Molecular dynamics (MD) simulations of uncoupling proteins (UCP), a class of transmembrane proteins relevant for proton transport across inner mitochondrial membranes, represent a complicated task due to the lack of available structural data. In this work, we use a combination of homology modelling and subsequent microsecond molecular dynamics simulations of UCP2 in the DOPC phospholipid bilayer, starting from the structure of the mitochondrial ATP/ADP carrier (ANT) as a template. We show that this protocol leads to a structure that is impermeable to water, in contrast to MD simulations of UCP2 structures based on the experimental NMR structure. We also show that ATP binding in the UCP2 cavity is tight in the homology modelled structure of UCP2 in agreement with experimental observations. Finally, we corroborate our results with conductance measurements in model membranes, which further suggest that the UCP2 structure modeled from ANT protein possesses additional key functional elements, such as a fatty acid-binding site at the R60 region of the protein, directly related to the proton transport mechanism across inner mitochondrial membranes.

• MeSH
• adenosintrifosfát chemie   metabolismus   MeSH
• iontový transport MeSH
• konformace proteinů * MeSH
• mastné kyseliny chemie   metabolismus   MeSH
• membránové proteiny chemie   MeSH
• mitochondriální proteiny chemie   metabolismus   MeSH
• myši MeSH
• sekvence aminokyselin MeSH
• simulace molekulární dynamiky * MeSH
• stabilita proteinů MeSH
• uncoupling protein 2 chemie   metabolismus   MeSH
• vazba proteinů MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

The rise in multidrug-resistant bacteria defines the need for identification of new antibacterial agents that are less prone to resistance acquisition. Compounds that simultaneously inhibit multiple bacterial targets are more likely to suppress the evolution of target-based resistance than monotargeting compounds. The structurally similar ATP binding sites of DNA gyrase and topoisomerase Ⅳ offer an opportunity to accomplish this goal. Here we present the design and structure-activity relationship analysis of balanced, low nanomolar inhibitors of bacterial DNA gyrase and topoisomerase IV that show potent antibacterial activities against the ESKAPE pathogens. For inhibitor 31c, a crystal structure in complex with Staphylococcus aureus DNA gyrase B was obtained that confirms the mode of action of these compounds. The best inhibitor, 31h, does not show any in vitro cytotoxicity and has excellent potency against Gram-positive (MICs: range, 0.0078-0.0625 μg/mL) and Gram-negative pathogens (MICs: range, 1-2 μg/mL). Furthermore, 31h inhibits GyrB mutants that can develop resistance to other drugs. Based on these data, we expect that structural derivatives of 31h will represent a step toward clinically efficacious multitargeting antimicrobials that are not impacted by existing antimicrobial resistance.

• MeSH
• adenosintrifosfát chemická syntéza   chemie   farmakologie   MeSH
• antibakteriální látky chemická syntéza   chemie   farmakologie   MeSH
• DNA gyráza metabolismus   MeSH
• DNA-topoisomerasa IV antagonisté a inhibitory   metabolismus   MeSH
• Escherichia coli účinky léků   enzymologie   patogenita   MeSH
• krystalografie rentgenová MeSH
• mikrobiální testy citlivosti MeSH
• molekulární struktura MeSH
• simulace molekulového dockingu MeSH
• Staphylococcus aureus účinky léků   enzymologie   patogenita   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND: The Hsp70 proteins maintain proteome integrity through the capacity of their nucleotide- and substrate-binding domains (NBD and SBD) to allosterically regulate substrate affinity in a nucleotide-dependent manner. Crystallographic studies showed that Hsp70 allostery relies on formation of contacts between ATP-bound NBD and an interdomain linker, accompanied by SBD subdomains docking onto distinct sites of the NBD leading to substrate release. However, the mechanics of ATP-induced SBD subdomains detachment is largely unknown. METHODS: Here, we investigated the structural and allosteric properties of human HSPA1A using hydrogen/deuterium exchange mass spectrometry, ATPase assays, surface plasmon resonance and fluorescence polarization-based substrate binding assays. RESULTS: Analysis of HSPA1A proteins bearing mutations at the interface of SBD subdomains close to the interdomain linker (amino acids L399, L510, I515, and D529) revealed that this region forms a folding unit stabilizing the structure of both SBD subdomains in the nucleotide-free state. The introduced mutations modulate HSPA1A allostery as they localize to the NBD-SBD interfaces in the ATP-bound protein. CONCLUSIONS: These findings show that residues forming the hydrophobic structural unit stabilizing the SBD structure are relocated during ATP-activated detachment of the SBD subdomains to different NBD-SBD docking interfaces enabling HSPA1A allostery. GENERAL SIGNIFICANCE: Mutation-induced perturbations tuned HSPA1A sensitivity to peptide/protein substrates and to Hsp40 in a way that is common for other Hsp70 proteins. Our results provide an insight into structural rearrangements in the SBD of Hsp70 proteins and highlight HSPA1A-specific allostery features, which is a prerequisite for selective targeting in Hsp-related pathologies.

