Knowledge of protein-ligand binding sites (LBSs) enables research ranging from protein function annotation to structure-based drug design. To this end, we have previously developed a stand-alone tool, P2Rank, and the web server PrankWeb (https://prankweb.cz/) for fast and accurate LBS prediction. Here, we present significant enhancements to PrankWeb. First, a new, more accurate evolutionary conservation estimation pipeline based on the UniRef50 sequence database and the HMMER3 package is introduced. Second, PrankWeb now allows users to enter UniProt ID to carry out LBS predictions in situations where no experimental structure is available by utilizing the AlphaFold model database. Additionally, a range of minor improvements has been implemented. These include the ability to deploy PrankWeb and P2Rank as Docker containers, support for the mmCIF file format, improved public REST API access, or the ability to batch download the LBS predictions for the whole PDB archive and parts of the AlphaFold database.
- MeSH
- databáze proteinů MeSH
- internet MeSH
- ligandy MeSH
- proteinové domény MeSH
- proteiny * chemie MeSH
- software * MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- ligandy MeSH
- proteiny * MeSH
Our study presents a novel germline c.1715G>T (p.G572V) mutation in the gene encoding Toll-like receptor 8 (TLR8) causing an autoimmune and autoinflammatory disorder in a family with monozygotic male twins, who suffer from severe autoimmune hemolytic anemia worsening with infections, and autoinflammation presenting as fevers, enteritis, arthritis, and CNS vasculitis. The pathogenicity of the mutation was confirmed by in vitro assays on transfected cell lines and primary cells. The p.G572V mutation causes impaired stability of the TLR8 protein, cross-reactivity to TLR7 ligands and reduced ability of TLR8 to attenuate TLR7 signaling. This imbalance toward TLR7-dependent signaling leads to increased pro-inflammatory responses, such as nuclear factor-κB (NF-κB) activation and production of pro-inflammatory cytokines IL-1β, IL-6, and TNFα. This unique TLR8 mutation with partial TLR8 protein loss and hyperinflammatory phenotype mediated by TLR7 ligands represents a novel inborn error of immunity with childhood-onset and a good response to TLR7 inhibition.
- MeSH
- autoimunitní hemolytická anemie genetika imunologie MeSH
- cytokiny genetika imunologie MeSH
- dvojčata monozygotní MeSH
- HEK293 buňky MeSH
- lidé MeSH
- mutace * MeSH
- posouzení stavu pacienta MeSH
- toll-like receptor 7 genetika imunologie MeSH
- toll-like receptor 8 genetika imunologie MeSH
- zánět genetika imunologie MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- studie na dvojčatech MeSH
- Názvy látek
- cytokiny MeSH
- TLR7 protein, human MeSH Prohlížeč
- TLR8 protein, human MeSH Prohlížeč
- toll-like receptor 7 MeSH
- toll-like receptor 8 MeSH
The ER membrane protein complex (EMC) is required for the biogenesis of a subset of tail anchored (TA) and polytopic membrane proteins, including Rhodopsin-1 (Rh1) and the TRP channel. To understand the physiological implications of EMC-dependent membrane protein biogenesis, we perform a bioinformatic identification of Drosophila TA proteins. From 254 predicted TA proteins, screening in larval eye discs identified two proteins that require EMC for their biogenesis: fan and Xport-A. Fan is required for male fertility in Drosophila and we show that EMC is also required for this process. Xport-A is essential for the biogenesis of both Rh1 and TRP, raising the possibility that disruption of Rh1 and TRP biogenesis in EMC mutants is secondary to the Xport-A defect. We show that EMC is required for Xport-A TMD membrane insertion and that EMC-independent Xport-A mutants rescue Rh1 and TRP biogenesis in EMC mutants. Finally, our work also reveals a role for Xport-A in a glycosylation-dependent triage mechanism during Rh1 biogenesis in the endoplasmic reticulum.
- Klíčová slova
- ER membrane protein complex, Rh1, TRP, Xport-A, tail anchored proteins,
- MeSH
- Drosophila genetika metabolismus MeSH
- endoplazmatické retikulum metabolismus MeSH
- membránové proteiny genetika metabolismus MeSH
- molekulární chaperony * genetika metabolismus MeSH
- proteiny Drosophily * genetika metabolismus MeSH
- represorové proteiny * genetika metabolismus MeSH
- rodopsin * genetika MeSH
- transkripční faktory bHLH * genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- emc protein, Drosophila MeSH Prohlížeč
- membránové proteiny MeSH
- molekulární chaperony * MeSH
- proteiny Drosophily * MeSH
- represorové proteiny * MeSH
- rodopsin * MeSH
- transkripční faktory bHLH * MeSH
- Xport-A protein, Drosophila MeSH Prohlížeč
Interactions among amino acid residues are the principal contributor to the stability of the three-dimensional structure of a protein. The Amino Acid Interactions (INTAA) web server (https://bioinfo.uochb.cas.cz/INTAA/) has established itself as a unique computational resource, which enables users to calculate the contribution of individual residues in a biomolecular structure to its total energy using a molecular mechanical scoring function. In this update, we describe major additions to the web server which help solidify its position as a robust, comprehensive resource for biomolecular structure analysis. Importantly, a new continuum solvation model was introduced, allowing more accurate representation of electrostatic interactions in aqueous media. In addition, a low-overhead pipeline for the estimation of evolutionary conservation in protein chains has been added. New visualization options were introduced as well, allowing users to easily switch between and interrelate the energetic and evolutionary views of the investigated structures.
- MeSH
- aminokyseliny chemie MeSH
- internet MeSH
- konformace proteinů * MeSH
- molekulární modely MeSH
- proteiny chemie MeSH
- software * MeSH
- statická elektřina MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- aminokyseliny MeSH
- proteiny MeSH
Interaction with the DNA minor groove is a significant contributor to specific sequence recognition in selected families of DNA-binding proteins. Based on a statistical analysis of 3D structures of protein-DNA complexes, we propose that distortion of the DNA minor groove resulting from interactions with hydrophobic amino acid residues is a universal element of protein-DNA recognition. We provide evidence to support this by associating each DNA minor groove-binding amino acid residue with the local dimensions of the DNA double helix using a novel algorithm. The widened DNA minor grooves are associated with high GC content. However, some AT-rich sequences contacted by hydrophobic amino acids (e.g., phenylalanine) display extreme values of minor groove width as well. For a number of hydrophobic amino acids, distinct secondary structure preferences could be identified for residues interacting with the widened DNA minor groove. These results hold even after discarding the most populous families of minor groove-binding proteins.
- Klíčová slova
- DNA shape, hydrophobic, indirect readout, minor groove, protein–DNA interaction, specific recognition,
- MeSH
- algoritmy MeSH
- aminokyselinové motivy MeSH
- aminokyseliny chemie MeSH
- Arabidopsis metabolismus MeSH
- DNA vazebné proteiny metabolismus MeSH
- DNA chemie MeSH
- fenylalanin chemie MeSH
- hydrofobní a hydrofilní interakce * MeSH
- konformace nukleové kyseliny MeSH
- kyselina glutamová chemie MeSH
- lidé MeSH
- proteiny chemie MeSH
- Saccharomyces cerevisiae metabolismus MeSH
- sekundární struktura proteinů MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- aminokyseliny MeSH
- DNA vazebné proteiny MeSH
- DNA MeSH
- fenylalanin MeSH
- kyselina glutamová MeSH
- proteiny MeSH
Many membrane proteins are thought to function as dimers or higher oligomers, but measuring membrane protein oligomerization in lipid membranes is particularly challenging. Förster resonance energy transfer (FRET) and fluorescence cross-correlation spectroscopy are noninvasive, optical methods of choice that have been applied to the analysis of dimerization of single-spanning membrane proteins. However, the effects inherent to such two-dimensional systems, such as the excluded volume of polytopic transmembrane proteins, proximity FRET, and rotational diffusion of fluorophore dipoles, complicate interpretation of FRET data and have not been typically accounted for. Here, using FRET and fluorescence cross-correlation spectroscopy, we introduce a method to measure surface protein density and to estimate the apparent Förster radius, and we use Monte Carlo simulations of the FRET data to account for the proximity FRET effect occurring in confined two-dimensional environments. We then use FRET to analyze the dimerization of human rhomboid protease RHBDL2 in giant plasma membrane vesicles. We find no evidence for stable oligomers of RHBDL2 in giant plasma membrane vesicles of human cells even at concentrations that highly exceed endogenous expression levels. This indicates that the rhomboid transmembrane core is intrinsically monomeric. Our findings will find use in the application of FRET and fluorescence correlation spectroscopy for the analysis of oligomerization of transmembrane proteins in cell-derived lipid membranes.
