De novo mutation hotspots in homologous protein domains identify function-altering mutations in neurodevelopmental disorders.

Variant interpretation remains a major challenge in medical genetics. We developed Meta-Domain HotSpot (MDHS) to identify mutational hotspots across homologous protein domains. We applied MDHS to a dataset of 45,221 de novo mutations (DNMs) from 31,058 individuals with neurodevelopmental disorders (NDDs) and identified three significantly enriched missense DNM hotspots in the ion transport protein domain family (PF00520). The 37 unique missense DNMs that drive enrichment affect 25 genes, 19 of which were previously associated with NDDs. 3D protein structure modeling supports the hypothesis of function-altering effects of these mutations. Hotspot genes have a unique expression pattern in tissue, and we used this pattern alongside in silico predictors and population constraint information to identify candidate NDD-associated genes. We also propose a lenient version of our method, which identifies 32 hotspot positions across 16 different protein domains. These positions are enriched for likely pathogenic variation in clinical databases and DNMs in other genetic disorders.

PredictProtein - Predicting Protein Structure and Function for 29 Years.

Since 1992 PredictProtein (https://predictprotein.org) is a one-stop online resource for protein sequence analysis with its main site hosted at the Luxembourg Centre for Systems Biomedicine (LCSB) and queried monthly by over 3,000 users in 2020. PredictProtein was the first Internet server for protein predictions. It pioneered combining evolutionary information and machine learning. Given a protein sequence as input, the server outputs multiple sequence alignments, predictions of protein structure in 1D and 2D (secondary structure, solvent accessibility, transmembrane segments, disordered regions, protein flexibility, and disulfide bridges) and predictions of protein function (functional effects of sequence variation or point mutations, Gene Ontology (GO) terms, subcellular localization, and protein-, RNA-, and DNA binding). PredictProtein's infrastructure has moved to the LCSB increasing throughput; the use of MMseqs2 sequence search reduced runtime five-fold (apparently without lowering performance of prediction methods); user interface elements improved usability, and new prediction methods were added. PredictProtein recently included predictions from deep learning embeddings (GO and secondary structure) and a method for the prediction of proteins and residues binding DNA, RNA, or other proteins. PredictProtein.org aspires to provide reliable predictions to computational and experimental biologists alike. All scripts and methods are freely available for offline execution in high-throughput settings.

MetaDome: Pathogenicity analysis of genetic variants through aggregation of homologous human protein domains.

The growing availability of human genetic variation has given rise to novel methods of measuring genetic tolerance that better interpret variants of unknown significance. We recently developed a concept based on protein domain homology in the human genome to improve variant interpretation. For this purpose, we mapped population variation from the Exome Aggregation Consortium (ExAC) and pathogenic mutations from the Human Gene Mutation Database (HGMD) onto Pfam protein domains. The aggregation of these variation data across homologous domains into meta-domains allowed us to generate amino acid resolution of genetic intolerance profiles for human protein domains. Here, we developed MetaDome, a fast and easy-to-use web server that visualizes meta-domain information and gene-wide profiles of genetic tolerance. We updated the underlying data of MetaDome to contain information from 56,319 human transcripts, 71,419 protein domains, 12,164,292 genetic variants from gnomAD, and 34,076 pathogenic mutations from ClinVar. MetaDome allows researchers to easily investigate their variants of interest for the presence or absence of variation at corresponding positions within homologous domains. We illustrate the added value of MetaDome by an example that highlights how it may help in the interpretation of variants of unknown significance. The MetaDome web server is freely accessible at https://stuart.radboudumc.nl/metadome.

Spatial Clustering of de Novo Missense Mutations Identifies Candidate Neurodevelopmental Disorder-Associated Genes.

Haploinsufficiency (HI) is the best characterized mechanism through which dominant mutations exert their effect and cause disease. Non-haploinsufficiency (NHI) mechanisms, such as gain-of-function and dominant-negative mechanisms, are often characterized by the spatial clustering of mutations, thereby affecting only particular regions or base pairs of a gene. Variants leading to haploinsufficency might occasionally cluster as well, for example in critical domains, but such clustering is on the whole less pronounced with mutations often spread throughout the gene. Here we exploit this property and develop a method to specifically identify genes with significant spatial clustering patterns of de novo mutations in large cohorts. We apply our method to a dataset of 4,061 de novo missense mutations from published exome studies of trios with intellectual disability and developmental disorders (ID/DD) and successfully identify 15 genes with clustering mutations, including 12 genes for which mutations are known to cause neurodevelopmental disorders. For 11 out of these 12, NHI mutation mechanisms have been reported. Additionally, we identify three candidate ID/DD-associated genes of which two have an established role in neuronal processes. We further observe a higher intolerance to normal genetic variation of the identified genes compared to known genes for which mutations lead to HI. Finally, 3D modeling of these mutations on their protein structures shows that 81% of the observed mutations are unlikely to affect the overall structural integrity and that they therefore most likely act through a mechanism other than HI.

Identification of a de novo variant in CHUK in a patient with an EEC/AEC syndrome-like phenotype and hypogammaglobulinemia.

The cardinal features of Ectrodactyly, Ectodermal dysplasia, Cleft lip/palate (EEC), and Ankyloblepharon-Ectodermal defects-Cleft lip/palate (AEC) syndromes are ectodermal dysplasia (ED), orofacial clefting, and limb anomalies. EEC and AEC are caused by heterozygous mutations in the transcription factor p63 encoded by TP63. Here, we report a patient with an EEC/AEC syndrome-like phenotype, including ankyloblepharon, ED, cleft palate, ectrodactyly, syndactyly, additional hypogammaglobulinemia, and growth delay. Neither pathogenic mutations in TP63 nor CNVs at the TP63 locus were identified. Exome sequencing revealed de novo heterozygous variants in CHUK (conserved helix-loop-helix ubiquitous kinase), PTGER4, and IFIT2. While the variant in PTGER4 might contribute to the immunodeficiency and growth delay, the variant in CHUK appeared to be most relevant for the EEC/AEC-like phenotype. CHUK is a direct target gene of p63 and encodes a component of the IKK complex that plays a key role in NF-κB pathway activation. The identified CHUK variant (g.101980394T>C; c.425A>G; p.His142Arg) is located in the kinase domain which is responsible for the phosphorylation activity of the protein. The variant may affect CHUK function and thus contribute to the disease phenotype in three ways: (1) the variant exhibits a dominant negative effect and results in an inactive IKK complex that affects the canonical NF-κB pathway; (2) it affects the feedback loop of the canonical and non-canonical NF-κB pathways that are CHUK kinase activity-dependent; and (3) it disrupts NF-κB independent epidermal development that is often p63-dependent. Therefore, we propose that the heterozygous CHUK variant is highly likely to be causative to the EEC/AEC-like and additional hypogammaglobulinemia phenotypes in the patient presented here.

3D-e-Chem-VM: Structural Cheminformatics Research Infrastructure in a Freely Available Virtual Machine.

3D-e-Chem-VM is an open source, freely available Virtual Machine ( http://3d-e-chem.github.io/3D-e-Chem-VM/ ) that integrates cheminformatics and bioinformatics tools for the analysis of protein-ligand interaction data. 3D-e-Chem-VM consists of software libraries, and database and workflow tools that can analyze and combine small molecule and protein structural information in a graphical programming environment. New chemical and biological data analytics tools and workflows have been developed for the efficient exploitation of structural and pharmacological protein-ligand interaction data from proteomewide databases (e.g., ChEMBLdb and PDB), as well as customized information systems focused on, e.g., G protein-coupled receptors (GPCRdb) and protein kinases (KLIFS). The integrated structural cheminformatics research infrastructure compiled in the 3D-e-Chem-VM enables the design of new approaches in virtual ligand screening (Chemdb4VS), ligand-based metabolism prediction (SyGMa), and structure-based protein binding site comparison and bioisosteric replacement for ligand design (KRIPOdb).

SMAD2 Mutations Are Associated with Arterial Aneurysms and Dissections.

We report three families with arterial aneurysms and dissections in which variants predicted to be pathogenic were identified in SMAD2. Moreover, one variant occurred de novo in a proband with unaffected parents. SMAD2 is a strong candidate gene for arterial aneurysms and dissections given its role in the TGF-ß signaling pathway. Furthermore, although SMAD2 and SMAD3 probably have functionally distinct roles in cell signaling, they are structurally very similar. Our findings indicate that SMAD2 mutations are associated with arterial aneurysms and dissections and are in accordance with the observation that patients with pathogenic variants in genes encoding proteins involved in the TGF-ß signaling pathway exhibit arterial aneurysms and dissections as key features.

Creating a specialist protein resource network: a meeting report for the protein bioinformatics and community resources retreat.

During 11-12 August 2014, a Protein Bioinformatics and Community Resources Retreat was held at the Wellcome Trust Genome Campus in Hinxton, UK. This meeting brought together the principal investigators of several specialized protein resources (such as CAZy, TCDB and MEROPS) as well as those from protein databases from the large Bioinformatics centres (including UniProt and RefSeq). The retreat was divided into five sessions: (1) key challenges, (2) the databases represented, (3) best practices for maintenance and curation, (4) information flow to and from large data centers and (5) communication and funding. An important outcome of this meeting was the creation of a Specialist Protein Resource Network that we believe will improve coordination of the activities of its member resources. We invite further protein database resources to join the network and continue the dialogue.

Generic GPCR residue numbers - aligning topology maps while minding the gaps.

Generic residue numbers facilitate comparisons of, for example, mutational effects, ligand interactions, and structural motifs. The numbering scheme by Ballesteros and Weinstein for residues within the class A GPCRs (G protein-coupled receptors) has more than 1100 citations, and the recent crystal structures for classes B, C, and F now call for a community consensus in residue numbering within and across these classes. Furthermore, the structural era has uncovered helix bulges and constrictions that offset the generic residue numbers. The use of generic residue numbers depends on convenient access by pharmacologists, chemists, and structural biologists. We review the generic residue numbering schemes for each GPCR class, as well as a complementary structure-based scheme, and provide illustrative examples and GPCR database (GPCRDB) web tools to number any receptor sequence or structure.

PredictProtein--an open resource for online prediction of protein structural and functional features.

PredictProtein is a meta-service for sequence analysis that has been predicting structural and functional features of proteins since 1992. Queried with a protein sequence it returns: multiple sequence alignments, predicted aspects of structure (secondary structure, solvent accessibility, transmembrane helices (TMSEG) and strands, coiled-coil regions, disulfide bonds and disordered regions) and function. The service incorporates analysis methods for the identification of functional regions (ConSurf), homology-based inference of Gene Ontology terms (metastudent), comprehensive subcellular localization prediction (LocTree3), protein-protein binding sites (ISIS2), protein-polynucleotide binding sites (SomeNA) and predictions of the effect of point mutations (non-synonymous SNPs) on protein function (SNAP2). Our goal has always been to develop a system optimized to meet the demands of experimentalists not highly experienced in bioinformatics. To this end, the PredictProtein results are presented as both text and a series of intuitive, interactive and visually appealing figures. The web server and sources are available at http://ppopen.rostlab.org.

GPCR-OKB: the G Protein Coupled Receptor Oligomer Knowledge Base.

