A single protein structure is rarely sufficient to capture the conformational variability of a protein. Both bound and unbound (holo and apo) forms of a protein are essential for understanding its geometry and making meaningful comparisons. Nevertheless, docking or drug design studies often still consider only single protein structures in their holo form, which are for the most part rigid. With the recent explosion in the field of structural biology, large, curated datasets are urgently needed. Here, we use a previously developed application (AHoJ) to perform a comprehensive search for apo-holo pairs for 468,293 biologically relevant protein-ligand interactions across 27,983 proteins. In each search, the binding pocket is captured and mapped across existing structures within the same UniProt, and the mapped pockets are annotated as apo or holo, based on the presence or absence of ligands. We assemble the results into a database, AHoJ-DB (www.apoholo.cz/db), that captures the variability of proteins with identical sequences, thereby exposing the agents responsible for the observed differences in geometry. We report several metrics for each annotated pocket, and we also include binding pockets that form at the interface of multiple chains. Analysis of the database shows that about 24% of the binding sites occur at the interface of two or more chains and that less than 50% of the total binding sites processed have an apo form in the PDB. These results can be used to train and evaluate predictors, discover potentially druggable proteins, and reveal protein- and ligand-specific relationships that were previously obscured by intermittent or partial data. Availability: www.apoholo.cz/db.
- MeSH
- apoproteiny chemie metabolismus MeSH
- databáze proteinů * MeSH
- konformace proteinů * MeSH
- lidé MeSH
- ligandy MeSH
- molekulární modely MeSH
- proteiny * chemie metabolismus MeSH
- vazba proteinů * MeSH
- vazebná místa MeSH
- výpočetní biologie metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
PrankWeb is an online resource providing an interface to P2Rank, a state-of-the-art method for ligand binding site prediction. P2Rank is a template-free machine learning method based on the prediction of local chemical neighborhood ligandability centered on points placed on a solvent-accessible protein surface. Points with a high ligandability score are then clustered to form the resulting ligand binding sites. In addition, PrankWeb provides a web interface enabling users to easily carry out the prediction and visually inspect the predicted binding sites via an integrated sequence-structure view. Moreover, PrankWeb can determine sequence conservation for the input molecule and use this in both the prediction and result visualization steps. Alongside its online visualization options, PrankWeb also offers the possibility of exporting the results as a PyMOL script for offline visualization. The web frontend communicates with the server side via a REST API. In high-throughput scenarios, therefore, users can utilize the server API directly, bypassing the need for a web-based frontend or installation of the P2Rank application. PrankWeb is available at http://prankweb.cz/, while the web application source code and the P2Rank method can be accessed at https://github.com/jendelel/PrankWebApp and https://github.com/rdk/p2rank, respectively.
- MeSH
- benchmarking MeSH
- datové soubory jako téma MeSH
- interakční proteinové domény a motivy MeSH
- internet MeSH
- konformace proteinů, alfa-helix MeSH
- konformace proteinů, beta-řetězec MeSH
- lidé MeSH
- ligandy MeSH
- proteiny chemie metabolismus MeSH
- sekvence aminokyselin MeSH
- software * MeSH
- strojové učení * MeSH
- termodynamika MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Mitochondria originated from proteobacterial endosymbionts, and their transition to organelles was tightly linked to establishment of the protein import pathways. The initial import of most proteins is mediated by the translocase of the outer membrane (TOM). Although TOM is common to all forms of mitochondria, an unexpected diversity of subunits between eukaryotic lineages has been predicted. However, experimental knowledge is limited to a few organisms, and so far, it remains unsettled whether the triplet-pore or the twin-pore structure is the generic form of TOM complex. Here, we analysed the TOM complex in hydrogenosomes, a metabolically specialised anaerobic form of mitochondria found in the excavate Trichomonas vaginalis. We demonstrate that the highly divergent β-barrel T. vaginalis TOM (TvTom)40-2 forms a translocation channel to conduct hydrogenosomal protein import. TvTom40-2 is present in high molecular weight complexes, and their analysis revealed the presence of four tail-anchored (TA) proteins. Two of them, Tom36 and Tom46, with heat shock protein (Hsp)20 and tetratricopeptide repeat (TPR) domains, can bind hydrogenosomal preproteins and most likely function as receptors. A third subunit, Tom22-like protein, has a short cis domain and a conserved Tom22 transmembrane segment but lacks a trans domain. The fourth protein, hydrogenosomal outer membrane protein 19 (Homp19) has no known homology. Furthermore, our data indicate that TvTOM is associated with sorting and assembly machinery (Sam)50 that is involved in β-barrel assembly. Visualisation of TvTOM by electron microscopy revealed that it forms three pores and has an unconventional skull-like shape. Although TvTOM seems to lack Tom7, our phylogenetic profiling predicted Tom7 in free-living excavates. Collectively, our results suggest that the triplet-pore TOM complex, composed of three conserved subunits, was present in the last common eukaryotic ancestor (LECA), while receptors responsible for substrate binding evolved independently in different eukaryotic lineages.
- MeSH
- fylogeneze MeSH
- membránové proteiny metabolismus MeSH
- membránové transportní proteiny metabolismus MeSH
- mitochondrie metabolismus MeSH
- organely MeSH
- transport proteinů fyziologie MeSH
- transportní proteiny mitochondriální membrány metabolismus MeSH
- transportní proteiny genetika metabolismus fyziologie MeSH
- Trichomonas vaginalis metabolismus patogenita fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Positive selection acting on Toll-like receptors (TLRs) has been recently investigated to reveal evolutionary mechanisms of host-pathogen molecular co-adaptation. Much of this research, however, has focused mainly on the identification of sites predicted to be under positive selection, bringing little insight into the functional differences and similarities among species and a limited understanding of convergent evolution in the innate immune molecules. In this study, we provide evidence of phenotypic variability in the avian TLR4 ligand-binding region (LBR), the direct interface between host and pathogen molecular structures. We show that 55 passerine species vary substantially in the distribution of electrostatic potential on the surface of the receptor, and based on these distinct patterns, we identified four species clusters. Seven of the 34 evolutionarily nonconservative and positively selected residues correspond topologically to sites previously identified as being important for lipopolysaccharide, lipid IVa or MD-2 binding. Five of these positions codetermine the identity of the charge clusters. Groups of species that host-related communities of pathogens were predicted to cluster based on their TLR4 LBR charge. Despite some evidence for convergence among taxa, there were no clear associations between the TLR4 LBR charge distribution and any of the general ecological characteristics compared (migration, latitudinal distribution and diet). Closely related species, however, mostly belonged to the same surface charge cluster indicating that phylogenetic constraints are key determinants shaping TLR4 adaptive evolution. Our results suggest that host innate immune evolution is consistent with Fahrenholz's rule on the cospeciation of hosts and their parasites.
- MeSH
- glykolipidy chemie genetika MeSH
- interakce hostitele a patogenu genetika MeSH
- konformace proteinů MeSH
- ligandy MeSH
- lipid A analogy a deriváty chemie genetika MeSH
- lipopolysacharidy chemie genetika MeSH
- lymfocytární antigen 96 chemie genetika MeSH
- mikrobiota genetika MeSH
- molekulární evoluce * MeSH
- molekulární modely MeSH
- přirozená imunita genetika MeSH
- ptáci genetika parazitologie MeSH
- sekvenční analýza DNA MeSH
- selekce (genetika) * genetika MeSH
- statická elektřina MeSH
- toll-like receptor 4 chemie genetika MeSH
- vazba proteinů MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
CAS is a docking protein downstream of the proto-oncogene Src with a role in invasion and metastasis of cancer cells. The CAS SH3 domain is indispensable for CAS-mediated signaling, but structural aspects of CAS SH3 ligand binding and regulation are not well understood. Here, we identified the consensus CAS SH3 binding motif and structurally characterized the CAS SH3 domain in complex with ligand. We revealed the requirement for an uncommon centrally localized lysine residue at position +2 of CAS SH3 ligands and two rather dissimilar optional anchoring residues, leucine and arginine, at position +5. We further expanded the knowledge of CAS SH3 ligand binding regulation by manipulating tyrosine 12 phosphorylation and confirmed the negative role of this phosphorylation on CAS SH3 ligand binding. Finally, by exploiting the newly identified binding requirements of the CAS SH3 domain, we predicted and experimentally verified two novel CAS SH3 binding partners, DOK7 and GLIS2.
- MeSH
- aminokyseliny metabolismus MeSH
- fosforylace fyziologie MeSH
- lidé MeSH
- ligandy MeSH
- sekvence aminokyselin MeSH
- signální transdukce fyziologie MeSH
- src homologní domény fyziologie MeSH
- substrátový protein asociovaný s Crk metabolismus MeSH
- vazba proteinů fyziologie MeSH
- vazebná místa fyziologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Splicing in S. cerevisiae has been shown to proceed cotranscriptionally, but the nature of the coupling remains a subject of debate. Here, we examine the effect of nineteen complex-related splicing factor Prp45 (a homolog of SNW1/SKIP) on cotranscriptional splicing. RNA-sequencing and RT-qPCR showed elevated pre-mRNA levels but only limited reduction of spliced mRNAs in cells expressing C-terminally truncated Prp45, Prp45(1-169). Assays with a series of reporters containing the AMA1 intron with regulatable splicing confirmed decreased splicing efficiency and showed the leakage of unspliced RNAs in prp45(1-169) cells. We also measured pre-mRNA accumulation of the meiotic MER2 gene, which depends on the expression of Mer1 factor for splicing. prp45(1-169) cells accumulated approximately threefold higher levels of MER2 pre-mRNA than WT cells only when splicing was induced. To monitor cotranscriptional splicing, we determined the presence of early spliceosome assembly factors and snRNP complexes along the ECM33 and ACT1 genes. We found that prp45(1-169) hampered the cotranscriptional recruitment of U2 and, to a larger extent, U5 and NTC, while the U1 profile was unaffected. The recruitment of Prp45(1-169) was impaired similarly to U5 snRNP and NTC. Our results imply that Prp45 is required for timely formation of complex A, prior to stable physical association of U5/NTC with the emerging pre-mRNA substrate. We suggest that Prp45 facilitates conformational rearrangements and/or contacts that couple U1 snRNP-recognition to downstream assembly events.
- MeSH
- introny MeSH
- malý jaderný ribonukleoprotein U1 metabolismus MeSH
- malý jaderný ribonukleoprotein U2 metabolismus MeSH
- Saccharomyces cerevisiae - proteiny genetika metabolismus MeSH
- Saccharomyces cerevisiae genetika metabolismus MeSH
- sestřih RNA * MeSH
- spliceozomy metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Yellow-related proteins (YRPs) present in sand fly saliva act as affinity binders of bioamines, and help the fly to complete a bloodmeal by scavenging the physiological signals of damaged cells. They are also the main antigens in sand fly saliva and their recombinant form is used as a marker of host exposure to sand flies. Moreover, several salivary proteins and plasmids coding these proteins induce strong immune response in hosts bitten by sand flies and are being used to design protecting vaccines against Leishmania parasites. In this study, thirty two 3D models of different yellow-related proteins from thirteen sand fly species of two genera were constructed based on the known protein structure from Lutzomyia longipalpis. We also studied evolutionary relationships among species based on protein sequences as well as sequence and structural variability of their ligand-binding site. All of these 33 sand fly YRPs shared a similar structure, including a unique tunnel that connects the ligand-binding site with the solvent by two independent paths. However, intraspecific modifications found among these proteins affects the charges of the entrances to the tunnel, the length of the tunnel and its hydrophobicity. We suggest that these structural and sequential differences influence the ligand-binding abilities of these proteins and provide sand flies with a greater number of YRP paralogs with more nuanced answers to bioamines. All these characteristics allow us to better evaluate these proteins with respect to their potential use as part of anti-Leishmania vaccines or as an antigen to measure host exposure to sand flies.
- MeSH
- fylogeneze MeSH
- glykosylace MeSH
- hmyzí proteiny chemie metabolismus MeSH
- konformace proteinů MeSH
- ligandy MeSH
- molekulární modely MeSH
- Psychodidae * MeSH
- sekvence aminokyselin MeSH
- sliny metabolismus MeSH
- statická elektřina MeSH
- vazebná místa MeSH
- vodíková vazba MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
N-methyl-D-aspartate receptors (NMDARs) mediate synaptic plasticity, and their dysfunction is implicated in multiple brain disorders. NMDARs can be allosterically modulated by numerous compounds, including endogenous neurosteroid pregnanolone sulfate. Here, we identify the molecular basis of the use-dependent and voltage-independent inhibitory effect of neurosteroids on NMDAR responses. The site of action is located at the extracellular vestibule of the receptor's ion channel pore and is accessible after receptor activation. Mutations in the extracellular vestibule in the SYTANLAAF motif disrupt the inhibitory effect of negatively charged steroids. In contrast, positively charged steroids inhibit mutated NMDAR responses in a voltage-dependent manner. These results, in combination with molecular modeling, characterize structure details of the open configuration of the NMDAR channel. Our results provide a unique opportunity for the development of new therapeutic neurosteroid-based ligands to treat diseases associated with dysfunction of the glutamate system.
- MeSH
- aminokyselinové motivy MeSH
- lidé MeSH
- mutace * MeSH
- pregnanolon * chemie metabolismus MeSH
- receptory N-methyl-D-aspartátu * antagonisté a inhibitory chemie genetika metabolismus MeSH
- vestibulární aparát * chemie metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: SH3 domains are eukaryotic protein domains that participate in a plethora of cellular processes including signal transduction, proliferation, and cellular movement. Several studies indicate that tyrosine phosphorylation could play a significant role in the regulation of SH3 domains. RESULTS: To explore the incidence of the tyrosine phosphorylation within SH3 domains we queried the PhosphoSite Plus database of phosphorylation sites. Over 100 tyrosine phosphorylations occurring on 20 different SH3 domain positions were identified. The tyrosine corresponding to c-Src Tyr-90 was by far the most frequently identified SH3 domain phosphorylation site. A comparison of sequences around this tyrosine led to delineation of a preferred sequence motif ALYD(Y/F). This motif is present in about 15% of human SH3 domains and is structurally well conserved. We further observed that tyrosine phosphorylation is more abundant than serine or threonine phosphorylation within SH3 domains and other adaptor domains, such as SH2 or WW domains. Tyrosine phosphorylation could represent an important regulatory mechanism of adaptor domains. CONCLUSIONS: While tyrosine phosphorylation typically promotes signaling protein interactions via SH2 or PTB domains, its role in SH3 domains is the opposite - it blocks or prevents interactions. The regulatory function of tyrosine phosphorylation is most likely achieved by the phosphate moiety and its charge interfering with binding of polyproline helices of SH3 domain interacting partners.
Crk-associated substrate (CAS) is a major tyrosine-phosphorylated protein in cells transformed by v-crk and v-src oncogenes and plays an important role in invasiveness of Src-transformed cells. A novel phosphorylation site on CAS, Tyr-12 (Y12) within the ligand-binding hydrophobic pocket of the CAS SH3 domain, was identified and found to be enriched in Src-transformed cells and invasive human carcinoma cells. To study the biological significance of CAS Y12 phosphorylation, phosphomimicking Y12E and nonphosphorylatable Y12F mutants of CAS were studied. The phosphomimicking mutation decreased interaction of the CAS SH3 domain with focal adhesion kinase (FAK) and PTP-PEST and reduced tyrosine phosphorylation of FAK. Live-cell imaging showed that green fluorescent protein-tagged CAS Y12E mutant is, in contrast to wild-type or Y12F CAS, excluded from focal adhesions but retains its localization to podosome-type adhesions. Expression of CAS-Y12F in cas-/- mouse embryonic fibroblasts resulted in hyperphosphorylation of the CAS substrate domain, and this was associated with slower turnover of focal adhesions and decreased cell migration. Moreover, expression of CAS Y12F in Src-transformed cells greatly decreased invasiveness when compared to wild-type CAS expression. These findings reveal an important role of CAS Y12 phosphorylation in the regulation of focal adhesion assembly, cell migration, and invasiveness of Src-transformed cells.
- MeSH
- fokální adheze metabolismus MeSH
- fokální adhezní tyrosinkinasy metabolismus MeSH
- fosforylace MeSH
- invazivní růst nádoru MeSH
- lidé MeSH
- molekuly buněčné adheze metabolismus MeSH
- mutace MeSH
- myši MeSH
- nádorová transformace buněk MeSH
- nádorové buněčné linie MeSH
- pohyb buněk MeSH
- signální transdukce MeSH
- src homologní domény MeSH
- substrátový protein asociovaný s Crk chemie genetika metabolismus MeSH
- transformované buněčné linie MeSH
- tyrosin metabolismus MeSH
- tyrosinfosfatasa nereceptorového typu 12 metabolismus MeSH
- zelené fluorescenční proteiny metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