• MeSH
• adenosintrifosfát chemie   genetika   MeSH
• alosterická regulace genetika   MeSH
• konformace proteinů * MeSH
• lidé MeSH
• mutace genetika   MeSH
• proteinové domény genetika   MeSH
• proteiny tepelného šoku HSP70 chemie   genetika   MeSH
• vazba proteinů genetika   MeSH
• vazebná místa genetika   MeSH
• vodík-deuteriová výměna MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Remdesivir was shown to inhibit RNA-dependent RNA-polymerases (RdRp) from distinct viral families such as from Filoviridae (Ebola) and Coronaviridae (SARS-CoV, SARS-CoV-2, MERS). In this study, we tested the ability of remdesivir to inhibit RdRps from the Flaviviridae family. Instead of remdesivir, we used the active species that is produced in cells from remdesivir, the appropriate triphosphate, which could be directly tested in vitro using recombinant flaviviral polymerases. Our results show that remdesivir can efficiently inhibit RdRps from viruses causing severe illnesses such as Yellow fever, West Nile fever, Japanese and Tick-borne encephalitis, Zika and Dengue. Taken together, this study demonstrates that remdesivir or its derivatives have the potential to become a broad-spectrum antiviral agent effective against many RNA viruses.

• MeSH
• adenosintrifosfát analogy a deriváty   chemie   farmakologie   MeSH
• antivirové látky chemie   farmakologie   MeSH
• Betacoronavirus účinky léků   enzymologie   MeSH
• COVID-19 MeSH
• Flavivirus účinky léků   enzymologie   MeSH
• inhibiční koncentrace 50 MeSH
• koronavirové infekce farmakoterapie   virologie   MeSH
• lidé MeSH
• pandemie MeSH
• RNA-dependentní RNA-polymerasa antagonisté a inhibitory   metabolismus   MeSH
• RNA-viry účinky léků   enzymologie   MeSH
• SARS-CoV-2 MeSH
• virová pneumonie farmakoterapie   virologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Adenosine triphosphate (ATP) is a crucial substrate and energy source commonly used in enzyme reactions. However, we demonstrated that the addition of this acidic compound to enzyme assay buffers can serve as a source of unnoticed pH changes. Even relatively low concentrations of ATP (up to 5 mM) shifted pH of reaction mixtures to acidic values. For example, Tris buffer lost buffering capacity at pH 7.46 by adding ATP at a concentration higher than 2 mM. In addition to the buffering capacity, the pH shifts differed with respect to the buffer concentration. High ATP concentrations are commonly used in hexokinase assays. We demonstrated how the presence of ATP affects pH of widely used enzyme assay buffers and inversely affected KM of human hexokinase 2 and S0.5 of human glucokinase. The pH optimum of human glucokinase was never reported before. We found that previously reported optimum of mammalian glucokinase was incorrect, affected by the ATP-induced pH shifts. The pH optimum of human glucokinase is at pH 8.5-8.7. Suggested is the full disclosure of reaction conditions, including the measurement of pH of the whole reaction mixtures instead of measuring pH prior to the addition of all the components.

