid being flooded with redundant information, a typical situation for intensely studied proteins. Furthermore, PPI3D provides a possibility to download user-defined sets of interaction interfaces and analyze them locally. The PPI3D web server is available at https://bioinformatics.lt/ppi3d.

The missing part: the Archaeoglobus fulgidus Argonaute forms a functional heterodimer with an N-L1-L2 domain protein.

Argonaute (Ago) proteins are present in all three domains of life (bacteria, archaea and eukaryotes). They use small (15-30 nucleotides) oligonucleotide guides to bind complementary nucleic acid targets and are responsible for gene expression regulation, mobile genome element silencing, and defence against viruses or plasmids. According to their domain organization, Agos are divided into long and short Agos. Long Agos found in prokaryotes (long-A and long-B pAgos) and eukaryotes (eAgos) comprise four major functional domains (N, PAZ, MID and PIWI) and two structural linker domains L1 and L2. The majority (∼60%) of pAgos are short pAgos, containing only the MID and inactive PIWI domains. Here we focus on the prokaryotic Argonaute AfAgo from Archaeoglobus fulgidus DSM4304. Although phylogenetically classified as a long-B pAgo, AfAgo contains only MID and catalytically inactive PIWI domains, akin to short pAgos. We show that AfAgo forms a heterodimeric complex with a protein encoded upstream in the same operon, which is a structural equivalent of the N-L1-L2 domains of long pAgos. This complex, structurally equivalent to a long PAZ-less pAgo, outperforms standalone AfAgo in guide RNA-mediated target DNA binding. Our findings provide a missing piece to one of the first and the most studied pAgos.

Ribosomal stalk-captured CARF-RelE ribonuclease inhibits translation following CRISPR signaling.

Prokaryotic type III CRISPR-Cas antiviral systems employ cyclic oligoadenylate (cAn) signaling to activate a diverse range of auxiliary proteins that reinforce the CRISPR-Cas defense. Here we characterize a class of cAn-dependent effector proteins named CRISPR-Cas-associated messenger RNA (mRNA) interferase 1 (Cami1) consisting of a CRISPR-associated Rossmann fold sensor domain fused to winged helix-turn-helix and a RelE-family mRNA interferase domain. Upon activation by cyclic tetra-adenylate (cA4), Cami1 cleaves mRNA exposed at the ribosomal A-site thereby depleting mRNA and leading to cell growth arrest. The structures of apo-Cami1 and the ribosome-bound Cami1-cA4 complex delineate the conformational changes that lead to Cami1 activation and the mechanism of Cami1 binding to a bacterial ribosome, revealing unexpected parallels with eukaryotic ribosome-inactivating proteins.

Prediction of protein assemblies by structure sampling followed by interface-focused scoring.

Proteins often function as part of permanent or transient multimeric complexes, and understanding function of these assemblies requires knowledge of their three-dimensional structures. While the ability of AlphaFold to predict structures of individual proteins with unprecedented accuracy has revolutionized structural biology, modeling structures of protein assemblies remains challenging. To address this challenge, we developed a protocol for predicting structures of protein complexes involving model sampling followed by scoring focused on the subunit-subunit interaction interface. In this protocol, we diversified AlphaFold models by varying construction and pairing of multiple sequence alignments as well as increasing the number of recycles. In cases when AlphaFold failed to assemble a full protein complex or produced unreliable results, additional diverse models were constructed by docking of monomers or subcomplexes. All the models were then scored using a newly developed method, VoroIF-jury, which relies only on structural information. Notably, VoroIF-jury is independent of AlphaFold self-assessment scores and therefore can be used to rank models originating from different structure prediction methods. We tested our protocol in CASP15 and obtained top results, significantly outperforming the standard AlphaFold-Multimer pipeline. Analysis of our results showed that the accuracy of our assembly models was capped mainly by structure sampling rather than model scoring. This observation suggests that better sampling, especially for the antibody-antigen complexes, may lead to further improvement. Our protocol is expected to be useful for modeling and/or scoring protein assemblies.

Investigating the composition and recruitment of the mycobacterial ImuA'-ImuB-DnaE2 mutasome.

