Microtubules, built from heterodimers of α- and β-tubulins, control cell shape, mediate intracellular transport, and power cell division. The concentration of αβ-tubulins is tightly controlled through a posttranscriptional mechanism involving selective and regulated degradation of tubulin-encoding mRNAs. Degradation is initiated by TTC5, which recognizes tubulin-synthesizing ribosomes and recruits downstream effectors to trigger mRNA deadenylation. Here, we investigate how cells regulate TTC5 activity. Biochemical and structural proteomic approaches reveal that under normal conditions, soluble αβ-tubulins bind to and sequester TTC5, preventing it from engaging nascent tubulins at translating ribosomes. We identify the flexible C-terminal tail of TTC5 as a molecular switch, toggling between soluble αβ-tubulin-bound and nascent tubulin-bound states. Loss of sequestration by soluble αβ-tubulins constitutively activates TTC5, leading to diminished tubulin mRNA levels and compromised microtubule-dependent chromosome segregation during cell division. Our findings provide a paradigm for how cells regulate the activity of a specificity factor to adapt posttranscriptional regulation of gene expression to cellular needs.

• MeSH
• lidé MeSH
• messenger RNA * metabolismus   genetika   MeSH
• mikrotubuly * metabolismus   MeSH
• proteiny asociované s mikrotubuly metabolismus   genetika   MeSH
• ribozomy metabolismus   MeSH
• segregace chromozomů MeSH
• stabilita RNA * MeSH
• tubulin * metabolismus   genetika   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• messenger RNA * MeSH
• proteiny asociované s mikrotubuly MeSH
• tubulin * MeSH

The ATP-independent chaperone SurA protects unfolded outer membrane proteins (OMPs) from aggregation in the periplasm of Gram-negative bacteria, and delivers them to the β-barrel assembly machinery (BAM) for folding into the outer membrane (OM). Precisely how SurA recognises and binds its different OMP clients remains unclear. Escherichia coli SurA comprises three domains: a core and two PPIase domains (P1 and P2). Here, by combining methyl-TROSY NMR, single-molecule Förster resonance energy transfer (smFRET), and bioinformatics analyses we show that SurA client binding is mediated by two binding hotspots in the core and P1 domains. These interactions are driven by aromatic-rich motifs in the client proteins, leading to SurA core/P1 domain rearrangements and expansion of clients from collapsed, non-native states. We demonstrate that the core domain is key to OMP expansion by SurA, and uncover a role for SurA PPIase domains in limiting the extent of expansion. The results reveal insights into SurA-OMP recognition and the mechanism of activation for an ATP-independent chaperone, and suggest a route to targeting the functions of a chaperone key to bacterial virulence and OM integrity.

• MeSH
• ABC transportéry metabolismus   chemie   genetika   MeSH
• adenosintrifosfát metabolismus   MeSH
• Escherichia coli * metabolismus   genetika   MeSH
• molekulární chaperony * metabolismus   MeSH
• molekulární modely MeSH
• peptidylprolylisomerasa * metabolismus   genetika   MeSH
• proteinové domény MeSH
• proteiny vnější bakteriální membrány metabolismus   genetika   chemie   MeSH
• proteiny z Escherichia coli * metabolismus   genetika   chemie   MeSH
• rezonanční přenos fluorescenční energie MeSH
• sbalování proteinů MeSH
• transportní proteiny * metabolismus   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• ABC transportéry MeSH
• adenosintrifosfát MeSH
• molekulární chaperony * MeSH
• peptidylprolylisomerasa * MeSH
• proteiny vnější bakteriální membrány MeSH
• proteiny z Escherichia coli * MeSH
• SurA protein, E coli MeSH Prohlížeč
• transportní proteiny * MeSH

Robust oxygenic photosynthesis requires a suite of accessory factors to ensure efficient assembly and repair of the oxygen-evolving photosystem two (PSII) complex. The highly conserved Ycf48 assembly factor binds to the newly synthesized D1 reaction center polypeptide and promotes the initial steps of PSII assembly, but its binding site is unclear. Here we use cryo-electron microscopy to determine the structure of a cyanobacterial PSII D1/D2 reaction center assembly complex with Ycf48 attached. Ycf48, a 7-bladed beta propeller, binds to the amino-acid residues of D1 that ultimately ligate the water-oxidising Mn4CaO5 cluster, thereby preventing the premature binding of Mn2+ and Ca2+ ions and protecting the site from damage. Interactions with D2 help explain how Ycf48 promotes assembly of the D1/D2 complex. Overall, our work provides valuable insights into the early stages of PSII assembly and the structural changes that create the binding site for the Mn4CaO5 cluster.