SUMMARY: Rapid expansion of available data about G Protein Coupled Receptor (GPCR) dimers/oligomers over the past few years requires an effective system to organize this information electronically. Based on an ontology derived from a community dialog involving colleagues using experimental and computational methodologies, we developed the GPCR-Oligomerization Knowledge Base (GPCR-OKB). GPCR-OKB is a system that supports browsing and searching for GPCR oligomer data. Such data were manually derived from the literature. While focused on GPCR oligomers, GPCR-OKB is seamlessly connected to GPCRDB, facilitating the correlation of information about GPCR protomers and oligomers. AVAILABILITY AND IMPLEMENTATION: The GPCR-OKB web application is freely available at http://www.gpcr-okb.org

Requirements and ontology for a G protein-coupled receptor oligomerization knowledge base.

BACKGROUND: G Protein-Coupled Receptors (GPCRs) are a large and diverse family of membrane proteins whose members participate in the regulation of most cellular and physiological processes and therefore represent key pharmacological targets. Although several bioinformatics resources support research on GPCRs, most of these have been designed based on the traditional assumption that monomeric GPCRs constitute the functional receptor unit. The increase in the frequency and number of reports about GPCR dimerization/oligomerization and the implication of oligomerization in receptor function makes necessary the ability to store and access information about GPCR dimers/oligomers electronically. RESULTS: We present here the requirements and ontology (the information scheme to describe oligomers and associated concepts and their relationships) for an information system that can manage the elements of information needed to describe comprehensively the phenomena of both homo- and hetero-oligomerization of GPCRs. The comprehensive information management scheme that we plan to use for the development of an intuitive and user-friendly GPCR-Oligomerization Knowledge Base (GPCR-OKB) is the result of a community dialog involving experimental and computational colleagues working on GPCRs. CONCLUSION: Our long term goal is to disseminate to the scientific community organized, curated, and detailed information about GPCR dimerization/oligomerization and its related structural context. This information will be reported as close to the data as possible so the user can make his own judgment on the conclusions drawn for a particular study. The requirements and ontology described here will facilitate the development of future information systems for GPCR oligomers that contain both computational and experimental information about GPCR oligomerization. This information is freely accessible at http://www.gpcr-okb.org.

The use of in vitro peptide binding profiles and in silico ligand-receptor interaction profiles to describe ligand-induced conformations of the retinoid X receptor alpha ligand-binding domain.

It is hypothesized that different ligand-induced conformational changes can explain the different interactions of nuclear receptors with regulatory proteins, resulting in specific biological activities. Understanding the mechanism of how ligands regulate cofactor interaction facilitates drug design. To investigate these ligand-induced conformational changes at the surface of proteins, we performed a time-resolved fluorescence resonance energy transfer assay with 52 different cofactor peptides measuring the ligand-induced cofactor recruitment to the retinoid X receptor-alpha (RXRalpha) in the presence of 11 compounds. Simultaneously we analyzed the binding modes of these compounds by molecular docking. An automated method converted the complex three-dimensional data of ligand-protein interactions into two-dimensional fingerprints, the so-called ligand-receptor interaction profiles. For a subset of compounds the conformational changes at the surface, as measured by peptide recruitment, correlate well with the calculated binding modes, suggesting that clustering of ligand-receptor interaction profiles is a very useful tool to discriminate compounds that may induce different conformations and possibly different effects in a cellular environment. In addition, we successfully combined ligand-receptor interaction profiles and peptide recruitment data to reveal structural elements that are possibly involved in the ligand-induced conformations. Interestingly, we could predict a possible binding mode of LG100754, a homodimer antagonist that showed no effect on peptide recruitment. Finally, the extensive analysis of the peptide recruitment profiles provided novel insight in the potential cellular effect of the compound; for the first time, we showed that in addition to the induction of coactivator peptide binding, all well-known RXRalpha agonists also induce binding of corepressor peptides to RXRalpha.

The nuclear receptor ligand-binding domain: a family-based structure analysis.