A DNA damage-inducible mutagenic gene cassette has been implicated in the emergence of drug resistance in Mycobacterium tuberculosis during anti-tuberculosis (TB) chemotherapy. However, the molecular composition and operation of the encoded 'mycobacterial mutasome' - minimally comprising DnaE2 polymerase and ImuA' and ImuB accessory proteins - remain elusive. Following exposure of mycobacteria to DNA damaging agents, we observe that DnaE2 and ImuB co-localize with the DNA polymerase III ß subunit (ß clamp) in distinct intracellular foci. Notably, genetic inactivation of the mutasome in an imuBAAAAGG mutant containing a disrupted ß clamp-binding motif abolishes ImuB-ß clamp focus formation, a phenotype recapitulated pharmacologically by treating bacilli with griselimycin and in biochemical assays in which this ß clamp-binding antibiotic collapses pre-formed ImuB-ß clamp complexes. These observations establish the essentiality of the ImuB-ß clamp interaction for mutagenic DNA repair in mycobacteria, identifying the mutasome as target for adjunctive therapeutics designed to protect anti-TB drugs against emerging resistance.

VoroIF-GNN: Voronoi tessellation-derived protein-protein interface assessment using a graph neural network.

We present VoroIF-GNN (Voronoi InterFace Graph Neural Network), a novel method for assessing inter-subunit interfaces in a structural model of a protein-protein complex, relying solely on the input structure without any additional information. Given a multimeric protein structural model, we derive interface contacts from the Voronoi tessellation of atomic balls, construct a graph of those contacts, and predict the accuracy of every contact using an attention-based GNN. The contact-level predictions are then summarized to produce whole interface-level scores. VoroIF-GNN was blindly tested for its ability to estimate the accuracy of protein complexes during CASP15 and showed strong performance in selecting the best multimeric model out of many. The method implementation is freely available at https://kliment-olechnovic.github.io/voronota/expansion_js/.

Discriminating physiological from non-physiological interfaces in structures of protein complexes: A community-wide study.

Reliably scoring and ranking candidate models of protein complexes and assigning their oligomeric state from the structure of the crystal lattice represent outstanding challenges. A community-wide effort was launched to tackle these challenges. The latest resources on protein complexes and interfaces were exploited to derive a benchmark dataset consisting of 1677 homodimer protein crystal structures, including a balanced mix of physiological and non-physiological complexes. The non-physiological complexes in the benchmark were selected to bury a similar or larger interface area than their physiological counterparts, making it more difficult for scoring functions to differentiate between them. Next, 252 functions for scoring protein-protein interfaces previously developed by 13 groups were collected and evaluated for their ability to discriminate between physiological and non-physiological complexes. A simple consensus score generated using the best performing score of each of the 13 groups, and a cross-validated Random Forest (RF) classifier were created. Both approaches showed excellent performance, with an area under the Receiver Operating Characteristic (ROC) curve of 0.93 and 0.94, respectively, outperforming individual scores developed by different groups. Additionally, AlphaFold2 engines recalled the physiological dimers with significantly higher accuracy than the non-physiological set, lending support to the reliability of our benchmark dataset annotations. Optimizing the combined power of interface scoring functions and evaluating it on challenging benchmark datasets appears to be a promising strategy.

RecA-NT homology motif in ImuB is essential for mycobacterial ImuA'-ImuB protein interaction and mutasome function.

The mycobacterial mutasome - minimally comprising ImuA', ImuB, and DnaE2 proteins - has been implicated in DNA damage-induced mutagenesis in Mycobacterium tuberculosis. ImuB, predicted to enable mutasome function via its interaction with the ß clamp, is a catalytically inactive member of the Y-family of DNA polymerases. Like other members of the Y family, ImuB features a recently identified amino acid motif with homology to the RecA-N-terminus (RecA-NT). In RecA, the motif mediates oligomerization of RecA monomers into RecA filaments. Given the role of ImuB in the mycobacterial mutasome, we hypothesized that the ImuB RecA-NT motif might mediate its interaction with ImuA', a RecA homolog of unknown function. To investigate this possibility, we constructed a panel of imuB alleles in which RecA-NT was removed, or mutated. Results from microbiological and biochemical assays indicate that RecA-NT is critical for the interaction of ImuB with ImuA'. A region downstream of RecA-NT (ImuB-C) also appears to stabilize the ImuB-ImuA' interaction, but its removal does not prevent complex formation. In contrast, replacing two key hydrophobic residues of RecA-NT, L378 and V383, is sufficient to disrupt ImuA'-ImuB interaction. To our knowledge, this constitutes the first experimental evidence showing the role of the RecA-NT motif in mediating the interaction between a Y-family member and a RecA homolog.