• MeSH
• elektronová kryomikroskopie MeSH
• fotosystém II (proteinový komplex) * metabolismus   MeSH
• kyslík metabolismus   MeSH
• mangan metabolismus   MeSH
• sinice * metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fotosystém II (proteinový komplex) * MeSH
• kyslík MeSH
• mangan MeSH

Plant precursor miRNAs (pre-miRNA) have conserved evolutionary footprints that correlate with mode of miRNA biogenesis. In plants, base to loop and loop to base modes of biogenesis have been reported. Conserved structural element(s) in pre-miRNA play a major role in turn over and abundance of mature miRNA. Pre-miR396c sequences and secondary structural characteristics across Oryza species are presented. Based on secondary structure, twelve Oryza pre-miR396c sequences are divided into three groups, with the precursor from halophytic Oryza coarctata forming a distinct group. The miRNA-miRNA* duplex region is completely conserved across eleven Oryza species as are other structural elements in the pre-miRNA, suggestive of an evolutionarily conserved base-to-loop mode of miRNA biogenesis. SNPs within O. coarctata mature miR396c sequence and miRNA* region have the potential to alter target specificity and association with the RNA-induced silencing complex. A conserved SNP variation, rs10234287911 (G/A), identified in O. sativa pre-miR396c sequences alters base pairing above the miRNA-miRNA* duplex. The more stable structure conferred by the 'A10234287911' allele may promote better processing vis-à-vis the structure conferred by 'G10234287911' allele. We also examine pri- and pre-miR396c expression in cultivated rice under heat and salinity and their correlation with miR396c expression.

• MeSH
• halotolerantní rostliny genetika   MeSH
• jednonukleotidový polymorfismus MeSH
• mikro RNA * genetika   MeSH
• rýže (rod) * genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mikro RNA * MeSH

Enterovirus 70 (EV70) is a human pathogen belonging to the family Picornaviridae. EV70 is transmitted by eye secretions and causes acute hemorrhagic conjunctivitis, a serious eye disease. Despite the severity of the disease caused by EV70, its structure is unknown. Here, we present the structures of the EV70 virion, altered particle, and empty capsid determined by cryo-electron microscopy. The capsid of EV70 is composed of the subunits VP1, VP2, VP3, and VP4. The partially collapsed hydrophobic pocket located in VP1 of the EV70 virion is not occupied by a pocket factor, which is commonly present in other enteroviruses. Nevertheless, we show that the pocket can be targeted by the antiviral compounds WIN51711 and pleconaril, which block virus infection. The inhibitors prevent genome release by stabilizing EV70 particles. Knowledge of the structures of complexes of EV70 with inhibitors will enable the development of capsid-binding therapeutics against this virus. IMPORTANCE Globally distributed enterovirus 70 (EV70) causes local outbreaks of acute hemorrhagic conjunctivitis. The discharge from infected eyes enables the high-efficiency transmission of EV70 in overcrowded areas with low hygienic standards. Currently, only symptomatic treatments are available. We determined the structures of EV70 in its native form, the genome release intermediate, and the empty capsid resulting from genome release. Furthermore, we elucidated the structures of EV70 in complex with two inhibitors that block virus infection, and we describe the mechanism of their binding to the virus capsid. These results enable the development of therapeutics against EV70.

• Klíčová slova
• Picornavirales, Picornaviridae, acute hemorrhagic conjunctivitis, antiviral, canyon, capsid, enterovirus, human, inhibitor, jelly roll, protein, structure, virion, virion structure, virus,
• MeSH
• akutní hemoragická konjunktivitida virologie   MeSH
• antivirové látky * farmakologie   MeSH
• elektronová kryomikroskopie MeSH
• kapsida * ultrastruktura   MeSH
• lidé MeSH
• lidský enterovirus D * účinky léků   ultrastruktura   MeSH
• oxadiazoly farmakologie   MeSH
• oxazoly farmakologie   MeSH
• virion účinky léků   ultrastruktura   MeSH
• virové plášťové proteiny MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antivirové látky * MeSH
• oxadiazoly MeSH
• oxazoly MeSH
• pleconaril MeSH Prohlížeč
• virové plášťové proteiny MeSH

Nitric oxide (NO) produced by mammalian nitric oxide synthases (mNOSs) is an important mediator in a variety of physiological functions. Crystal structures of mNOSs have shown strong conservation of the active-site residue Val567 (numbering for rat neuronal NOS, nNOS). NOS-like proteins have been identified in several bacterial pathogens, and these display striking sequence identity to the oxygenase domain of mNOS (NOSoxy), with the exception of a Val to Ile mutation at the active site. Preliminary studies have highlighted the importance of this Val residue in NO-binding, substrate recognition, and oxidation in mNOSs. To further elucidate the role of this valine in substrate and substrate analogue recognition, we generated five Val567 mutants of the oxygenase domain of the neuronal NOS (nNOSoxy) and used UV-visible and EPR spectroscopy to investigate the effects of these mutations on the heme distal environment, the stability of the heme-FeII-CO complexes, and the binding of a series of substrate analogues. Our results are consistent with Val567 playing an important role in preserving the integrity of the active site for substrate binding, stability of heme-bound gaseous ligands, and potential NO production.