• MeSH
• adenosintrifosfát chemie   metabolismus   MeSH
• enzymatické testy metody   MeSH
• hexokinasa chemie   genetika   izolace a purifikace   metabolismus   MeSH
• koncentrace vodíkových iontů * MeSH
• ověření koncepční studie MeSH
• rekombinantní proteiny chemie   genetika   izolace a purifikace   metabolismus   MeSH
• substrátová specifita MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Transport of proteins across membranes is a fundamental process, achieved in every cell by the 'Sec' translocon. In prokaryotes, SecYEG associates with the motor ATPase SecA to carry out translocation for pre-protein secretion. Previously, we proposed a Brownian ratchet model for transport, whereby the free energy of ATP-turnover favours the directional diffusion of the polypeptide (Allen et al., 2016). Here, we show that ATP enhances this process by modulating secondary structure formation within the translocating protein. A combination of molecular simulation with hydrogendeuterium-exchange mass spectrometry and electron paramagnetic resonance spectroscopy reveal an asymmetry across the membrane: ATP-induced conformational changes in the cytosolic cavity promote unfolded pre-protein structure, while the exterior cavity favours its formation. This ability to exploit structure within a pre-protein is an unexplored area of protein transport, which may apply to other protein transporters, such as those of the endoplasmic reticulum and mitochondria.

• MeSH
• adenosintrifosfát chemie   metabolismus   MeSH
• adenosintrifosfatasy chemie   metabolismus   MeSH
• Escherichia coli metabolismus   MeSH
• membránové transportní proteiny chemie   metabolismus   MeSH
• molekulární modely MeSH
• proteinové prekurzory metabolismus   MeSH
• proteiny SecA chemie   metabolismus   MeSH
• proteiny z Escherichia coli chemie   metabolismus   MeSH
• sbalování proteinů * MeSH
• translokační kanály SEC chemie   metabolismus   MeSH
• transport proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Type I restriction-modification enzymes differ significantly from the type II enzymes commonly used as molecular biology reagents. On hemi-methylated DNAs type I enzymes like the EcoR124I restriction-modification complex act as conventional adenine methylases at their specific target sequences, but unmethylated targets induce them to translocate thousands of base pairs through the stationary enzyme before cleaving distant sites nonspecifically. EcoR124I is a superfamily 2 DEAD-box helicase like eukaryotic double-strand DNA translocase Rad54, with two RecA-like helicase domains and seven characteristic sequence motifs that are implicated in translocation. In Rad54 a so-called extended region adjacent to motif III is involved in ATPase activity. Although the EcoR124I extended region bears sequence and structural similarities with Rad54, it does not influence ATPase or restriction activity as shown in this work, but mutagenesis of the conserved glycine residue of its motif III does alter ATPase and DNA cleavage activity. Through the lens of molecular dynamics, a full model of HsdR of EcoR124I based on available crystal structures allowed interpretation of functional effects of mutants in motif III and its extended region. The results indicate that the conserved glycine residue of motif III has a role in positioning the two helicase domains.

• MeSH
• adenosintrifosfát chemie   MeSH
• aktivace enzymů MeSH
• analýza hlavních komponent MeSH
• DNA-helikasy chemie   genetika   metabolismus   MeSH
• hydrolýza MeSH
• interakční proteinové domény a motivy * MeSH
• konformace proteinů MeSH
• multienzymové komplexy chemie   MeSH
• mutace MeSH
• podjednotky proteinů chemie   genetika   metabolismus   MeSH
• restrikční endonukleasy typu I chemie   genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• simulace molekulární dynamiky MeSH
• Publikační typ
• časopisecké články MeSH

We describe the expression and purification of an active recombinant Zika virus RNA-dependent RNA polymerase (RdRp). Next, we present the development and optimization of an in vitro assay to measure its activity. We then applied the assay to selected triphosphate analogs and discovered that 2'-C-methylated nucleosides exhibit strong inhibitory activity. Surprisingly, also carbocyclic derivatives with the carbohydrate locked in a North-like conformation as well as a ribonucleotide with a South conformation exhibited strong activity. Our results suggest that the traditional 2'-C-methylated nucleosides pursued in the race for anti-HCV treatment can be superseded by brand new scaffolds in the case of the Zika virus.