TnpB structure reveals minimal functional core of Cas12 nuclease family.

The widespread TnpB proteins of IS200/IS605 transposon family have recently emerged as the smallest RNA-guided nucleases capable of targeted genome editing in eukaryotic cells1,2. Bioinformatic analysis identified TnpB proteins as the likely predecessors of Cas12 nucleases3-5, which along with Cas9 are widely used for targeted genome manipulation. Whereas Cas12 family nucleases are well characterized both biochemically and structurally6, the molecular mechanism of TnpB remains unknown. Here we present the cryogenic-electron microscopy structures of the Deinococcus radiodurans TnpB-reRNA (right-end transposon element-derived RNA) complex in DNA-bound and -free forms. The structures reveal the basic architecture of TnpB nuclease and the molecular mechanism for DNA target recognition and cleavage that is supported by biochemical experiments. Collectively, these results demonstrate that TnpB represents the minimal structural and functional core of the Cas12 protein family and provide a framework for developing TnpB-based genome editing tools.

Short prokaryotic Argonautes provide defence against incoming mobile genetic elements through NAD+ depletion.

Argonaute (Ago) proteins are found in all three domains of life. The so-called long Agos are composed of four major domains (N, PAZ, MID and PIWI) and contribute to RNA silencing in eukaryotes (eAgos) or defence against invading mobile genetic elements in prokaryotes (pAgos). The majority (~60%) of pAgos identified bioinformatically are shorter (comprising only MID and PIWI domains) and are typically associated with Sir2, Mrr or TIR domain-containing proteins. The cellular function and mechanism of short pAgos remain enigmatic. Here we show that Geobacter sulfurreducens short pAgo and the NAD+-bound Sir2 protein form a stable heterodimeric complex. The GsSir2/Ago complex presumably recognizes invading plasmid or phage DNA and activates the Sir2 subunit, which triggers endogenous NAD+ depletion and cell death, and prevents the propagation of invading DNA. We reconstituted NAD+ depletion activity in vitro and showed that activated GsSir2/Ago complex functions as a NADase that hydrolyses NAD+ to ADPR. Thus, short Sir2-associated pAgos provide defence against phages and plasmids, underscoring the diversity of mechanisms of prokaryotic Agos.

Transposon-associated TnpB is a programmable RNA-guided DNA endonuclease.

Transposition has a key role in reshaping genomes of all living organisms1. Insertion sequences of IS200/IS605 and IS607 families2 are among the simplest mobile genetic elements and contain only the genes that are required for their transposition and its regulation. These elements encode tnpA transposase, which is essential for mobilization, and often carry an accessory tnpB gene, which is dispensable for transposition. Although the role of TnpA in transposon mobilization of IS200/IS605 is well documented, the function of TnpB has remained largely unknown. It had been suggested that TnpB has a role in the regulation of transposition, although no mechanism for this has been established3-5. A bioinformatic analysis indicated that TnpB might be a predecessor of the CRISPR-Cas9/Cas12 nucleases6-8. However, no biochemical activities have been ascribed to TnpB. Here we show that TnpB of Deinococcus radiodurans ISDra2 is an RNA-directed nuclease that is guided by an RNA, derived from the right-end element of a transposon, to cleave DNA next to the 5'-TTGAT transposon-associated motif. We also show that TnpB could be reprogrammed to cleave DNA target sites in human cells. Together, this study expands our understanding of transposition mechanisms by highlighting the role of TnpB in transposition, experimentally confirms that TnpB is a functional progenitor of CRISPR-Cas nucleases and establishes TnpB as a prototype of a new system for genome editing.

Modeling SARS-CoV-2 proteins in the CASP-commons experiment.

Critical Assessment of Structure Prediction (CASP) is an organization aimed at advancing the state of the art in computing protein structure from sequence. In the spring of 2020, CASP launched a community project to compute the structures of the most structurally challenging proteins coded for in the SARS-CoV-2 genome. Forty-seven research groups submitted over 3000 three-dimensional models and 700 sets of accuracy estimates on 10 proteins. The resulting models were released to the public. CASP community members also worked together to provide estimates of local and global accuracy and identify structure-based domain boundaries for some proteins. Subsequently, two of these structures (ORF3a and ORF8) have been solved experimentally, allowing assessment of both model quality and the accuracy estimates. Models from the AlphaFold2 group were found to have good agreement with the experimental structures, with main chain GDT_TS accuracy scores ranging from 63 (a correct topology) to 87 (competitive with experiment).

Modeling of protein complexes in CASP14 with emphasis on the interaction interface prediction.

The goal of CASP experiments is to monitor the progress in the protein structure prediction field. During the 14th CASP edition we aimed to test our capabilities of predicting structures of protein complexes. Our protocol for modeling protein assemblies included both template-based modeling and free docking. Structural templates were identified using sensitive sequence-based searches. If sequence-based searches failed, we performed structure-based template searches using selected CASP server models. In the absence of reliable templates we applied free docking starting from monomers generated by CASP servers. We evaluated and ranked models of protein complexes using an improved version of our protein structure quality assessment method, VoroMQA, taking into account both interaction interface and global structure scores. If reliable templates could be identified, generally accurate models of protein assemblies were generated with the exception of an antibody-antigen interaction. The success of free docking mainly depended on the accuracy of initial subunit models and on the scoring of docking solutions. To put our overall results in perspective, we analyzed our performance in the context of other CASP groups. Although the subunits in our assembly models often were not of the top quality, these models had, overall, the best-predicted intersubunit interfaces according to several accuracy measures. We attribute our relative success primarily to the emphasis on the interaction interface when modeling and scoring.

VoroContacts: a tool for the analysis of interatomic contacts in macromolecular structures.

SUMMARY: VoroContacts is a versatile tool for computing and analyzing contact surface areas (CSAs) and solvent accessible surface areas (SASAs) for three-dimensional (3D) structures of proteins, nucleic acids and their complexes at the atomic resolution. CSAs and SASAs are derived using Voronoi tessellation of 3D structure, represented as a collection of atomic balls. VoroContacts web server features a highly configurable query interface, which enables on-the-fly analysis of contacts for selected set of atoms and allows filtering interatomic contacts by their type, surface areas, distance between contacting atoms and sequence separation between contacting residues. The VoroContacts functionality is also implemented as part of the standalone Voronota package, enabling batch processing. AVAILABILITY AND IMPLEMENTATION: https://bioinformatics.lt/wtsam/vorocontacts. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

VoroCNN: deep convolutional neural network built on 3D Voronoi tessellation of protein structures.

MOTIVATION: Effective use of evolutionary information has recently led to tremendous progress in computational prediction of three-dimensional (3D) structures of proteins and their complexes. Despite the progress, the accuracy of predicted structures tends to vary considerably from case to case. Since the utility of computational models depends on their accuracy, reliable estimates of deviation between predicted and native structures are of utmost importance. RESULTS: For the first time, we present a deep convolutional neural network (CNN) constructed on a Voronoi tessellation of 3D molecular structures. Despite the irregular data domain, our data representation allows us to efficiently introduce both convolution and pooling operations and train the network in an end-to-end fashion without precomputed descriptors. The resultant model, VoroCNN, predicts local qualities of 3D protein folds. The prediction results are competitive to state of the art and superior to the previous 3D CNN architectures built for the same task. We also discuss practical applications of VoroCNN, for example, in recognition of protein binding interfaces. AVAILABILITY AND IMPLEMENTATION: The model, data and evaluation tests are available at https://team.inria.fr/nano-d/software/vorocnn/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

NR5A1 c.991-1G > C splice-site variant causes familial 46,XY partial gonadal dysgenesis with incomplete penetrance.

OBJECTIVE: The study aimed to identify the genetic basis of partial gonadal dysgenesis (PGD) in a non-consanguineous family from Estonia. PATIENTS: Cousins P (proband) 1 (12 years; 46,XY) and P2 (18 years; 46,XY) presented bilateral cryptorchidism, severe penoscrotal hypospadias, low bitesticular volume and azoospermia in P2. Their distant relative, P3 (30 years; 46,XY), presented bilateral cryptorchidism and cryptozoospermia. DESIGN: Exome sequencing was targeted to P1-P3 and five unaffected family members. RESULTS: P1-P2 were identified as heterozygous carriers of NR5A1 c.991-1G > C. NR5A1 encodes the steroidogenic factor-1 essential in gonadal development and specifically expressed in adrenal, spleen, pituitary and testes. Together with a previous PGD case from Belgium (Robevska et al 2018), c.991-1G > C represents the first recurrent NR5A1 splice-site mutation identified in patients. The majority of previous reports on NR5A1 mutation carriers have not included phenotype-genotype data of the family members. Segregation analysis across three generations showed incomplete penetrance (<50%) and phenotypic variability among the carriers of NR5A1 c.991-1G > C. The variant pathogenicity was possibly modulated by rare heterozygous variants inherited from the other parent, OTX2 p.P134R (P1) or PROP1 c.301_302delAG (P2). For P3, the pedigree structure supported a distinct genetic cause. He carries a previously undescribed likely pathogenic variant SOS1 p.Y136H. SOS1, critical in Ras/MAPK signalling and foetal development, is a strong novel candidate gene for cryptorchidism. CONCLUSIONS: Detailed genetic profiling facilitates counselling and clinical management of the probands, and supports unaffected mutation carriers in the family for their reproductive decision making.

HEPN-MNT Toxin-Antitoxin System: The HEPN Ribonuclease Is Neutralized by OligoAMPylation.

Prokaryotic toxin-antitoxin (TA) systems are composed of a toxin capable of interfering with key cellular processes and its neutralizing antidote, the antitoxin. Here, we focus on the HEPN-MNT TA system encoded in the vicinity of a subtype I-D CRISPR-Cas system in the cyanobacterium Aphanizomenon flos-aquae. We show that HEPN acts as a toxic RNase, which cleaves off 4 nt from the 3' end in a subset of tRNAs, thereby interfering with translation. Surprisingly, we find that the MNT (minimal nucleotidyltransferase) antitoxin inhibits HEPN RNase through covalent di-AMPylation (diadenylylation) of a conserved tyrosine residue, Y109, in the active site loop. Furthermore, we present crystallographic snapshots of the di-AMPylation reaction at different stages that explain the mechanism of HEPN RNase inactivation. Finally, we propose that the HEPN-MNT system functions as a cellular ATP sensor that monitors ATP homeostasis and, at low ATP levels, releases active HEPN toxin.

A catalogue of biochemically diverse CRISPR-Cas9 orthologs.

Bacterial Cas9 nucleases from type II CRISPR-Cas antiviral defence systems have been repurposed as genome editing tools. Although these proteins are found in many microbes, only a handful of variants are used for these applications. Here, we use bioinformatic and biochemical analyses to explore this largely uncharacterized diversity. We apply cell-free biochemical screens to assess the protospacer adjacent motif (PAM) and guide RNA (gRNA) requirements of 79 Cas9 proteins, thus identifying at least 7 distinct gRNA classes and 50 different PAM sequence requirements. PAM recognition spans the entire spectrum of T-, A-, C-, and G-rich nucleotides, from single nucleotide recognition to sequence strings longer than 4 nucleotides. Characterization of a subset of Cas9 orthologs using purified components reveals additional biochemical diversity, including both narrow and broad ranges of temperature dependence, staggered-end DNA target cleavage, and a requirement for long stretches of homology between gRNA and DNA target. Our results expand the available toolset of RNA-programmable CRISPR-associated nucleases.

Diversity and evolution of B-family DNA polymerases.

B-family DNA polymerases (PolBs) represent the most common replicases. PolB enzymes that require RNA (or DNA) primed templates for DNA synthesis are found in all domains of life and many DNA viruses. Despite extensive research on PolBs, their origins and evolution remain enigmatic. Massive accumulation of new genomic and metagenomic data from diverse habitats as well as availability of new structural information prompted us to conduct a comprehensive analysis of the PolB sequences, structures, domain organizations, taxonomic distribution and co-occurrence in genomes. Based on phylogenetic analysis, we identified a new, widespread group of bacterial PolBs that are more closely related to the catalytically active N-terminal half of the eukaryotic PolEpsilon (PolEpsilonN) than to Escherichia coli Pol II. In Archaea, we characterized six new groups of PolBs. Two of them show close relationships with eukaryotic PolBs, the first one with PolEpsilonN, and the second one with PolAlpha, PolDelta and PolZeta. In addition, structure comparisons suggested common origin of the catalytically inactive C-terminal half of PolEpsilon (PolEpsilonC) and PolAlpha. Finally, in certain archaeal PolBs we discovered C-terminal Zn-binding domains closely related to those of PolAlpha and PolEpsilonC. Collectively, the obtained results allowed us to propose a scenario for the evolution of eukaryotic PolBs.