Nuclear receptors (NRs) are ligand-dependent transcription factors that play a central role in various physiological processes. The pharmaceutical industry has great interest in this gene-family for the discovery of novel or improved drugs for treatment of, for example, cancer, infertility, or diabetes. The usage of three-dimensional coordinates of protein structures to analyse and predict interactions with ligands is an important aspect of this process. All NR ligand-binding domains have a similar fold, which allows for comparison of the structures of their three main functional sites: the ligand-binding pocket, the cofactor-binding groove, and the dimerization interface. We performed an analysis of nearly one hundred NR ligand-binding domain structures, and identified the functionally important residues. The combined knowledge about the shape of the binding sites and the residues involved in the binding is important for drug design in two ways. First, knowledge about the location of residues that interact with a ligand in all crystal structures or in certain subfamilies assists in the design and docking of drugs. Second, similarities and differences in the residue types of the most frequent ligand- and cofactor-binding residues provide insight about potential cross-reactivity of ligands or cofactors.

A family-based approach reveals the function of residues in the nuclear receptor ligand-binding domain.

Literature studies, 3D structure data, and a series of sequence analysis techniques were combined to reveal important residues in the structure and function of the ligand-binding domain of nuclear hormone receptors. A structure-based multiple sequence alignment allowed for the seamless combination of data from many different studies on different receptors into one single functional model. It was recently shown that a combined analysis of sequence entropy and variability can divide residues in five classes; (1) the main function or active site, (2) support for the main function, (3) signal transduction, (4) modulator or ligand binding and (5) the rest. Mutation data extracted from the literature and intermolecular contacts observed in nuclear receptor structures were analyzed in view of this classification and showed that the main function or active site residues of the nuclear receptor ligand-binding domain are involved in cofactor recruitment. Furthermore, the sequence entropy-variability analysis identified the presence of signal transduction residues that are located between the ligand, cofactor and dimer sites, suggesting communication between these regulatory binding sites. Experimental and computational results agreed well for most residues for which mutation data and intermolecular contact data were available. This allows us to predict the role of the residues for which no functional data is available yet. This study illustrates the power of family-based approaches towards the analysis of protein function, and it points out the problems and possibilities presented by the massive amounts of data that are becoming available in the "omics era". The results shed light on the nuclear receptor family that is involved in processes ranging from cancer to infertility, and that is one of the more important targets in the pharmaceutical industry.

Identification of functionally conserved residues with the use of entropy-variability plots.

We introduce sequence entropy-variability plots as a method of analyzing families of protein sequences, and demonstrate this for three well-known sequence families: globins, ras-like proteins, and serine-proteases. The location of an aligned residue position in the entropy-variability plot correlates with structural characteristics, and with known facts about the roles of individual amino acids in the function of these proteins. The large numbers of known sequences in these families allowed us to introduce new filtering methods for variability patterns. The results are discussed in terms of a simple evolutionary model for functional proteins.

Sequence analysis reveals how G protein-coupled receptors transduce the signal to the G protein.

Sequence entropy-variability plots based on alignments of very large numbers of sequences-can indicate the location in proteins of the main active site and modulator sites. In the previous article in this issue, we applied this observation to a series of well-studied proteins and concluded that it was possible to detect most of the residues with a known functional role. Here, we apply the method to rhodopsin-like G protein-coupled receptors. Our conclusion is that G protein binding is the main evolutionary constraint on these receptors, and that other ligands, such as agonists, act as modulators. The activation of the receptors can be described as a simple, two-step process, and the residues involved in signal transduction can be identified.

GPCRDB information system for G protein-coupled receptors.

The GPCRDB is a molecular class-specific information system that collects, combines, validates and disseminates heterogeneous data on G protein-coupled receptors (GPCRs). The database stores data on sequences, ligand binding constants and mutations. The system also provides computationally derived data such as sequence alignments, homology models, and a series of query and visualization tools. The GPCRDB is updated automatically once every 4-5 months and is freely accessible at http://www.gpcr.org/7tm/.