• MeSH
• adenosintrifosfát analogy a deriváty   chemie   MeSH
• antivirové látky farmakologie   MeSH
• inhibitory enzymů farmakologie   MeSH
• lidé MeSH
• molekulární konformace MeSH
• nukleosidy chemie   farmakologie   MeSH
• objevování léků MeSH
• RNA-dependentní RNA-polymerasa antagonisté a inhibitory   genetika   izolace a purifikace   MeSH
• virus zika účinky léků   enzymologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

1. Purine cyclin-dependent kinase inhibitors have recently been recognised as promising candidates for the treatment of various cancers. While pharmacodynamic properties of these compounds are relatively well understood, their pharmacokinetics including possible interactions with placental transport systems have not been characterised to date. 2. In this study, we investigated transplacental passage of olomoucine II and purvalanol A in rat focusing on possible role of p-glycoprotein (ABCB1), breast cancer resistance protein (ABCG2) and/or multidrug resistance-associated proteins (ABCCs). Employing the in situ method of dually perfused rat term placenta, we demonstrate transplacental passage of both olomoucine II and purvalanol A against the concentration gradient in foetus-to-mother direction. Using several ATP-binding cassette (ABC) drug transporter inhibitors, we confirm the participation of ABCB1, ABCG2 and ABCCs transporters in the placental passage of olomoucine II, but not purvalanol A. 3. Transplacental passage of olomoucine II and purvalanol A from mother to foetus is significantly reduced by active transporters, restricting thereby foetal exposure and providing protection against harmful effects of these xenobiotics. Importantly, we demonstrate that in spite of their considerable structural similarity, the two molecules utilise distinct placental transport systems. These facts should be kept in mind when introducing these prospective anticancer candidates and/or their analogues into the clinical area.

• MeSH
• ABC transportéry metabolismus   MeSH
• adenosintrifosfát chemie   MeSH
• aktivní transport MeSH
• krysa rodu rattus MeSH
• matka - expozice noxám MeSH
• P-glykoproteiny metabolismus   MeSH
• placenta účinky léků   metabolismus   MeSH
• potkani Wistar MeSH
• proteiny spojené s mnohočetnou rezistencí k lékům metabolismus   MeSH
• puriny aplikace a dávkování   farmakokinetika   MeSH
• těhotenství u zvířat MeSH
• těhotenství MeSH
• trofoblasty účinky léků   MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• xenobiotika chemie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The allosteric influence of adenosine triphosphate (ATP) on the binding effectiveness of a series of peptide inhibitors with the catalytic subunit of 3'5'-cyclic adenosine monophosphate dependent protein kinase was investigated, and the dependence of this effect on peptide structure was analyzed. The allosteric effect was calculated as ratio of peptide binding effectiveness with the enzyme-ATP complex and with the free enzyme, quantified by the competitive inhibition of the enzyme in the presence of ATP excess, and by the enzyme-peptide complex denaturation assay, respectively It was found that the principle "better binding-stronger allostery" holds for interactions of the studied peptides with the enzyme, indicating that allostery and peptide binding with the free enzyme are governed by the same specificity pattern. This means that the allosteric regulation does not include new ligand-protein interactions, but changes the intensity (strength) of the interatomic forces that govern the complex formation in the case of each individual ligand. We propose that the allosteric regulation can be explained by the alteration of the intrinsic dynamics of the protein by ligand binding, and that this phenomenon, in turn, modulates the ligand off-rate from its binding site as well as the binding affinity. The positive allostery could therefore be induced by a reduction in the enzyme's overall intrinsic dynamics.

• MeSH
• 2-naftylamin analogy a deriváty   chemie   MeSH
• adenosintrifosfát chemie   metabolismus   MeSH
• alosterická regulace MeSH
• alosterické místo MeSH
• AMP cyklický chemie   metabolismus   MeSH
• barvení a značení metody   MeSH
• fluorescenční barviva chemie   MeSH
• inhibitory proteinkinas chemie   metabolismus   MeSH
• katalytická doména MeSH
• kinetika MeSH
• lidé MeSH
• ligandy MeSH
• peptidy chemie   metabolismus   MeSH
• proteinkinasy závislé na cyklickém AMP chemie   metabolismus   MeSH
• sekvence aminokyselin MeSH
• termodynamika MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH