Influenza A virus (IAV) encodes a polymerase composed of three subunits: PA, with endonuclease activity, PB1 with polymerase activity and PB2 with host RNA five-prime cap binding site. Their cooperation and stepwise activation include a process called cap-snatching, which is a crucial step in the IAV life cycle. Reproduction of IAV can be blocked by disrupting the interaction between the PB2 domain and the five-prime cap. An inhibitor of this interaction called pimodivir (VX-787) recently entered the third phase of clinical trial; however, several mutations in PB2 that cause resistance to pimodivir were observed. First major mutation, F404Y, causing resistance was identified during preclinical testing, next the mutation M431I was identified in patients during the second phase of clinical trials. The mutation H357N was identified during testing of IAV strains at Centers for Disease Control and Prevention. We set out to provide a structural and thermodynamic analysis of the interactions between cap-binding domain of PB2 wild-type and PB2 variants bearing these mutations and pimodivir. Here we present four crystal structures of PB2-WT, PB2-F404Y, PB2-M431I and PB2-H357N in complex with pimodivir. We have thermodynamically analysed all PB2 variants and proposed the effect of these mutations on thermodynamic parameters of these interactions and pimodivir resistance development. These data will contribute to understanding the effect of these missense mutations to the resistance development and help to design next generation inhibitors.
- MeSH
- krystalografie rentgenová MeSH
- kvantová teorie MeSH
- molekulární modely MeSH
- mutace genetika MeSH
- mutantní proteiny metabolismus MeSH
- podjednotky proteinů antagonisté a inhibitory chemie metabolismus MeSH
- proteinové domény MeSH
- pyridiny chemie farmakologie MeSH
- pyrimidiny chemie farmakologie MeSH
- pyrroly chemie farmakologie MeSH
- RNA-dependentní RNA-polymerasa antagonisté a inhibitory chemie metabolismus MeSH
- termodynamika MeSH
- virová léková rezistence účinky léků MeSH
- virové proteiny antagonisté a inhibitory chemie metabolismus MeSH
- virus chřipky A účinky léků enzymologie MeSH
- Publikační typ
- časopisecké články MeSH
N-methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors that play an essential role in mediating excitatory neurotransmission in the mammalian central nervous system (CNS). Functional NMDARs are tetramers composed of GluN1, GluN2A-D, and/or GluN3A-B subunits, giving rise to a wide variety of NMDAR subtypes with unique functional properties. Here, we examined the surface delivery and functional properties of NMDARs containing mutations in the glycine-binding sites in GluN1 and GluN3A subunits expressed in mammalian cell lines and primary rat hippocampal neurons. We found that the structural features of the glycine-binding sites in both GluN1 and GluN3A subunits are correlated with receptor forward trafficking to the cell surface. In addition, we found that a potentially clinically relevant mutation in the glycine-binding site of the human GluN3A subunit significantly reduces surface delivery of NMDARs. Taken together, these findings provide novel insight into how NMDARs are regulated by their glycine-binding sites and may provide important information regarding the role of NMDARs in both physiological and pathophysiological processes in the mammalian CNS.
- MeSH
- buněčná membrána metabolismus MeSH
- Cercopithecus aethiops MeSH
- COS buňky MeSH
- glycin metabolismus MeSH
- HEK293 buňky MeSH
- hipokampus cytologie MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- membránové glykoproteiny chemie metabolismus MeSH
- mutace genetika MeSH
- neurony metabolismus MeSH
- podjednotky proteinů chemie metabolismus MeSH
- proteinové domény MeSH
- receptory N-methyl-D-aspartátu chemie metabolismus MeSH
- sekvence aminokyselin MeSH
- vazebná místa MeSH
- vztahy mezi strukturou a aktivitou MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The vanilloid transient receptor potential channel TRPV3 is a putative molecular thermosensor widely considered to be involved in cutaneous sensation, skin homeostasis, nociception, and pruritus. Repeated stimulation of TRPV3 by high temperatures above 50 °C progressively increases its responses and shifts the activation threshold to physiological temperatures. This use-dependence does not occur in the related heat-sensitive TRPV1 channel in which responses decrease, and the activation threshold is retained above 40 °C during activations. By combining structure-based mutagenesis, electrophysiology, and molecular modeling, we showed that chimeric replacement of the residues from the TRPV3 cytoplasmic inter-subunit interface (N251-E257) with the homologous residues of TRPV1 resulted in channels that, similarly to TRPV1, exhibited a lowered thermal threshold, were sensitized, and failed to close completely after intense stimulation. Crosslinking of this interface by the engineered disulfide bridge between substituted cysteines F259C and V385C (or, to a lesser extent, Y382C) locked the channel in an open state. On the other hand, mutation of a single residue within this region (E736) resulted in heat resistant channels. We propose that alterations in the cytoplasmic inter-subunit interface produce shifts in the channel gating equilibrium and that this domain is critical for the use-dependence of the heat sensitivity of TRPV3.
- MeSH
- cytoplazma metabolismus MeSH
- HEK293 buňky MeSH
- kationtové kanály TRPV chemie genetika metabolismus MeSH
- lidé MeSH
- mutace MeSH
- podjednotky proteinů chemie genetika metabolismus MeSH
- proteinové domény MeSH
- simulace molekulární dynamiky MeSH
- vysoká teplota MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Although EcoR124 is one of the better-studied Type I restriction-modification enzymes, it still presents many challenges to detailed analyses because of its structural and functional complexity and missing structural information. In all available structures of its motor subunit HsdR, responsible for DNA translocation and cleavage, a large part of the HsdR C terminus remains unresolved. The crystal structure of the C terminus of HsdR, obtained with a crystallization chaperone in the form of pHluorin fusion and refined to 2.45 Å, revealed that this part of the protein forms an independent domain with its own hydrophobic core and displays a unique α-helical fold. The full-length HsdR model, based on the WT structure and the C-terminal domain determined here, disclosed a proposed DNA-binding groove lined by positively charged residues. In vivo and in vitro assays with a C-terminal deletion mutant of HsdR supported the idea that this domain is involved in complex assembly and DNA binding. Conserved residues identified through sequence analysis of the C-terminal domain may play a key role in protein-protein and protein-DNA interactions. We conclude that the motor subunit of EcoR124 comprises five structural and functional domains, with the fifth, the C-terminal domain, revealing a unique fold characterized by four conserved motifs in the IC subfamily of Type I restriction-modification systems. In summary, the structural and biochemical results reported here support a model in which the C-terminal domain of the motor subunit HsdR of the endonuclease EcoR124 is involved in complex assembly and DNA binding.
- MeSH
- biofyzikální jevy MeSH
- DNA vazebné proteiny chemie genetika MeSH
- Escherichia coli chemie genetika MeSH
- konformace proteinů MeSH
- krystalografie rentgenová MeSH
- multiproteinové komplexy chemie genetika MeSH
- podjednotky proteinů chemie genetika MeSH
- proteinové domény genetika MeSH
- proteiny z Escherichia coli chemie genetika MeSH
- restrikční endonukleasy typu I chemie genetika MeSH
- sekvence aminokyselin 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
Activating mutations in NT5C2, a gene encoding cytosolic purine 5'-nucleotidase (cN-II), confer chemoresistance in relapsed acute lymphoblastic leukemia. Here we show that all mutants became independent of allosteric effects of ATP and thus constitutively active. Structural mapping of mutations described in patients demonstrates that 90% of leukemia-specific allelles directly affect two regulatory hotspots within the cN-II molecule-the helix A region: residues 355-365, and the intersubunit interface: helix B (232-242) and flexible interhelical loop L (400-418). Furthermore, analysis of hetero-oligomeric complexes combining wild-type (WT) and mutant subunits showed that the activation is transmitted from the mutated to the WT subunit. This intersubunit interaction forms structural basis of hyperactive NT5C2 in drug-resistant leukemia in which heterozygous NT5C2 mutation gave rise to hetero-tetramer mutant and WT proteins. This enabled us to define criteria to aid the prediction of NT5C2 drug resistance mutations in leukemia.
- MeSH
- 5'-nukleotidasa chemie genetika MeSH
- akutní lymfatická leukemie farmakoterapie genetika MeSH
- chemorezistence MeSH
- konformace proteinů MeSH
- lidé MeSH
- mutace * MeSH
- podjednotky proteinů chemie MeSH
- recidiva MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Biogenesis of the plant secondary cell wall involves many important aspects, such as phenolic compound deposition and often silica encrustation. Previously, we demonstrated the importance of the exocyst subunit EXO70H4 for biogenesis of the trichome secondary cell wall, namely for deposition of the autofluorescent and callose-rich cell wall layer. Here, we reveal that EXO70H4-driven cell wall biogenesis is constitutively active in the mature trichome, but also can be activated elsewhere upon pathogen attack, giving this study a broader significance with an overlap into phytopathology. To address the specificity of EXO70H4 among the EXO70 family, we complemented the exo70H4-1 mutant by 18 different Arabidopsis (Arabidopsis thaliana) EXO70 paralogs subcloned under the EXO70H4 promoter. Only EXO70H4 had the capacity to rescue the exo70H4-1 trichome phenotype. Callose deposition phenotype of exo70H4-1 mutant is caused by impaired secretion of PMR4, a callose synthase responsible for the synthesis of callose in the trichome. PMR4 colocalizes with EXO70H4 on plasma membrane microdomains that do not develop in the exo70H4-1 mutant. Using energy-dispersive x-ray microanalysis, we show that both EXO70H4- and PMR4-dependent callose deposition in the trichome are essential for cell wall silicification.
- MeSH
- Arabidopsis účinky léků genetika metabolismus MeSH
- buněčná membrána účinky léků metabolismus MeSH
- buněčná stěna účinky léků metabolismus MeSH
- epidermis rostlin cytologie účinky léků metabolismus MeSH
- fenotyp MeSH
- flagelin farmakologie MeSH
- glukany MeSH
- glukosyltransferasy metabolismus MeSH
- mutace genetika MeSH
- oxid křemičitý metabolismus MeSH
- podjednotky proteinů chemie metabolismus MeSH
- proteinové domény MeSH
- proteiny huseníčku chemie metabolismus MeSH
- regulace genové exprese u rostlin účinky léků MeSH
- trichomy metabolismus MeSH
- upregulace účinky léků MeSH
- vezikulární transportní proteiny chemie metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Accumulating evidence indicates that hypertension is associated with "ion channel remodeling" of vascular smooth muscle cells (VSMCs). The objective of this study was to determine the effects of exercise intensity/volume on hypertension-associated changes in large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels in mesenteric arteries (MAs) from spontaneously hypertensive rats (SHR). Male SHRs were randomly assigned to three groups: a low-intensity aerobic exercise group (SHR-L: 14 m/min), a moderate-intensity aerobic exercise group (SHR-M: 20 m/min), and a sedentary group (SHR). Age-matched Wistar-Kyoto rats (WKYs) were used as normotensive controls. Exercise groups completed an 8-week exercise program. Elevation of the alpha and beta1 proteins was unequal in MA myocytes from SHRs, with the beta1 subunit increasing more than the alpha subunit. BK(Ca) contribution to vascular tone regulation was higher in the myocytes and arteries of SHRs compared to WKYs. SHR BK(Ca) channel subunit protein expression, beta1/alpha ratio, whole cell current density and single-channel open probability was also increased compared with WKYs. Aerobic exercise lowered systemic blood pressure and normalized hypertension-associated BK(Ca) alterations to normotensive control levels in the SHRs. These effects were more pronounced in the moderate-intensity group than in the low-intensity group. There is a dose-effect for aerobic exercise training in the range of low to moderate-intensity and accompanying volume for the correction of the pathological adaptation of BK(Ca) channels in myocytes of MAs from SHR.
- MeSH
- aerobióza MeSH
- arteriae mesentericae patofyziologie MeSH
- gating iontového kanálu MeSH
- hypertenze patofyziologie terapie MeSH
- kondiční příprava zvířat * MeSH
- krysa rodu rattus MeSH
- podjednotky proteinů chemie metabolismus MeSH
- potkani inbrední SHR MeSH
- potkani inbrední WKY MeSH
- regulace genové exprese MeSH
- tělesná námaha MeSH
- terapie cvičením metody MeSH
- vápníkem aktivované draslíkové kanály s vysokou vodivostí chemie metabolismus MeSH
- vápníková signalizace * MeSH
- výsledek terapie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Protein synthesis is mediated via numerous molecules including the ribosome, mRNA, tRNAs, as well as translation initiation, elongation and release factors. Some of these factors play several roles throughout the entire process to ensure proper assembly of the preinitiation complex on the right mRNA, accurate selection of the initiation codon, errorless production of the encoded polypeptide and its proper termination. Perhaps, the most intriguing of these multitasking factors is the eukaryotic initiation factor eIF3. Recent evidence strongly suggests that this factor, which coordinates the progress of most of the initiation steps, does not come off the initiation complex upon subunit joining, but instead it remains bound to 80S ribosomes and gradually falls off during the first few elongation cycles to: (1) promote resumption of scanning on the same mRNA molecule for reinitiation downstream-in case of translation of upstream ORFs short enough to preserve eIF3 bound; or (2) come back during termination on long ORFs to fine tune its fidelity or, if signaled, promote programmed stop codon readthrough. Here, we unite recent structural views of the eIF3-40S complex and discus all known eIF3 roles to provide a broad picture of the eIF3's impact on translational control in eukaryotic cells.
- MeSH
- eukaryotický iniciační faktor 3 chemie genetika metabolismus MeSH
- konformace proteinů * MeSH
- lidé MeSH
- messenger RNA genetika metabolismus MeSH
- molekulární modely MeSH
- podjednotky proteinů chemie genetika metabolismus MeSH
- proteosyntéza * MeSH
- ribozomy genetika metabolismus MeSH
- Saccharomyces cerevisiae - proteiny chemie genetika metabolismus MeSH
- vazba proteinů MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
High pressure methods have become a useful tool for studying protein structure and stability. Using them, various physico-chemical processes including protein unfolding, aggregation, oligomer dissociation or enzyme-activity decrease were studied on many different proteins. Oligomeric protein dissociation is a process that can perfectly utilize the potential of high-pressure techniques, as the high pressure shifts the equilibria to higher concentrations making them better observable by spectroscopic methods. This can be especially useful when the oligomeric form is highly stable at atmospheric pressure. These applications may be, however, hindered by less intensive experimental response as well as interference of the oligomerization equilibria with unfolding or aggregation of the subunits, but also by more complex theoretical description. In this study we develop mathematical models describing different kinds of oligomerization equilibria, both closed (equilibrium of monomer and the highest possible oligomer without any intermediates) and consecutive. Closed homooligomer equilibria are discussed for any oligomerization degree, while the more complex heterooligomer equilibria and the consecutive equilibria in both homo- and heterooligomers are taken into account only for dimers and trimers. In all the cases, fractions of all the relevant forms are evaluated as functions of pressure and concentration. Significant points (inflection points and extremes) of the resulting transition curves, that can be determined experimentally, are evaluated as functions of pressure and/or concentration. These functions can be further used in order to evaluate the thermodynamic parameters of the system, i.e. atmospheric-pressure equilibrium constants and volume changes of the individual steps of the oligomer-dissociation processes.
