SUMMARY: MOLEonline is an interactive, web-based tool designed to detect and analyse channels (pores and tunnels) within protein structures. The latest version of MOLEonline addresses the limitations of its predecessor by integrating the Mol* viewer for visualization and offering a streamlined, fully interactive user experience. The new features include colouring tunnels in the 3D viewer based on their physicochemical properties. A 2D representation of the protein structure and calculated tunnels is generated using 2DProts. Users can now store tunnels directly in the mmCIF file format, facilitating sharing via the community-standard FAIR format for structural data. In addition, the ability to store and load computation settings ensures the reproducibility of tunnel computation results. Integration with the ChannelsDB 2.0 database allows users to access precomputed tunnels. AVAILABILITY AND IMPLEMENTATION: The MOLEonline application is freely available at https://moleonline.cz with no login requirement, its source code is stored at GitHub under the MIT licence at https://github.com/sb-ncbr/moleonline-web, and archived at Figshare at https://doi.org/10.6084/m9.figshare.29816174.

• MeSH
• databáze proteinů MeSH
• internet MeSH
• konformace proteinů MeSH
• proteiny * chemie   MeSH
• software * MeSH
• uživatelské rozhraní počítače MeSH
• výpočetní biologie * metody   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• proteiny * MeSH

Enzymes with buried active sites utilize molecular tunnels to exchange substrates, products, and solvent molecules with the surface. These transport mechanisms are crucial for protein function and influence various properties. As proteins are inherently dynamic, their tunnels also vary structurally. Understanding these dynamics is essential for elucidating structure-function relationships, drug discovery, and bioengineering. Caver Web 2.0 is a user-friendly web server that retains all Caver Web 1.0 functionalities while introducing key improvements: (i) generation of dynamic ensembles via automated molecular dynamics with YASARA, (ii) analysis of dynamic tunnels with CAVER 3.0, (iii) prediction of ligand trajectories in multiple snapshots with CaverDock 1.2, and (iv) customizable ligand libraries for virtual screening. Users can assess protein flexibility, identify and characterize tunnels, and predict ligand trajectories and energy profiles in both static and dynamic structures. Additionally, the platform supports virtual screening with FDA/EMA-approved drugs and user-defined datasets. Caver Web 2.0 is a versatile tool for biological research, protein engineering, and drug discovery, aiding the identification of strong inhibitors or new substrates to bind to the active sites or tunnels, and supporting drug repurposing efforts. The server is freely accessible at https://loschmidt.chemi.muni.cz/caverweb.

• MeSH
• internet MeSH
• katalytická doména MeSH
• konformace proteinů MeSH
• ligandy MeSH
• objevování léků MeSH
• proteiny * chemie   metabolismus   MeSH
• simulace molekulární dynamiky MeSH
• software * MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• ligandy MeSH
• proteiny * MeSH

The engineering of efficient enzymes for large-scale production of industrially relevant compounds is a challenging task. Utilizing rational protein design, which relies on a comprehensive understanding of mechanistic information, holds significant promise for achieving success in this endeavor. Pre-steady-state kinetic measurements, obtained either through fast-mixing techniques or photoswitchable substrates, provide crucial mechanistic insights. The latter approach not only furnishes mechanistic clarity but also affords real-time structural elucidation of reaction intermediates via time-resolved femtosecond crystallography. Unfortunately, only a limited number of such valuable mechanistic probes are available. To address this gap, we applied a multidisciplinary approach, including computational analysis, chemical synthesis, physicochemical property screening, and enzyme kinetics to identify promising candidates for photoswitchable probes. We demonstrate the approach by designing an azobenzene-based photoswitchable substrate tailored for haloalkane dehalogenases, a prototypic class of enzymes pivotal in developing computational tools for rational protein design. The probe was subjected to steady-state and pre-steady-state kinetic analysis, which revealed new insights about the catalytic behavior of the model biocatalysts. We employed laser-triggered Z-to-E azobenzene photoswitching to generate the productive isomer in situ, opening avenues for advanced mechanistic studies using time-resolved femtosecond crystallography. Our results not only pave the way for the mechanistic understanding of this model enzyme family, incorporating both kinetic and structural dimensions, but also propose a systematic approach to the rational design of photoswitchable enzymatic substrates.

• Publikační typ
• časopisecké články MeSH

UNLABELLED: Staphylococcus aureus is a lethal pathogen that can cause various bacterial infections. This study targets the CrtM enzyme of S. aureus, which is crucial for synthesizing golden carotenoid pigment: staphyloxanthin, which provides anti-oxidant activity to this bacterium for combating antimicrobial resistance inside the host cell. The present investigation quests for human SQS inhibitors against the CrtM enzyme by employing structure-based drug design approaches including induced fit docking (IFD), molecular dynamic (MD) simulations, and binding free energy calculations. Depending upon the docking scores, two compounds, lapaquistat acetate and squalestatin analog 20, were identified as the lead molecules exhibit higher affinity toward the CrtM enzyme. These docked complexes were further subjected to 100 ns MD simulation and several thermodynamics parameters were analyzed. Further, the binding free energies (ΔG) were calculated for each simulated protein-ligand complex to study the stability of molecular contacts using the MM-GBSA approach. Pre-ADMET analysis was conducted for systematic evaluation of physicochemical and medicinal chemistry properties of these compounds. The above study suggested that lapaquistat acetate and squalestatin analog 20 can be selected as potential lead candidates with promising binding affinity for the S. aureus CrtM enzyme. This study might provide insights into the discovery of potential drug candidates for S. aureus with a high therapeutic index. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-023-03862-y.

• Klíčová slova
• Anti-virulence, CrtM, Docking, MM-GBSA, Molecular dynamics, Staphyloxanthin virtual screening,
• Publikační typ
• časopisecké články MeSH

ChannelsDB 2.0 is an updated database providing structural information about the position, geometry and physicochemical properties of protein channels-tunnels and pores-within deposited biomacromolecular structures from PDB and AlphaFoldDB databases. The newly deposited information originated from several sources. Firstly, we included data calculated using a popular CAVER tool to complement the data obtained using original MOLE tool for detection and analysis of protein tunnels and pores. Secondly, we added tunnels starting from cofactors within the AlphaFill database to enlarge the scope of the database to protein models based on Uniprot. This has enlarged available channel annotations ∼4.6 times as of 1 September 2023. The database stores information about geometrical features, e.g. length and radius, and physico-chemical properties based on channel-lining amino acids. The stored data are interlinked with the available UniProt mutation annotation data. ChannelsDB 2.0 provides an excellent resource for deep analysis of the role of biomacromolecular tunnels and pores. The database is available free of charge: https://channelsdb2.biodata.ceitec.cz.

• MeSH
• aminokyseliny MeSH
• databáze proteinů * MeSH
• konformace proteinů MeSH
• proteiny * chemie   MeSH
• software * MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• aminokyseliny MeSH
• proteiny * MeSH

NanoLuc, a superior β-barrel fold luciferase, was engineered 10 years ago but the nature of its catalysis remains puzzling. Here experimental and computational techniques are combined, revealing that imidazopyrazinone luciferins bind to an intra-barrel catalytic site but also to an allosteric site shaped on the enzyme surface. Structurally, binding to the allosteric site prevents simultaneous binding to the catalytic site, and vice versa, through concerted conformational changes. We demonstrate that restructuration of the allosteric site can boost the luminescent reaction in the remote active site. Mechanistically, an intra-barrel arginine coordinates the imidazopyrazinone component of luciferin, which reacts with O2 via a radical charge-transfer mechanism, and then it also protonates the resulting excited amide product to form a light-emitting neutral species. Concomitantly, an aspartate, supported by two tyrosines, fine-tunes the blue color emitter to secure a high emission intensity. This information is critical to engineering the next-generation of ultrasensitive bioluminescent reporters.

• MeSH
• katalytická doména MeSH
• luciferasy metabolismus   MeSH
• luminiscenční měření * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• luciferasy MeSH
• nanoluc MeSH Prohlížeč

PredictONCO 1.0 is a unique web server that analyzes effects of mutations on proteins frequently altered in various cancer types. The server can assess the impact of mutations on the protein sequential and structural properties and apply a virtual screening to identify potential inhibitors that could be used as a highly individualized therapeutic approach, possibly based on the drug repurposing. PredictONCO integrates predictive algorithms and state-of-the-art computational tools combined with information from established databases. The user interface was carefully designed for the target specialists in precision oncology, molecular pathology, clinical genetics and clinical sciences. The tool summarizes the effect of the mutation on protein stability and function and currently covers 44 common oncological targets. The binding affinities of Food and Drug Administration/ European Medicines Agency -approved drugs with the wild-type and mutant proteins are calculated to facilitate treatment decisions. The reliability of predictions was confirmed against 108 clinically validated mutations. The server provides a fast and compact output, ideal for the often time-sensitive decision-making process in oncology. Three use cases of missense mutations, (i) K22A in cyclin-dependent kinase 4 identified in melanoma, (ii) E1197K mutation in anaplastic lymphoma kinase 4 identified in lung carcinoma and (iii) V765A mutation in epidermal growth factor receptor in a patient with congenital mismatch repair deficiency highlight how the tool can increase levels of confidence regarding the pathogenicity of the variants and identify the most effective inhibitors. The server is available at https://loschmidt.chemi.muni.cz/predictonco.

• Klíčová slova
• cancer, oncology, personalized medicine, single-nucleotide polymorphism, targeted therapy,
• MeSH
• individualizovaná medicína * MeSH
• lidé MeSH
• melanom * MeSH
• mutace MeSH
• proteiny MeSH
• reprodukovatelnost výsledků MeSH
• strojové učení MeSH
• výpočetní biologie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteiny MeSH

Pregnenolone (P5) is synthesized as the first bioactive steroid in the mitochondria from cholesterol. Clusters of differentiation 4 (CD4+) and Clusters of differentiation 8 (CD8+) immune cells synthesize P5 de novo; P5, in turn, play important role in immune homeostasis and regulation. However, P5's biochemical mode of action in immune cells is still emerging. We envisage that revealing the complete spectrum of P5 target proteins in immune cells would have multifold applications, not only in basic understanding of steroids biochemistry in immune cells but also in developing new therapeutic applications. We employed a CLICK-enabled probe to capture P5-binding proteins in live T helper cell type 2 (Th2) cells. Subsequently, using high-throughput quantitative proteomics, we identified the P5 interactome in CD4+ Th2 cells. Our study revealed P5's mode of action in CD4+ immune cells. We identified novel proteins from mitochondrial and endoplasmic reticulum membranes to be the primary mediators of P5's biochemistry in CD4+ and to concur with our earlier finding in CD8+ immune cells. Applying advanced computational algorithms and molecular simulations, we were able to generate near-native maps of P5-protein key molecular interactions. We showed bonds and interactions between key amino acids and P5, which revealed the importance of ionic bond, hydrophobic interactions, and water channels. We point out that our results can lead to designing of novel molecular therapeutics strategies.

• Klíčová slova
• TH2, chemoproteomics, click chemistry, lymphosteroid, pregnenolone,
• MeSH
• pregnenolon * metabolismus   farmakologie   MeSH
• simulace molekulární dynamiky MeSH
• steroidy MeSH
• Th2 buňky * metabolismus   MeSH
• transportní proteiny metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• pregnenolon * MeSH
• steroidy MeSH
• transportní proteiny MeSH

Catalase-peroxidases (KatGs) are unique bifunctional oxidoreductases that contain heme in their active centers allowing both the peroxidatic and catalatic reaction modes. These originally bacterial enzymes are broadly distributed among various fungi allowing them to cope with reactive oxygen species present in the environment or inside the cells. We used various biophysical, biochemical, and bioinformatics methods to investigate differences between catalase-peroxidases originating in thermophilic and mesophilic fungi from different habitats. Our results indicate that the architecture of the active center with a specific post-translational modification is highly similar in mesophilic and thermophilic KatG and also the peroxidatic acitivity with ABTS, guaiacol, and L-DOPA. However, only the thermophilic variant CthedisKatG reveals increased manganese peroxidase activity at elevated temperatures. The catalatic activity releasing molecular oxygen is comparable between CthedisKatG and mesophilic MagKatG1 over a broad temperature range. Two constructed point mutations in the active center were performed selectively blocking the formation of described post-translational modification in the active center. They exhibited a total loss of catalatic activity and changes in the peroxidatic activity. Our results indicate the capacity of bifunctional heme enzymes in the variable reactivity for potential biotech applications.

• Klíčová slova
• bifunctional enzyme, heme catalase, oxidative stress, peroxidase–catalase superfamily, reactive oxygen species,
• Publikační typ
• časopisecké články MeSH

The biodegradative capacity of bacteria in their natural habitats is affected by water availability. In this work, we have examined the activity and effector specificity of the transcriptional regulator XylR of the TOL plasmid pWW0 of Pseudomonas putida mt-2 for biodegradation of m-xylene when external water potential was manipulated with polyethylene glycol PEG8000. By using non-disruptive luxCDEAB reporter technology, we noticed that the promoter activated by XylR (Pu) restricted its activity and the regulator became more effector-specific towards head TOL substrates when cells were grown under water subsaturation. Such a tight specificity brought about by water limitation was relaxed when intracellular osmotic stress was counteracted by the external addition of the compatible solute glycine betaine. With these facts in hand, XylR variants isolated earlier as effector-specificity responders to the non-substrate 1,2,4-trichlorobenzene under high matric stress were re-examined and found to be unaffected by water potential in vivo. All these phenomena could be ultimately explained as the result of water potential-dependent conformational changes in the A domain of XylR and its effector-binding pocket, as suggested by AlphaFold prediction of protein structures. The consequences of this scenario for the evolution of specificities in regulators and the emergence of catabolic pathways are discussed.

• MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• DNA vazebné proteiny metabolismus   MeSH
• plazmidy MeSH
• promotorové oblasti (genetika) MeSH
• Pseudomonas putida * genetika   metabolismus   MeSH
• regulace genové exprese u bakterií MeSH
• transkripční faktory * genetika   metabolismus   MeSH
• xyleny metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• DNA vazebné proteiny MeSH
• transkripční faktory * MeSH
• xyleny MeSH