- MeSH
- buněčná membrána metabolismus MeSH
- dimerizace MeSH
- lidé MeSH
- membránové lipidy metabolismus MeSH
- membránové proteiny * metabolismus MeSH
- multimerizace proteinu MeSH
- rezonanční přenos fluorescenční energie * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- membránové lipidy MeSH
- membránové proteiny * MeSH
Estimation of binding free energies is one of the central aims of simulations of biomolecular complexes. We explore the accuracy and efficiency of setups based on nonequilibrium pulling simulations applied to the estimation of binding affinities of DNA-binding proteins. Absolute binding free energies are calculated over a range of temperatures and compared to results obtained previously using an equilibrium method. We show that realistic binding affinities can be obtained with the presented nonequilibrium approach, which also entails lower computational requirements. Errors of the binding free energy estimates are investigated and are shown to be comparable to those observed previously. Bounds are provided on the convergence of the errors with respect to the number of pulling simulations performed and with respect to the applied pull rate.
- MeSH
- DNA vazebné proteiny chemie MeSH
- DNA chemie MeSH
- entropie MeSH
- počítačová simulace MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- DNA vazebné proteiny MeSH
- DNA MeSH
We systematically investigate the applicability of a molecular dynamics-based setup for the calculations of standard binding free energies of biologically relevant protein-DNA complexes. The free energies are extracted from a potential of mean force calculated using umbrella sampling simulations. Two protein-DNA systems derived from a homeodomain transcription factor complex are studied in order to investigate the binding of both disordered and globular proteins. Free energies and trajectories obtained using two modern molecular mechanical force fields are compared to each other and to experimental data. The temperature dependence of the calculated standard binding free energies is investigated by performing all simulations over a range of temperatures. We show that the values of standard binding free energies obtained from these simulations are overestimated compared to experimental results. Significant differences are observed between the two protein-DNA systems and between the two force fields, which are explained by different propensities to form inter- and intramolecular contacts. The number of protein-DNA contacts increases with increasing temperature, in agreement with the experimentally known temperature dependence of enthalpies of binding. However, conclusions about the temperature dependence of the standard binding free energies cannot be made with confidence, as the differences among the values are on the order of statistical uncertainty.
- MeSH
- DNA chemie metabolismus MeSH
- entropie MeSH
- homeoboxový protein Nkx-2.5 chemie metabolismus MeSH
- homeodoménové proteiny chemie metabolismus MeSH
- lidé MeSH
- simulace molekulární dynamiky MeSH
- simulace molekulového dockingu MeSH
- teplota MeSH
- termodynamika * MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- DNA MeSH
- homeoboxový protein Nkx-2.5 MeSH
- homeodoménové proteiny MeSH
- NKX2-5 protein, human MeSH Prohlížeč
SUMMARY: Amino acid residues showing above background levels of conservation are often indicative of functionally significant regions within a protein. Understanding how the sequence conservation profile relates in space requires projection onto a protein structure, a potentially time-consuming process. 3DPatch is a web application that streamlines this task by automatically generating multiple sequence alignments (where appropriate) and finding structural homologs, presenting the user with a choice of structures matching their query, annotated with residue conservation scores in a matter of seconds. AVAILABILITY AND IMPLEMENTATION: 3DPatch is written in JavaScript and is freely available at http://www.skylign.org/3DPatch/. Mozilla Firefox, Google Chrome, and Safari web browsers are supported. Source code is available under MIT license at https://github.com/davidjakubec/3DPatch. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
- MeSH
- databáze proteinů MeSH
- internetový prohlížeč MeSH
- konformace proteinů * MeSH
- lidé MeSH
- sekvenční seřazení * MeSH
- software * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Domains are distinct units within proteins that typically can fold independently into recognizable three-dimensional structures to facilitate their functions. The structural and functional independence of protein domains is reflected by their apparent modularity in the context of multi-domain proteins. In this work, we examined the coupling of evolution of domain sequences co-occurring within multi-domain proteins to see if it proceeds independently, or in a coordinated manner. We used continuous information theory measures to assess the extent of correlated mutations among domains in multi-domain proteins from organisms across the tree of life. In all multi-domain architectures we examined, domains co-occurring within protein sequences had to some degree undergone concerted evolution. This finding challenges the notion of complete modularity and independence of protein domains, providing new perspective on the evolution of protein sequence and function.
- MeSH
- biologické modely * MeSH
- informační teorie MeSH
- molekulární evoluce * MeSH
- proteinové domény * MeSH
- proteiny genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- proteiny MeSH