• Klíčová slova
• UV‐visible spectroscopy, active‐site mutations, heme, nitric oxide, nitric oxide synthase, substrate analogues binding,
• Publikační typ
• časopisecké články MeSH

Tick-borne encephalitis virus (TBEV) causes 13,000 cases of human meningitis and encephalitis annually. However, the structure of the TBEV virion and its interactions with antibodies are unknown. Here, we present cryo-EM structures of the native TBEV virion and its complex with Fab fragments of neutralizing antibody 19/1786. Flavivirus genome delivery depends on membrane fusion that is triggered at low pH. The virion structure indicates that the repulsive interactions of histidine side chains, which become protonated at low pH, may contribute to the disruption of heterotetramers of the TBEV envelope and membrane proteins and induce detachment of the envelope protein ectodomains from the virus membrane. The Fab fragments bind to 120 out of the 180 envelope glycoproteins of the TBEV virion. Unlike most of the previously studied flavivirus-neutralizing antibodies, the Fab fragments do not lock the E-proteins in the native-like arrangement, but interfere with the process of virus-induced membrane fusion.

• MeSH
• elektronová kryomikroskopie MeSH
• exprese genu MeSH
• fúze membrán genetika   MeSH
• imunoglobuliny - Fab fragmenty biosyntéza   chemie   MeSH
• internalizace viru MeSH
• koncentrace vodíkových iontů MeSH
• lidé MeSH
• multimerizace proteinu MeSH
• nádorové buněčné linie MeSH
• neurony patologie   virologie   MeSH
• neutralizující protilátky biosyntéza   chemie   MeSH
• proteinové domény MeSH
• protilátky virové biosyntéza   chemie   MeSH
• virion genetika   metabolismus   ultrastruktura   MeSH
• virové proteiny chemie   genetika   metabolismus   MeSH
• viry klíšťové encefalitidy genetika   metabolismus   ultrastruktura   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• imunoglobuliny - Fab fragmenty MeSH
• neutralizující protilátky MeSH
• protilátky virové MeSH
• virové proteiny MeSH

There is great interest in increasing proteins' stability to enhance their utility as biocatalysts, therapeutics, diagnostics and nanomaterials. Directed evolution is a powerful, but experimentally strenuous approach. Computational methods offer attractive alternatives. However, due to the limited reliability of predictions and potentially antagonistic effects of substitutions, only single-point mutations are usually predicted in silico, experimentally verified and then recombined in multiple-point mutants. Thus, substantial screening is still required. Here we present FireProt, a robust computational strategy for predicting highly stable multiple-point mutants that combines energy- and evolution-based approaches with smart filtering to identify additive stabilizing mutations. FireProt's reliability and applicability was demonstrated by validating its predictions against 656 mutations from the ProTherm database. We demonstrate that thermostability of the model enzymes haloalkane dehalogenase DhaA and γ-hexachlorocyclohexane dehydrochlorinase LinA can be substantially increased (ΔTm = 24°C and 21°C) by constructing and characterizing only a handful of multiple-point mutants. FireProt can be applied to any protein for which a tertiary structure and homologous sequences are available, and will facilitate the rapid development of robust proteins for biomedical and biotechnological applications.

• MeSH
• bodová mutace genetika   fyziologie   MeSH
• databáze genetické MeSH
• lyasy chemie   genetika   metabolismus   MeSH
• molekulární modely MeSH
• počítačová simulace MeSH
• proteinové inženýrství metody   MeSH
• stabilita enzymů genetika   MeSH
• teplota MeSH
• výpočetní biologie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• lyasy MeSH

In Bacillus subtilis, the arabinose repressor AraR negatively controls the expression of genes in the metabolic pathway of arabinose-containing polysaccharides. The protein is composed of two domains of different phylogenetic origin and function: an N-terminal DNA-binding domain belonging to the GntR family and a C-terminal effector-binding domain that shows similarity to members of the GalR/LacI family. The crystal structure of the C-terminal effector-binding domain of AraR in complex with the effector L-arabinose has been determined at 2.2 Å resolution. The L-arabinose binding affinity was characterized by isothermal titration calorimetry and differential scanning fluorimetry; the K(d) value was 8.4 ± 0.4 µM. The effect of L-arabinose on the protein oligomeric state was investigated in solution and detailed analysis of the crystal identified a dimer organization which is distinctive from that of other members of the GalR/LacI family.

• MeSH
• arabinosa metabolismus   MeSH
• Bacillus subtilis chemie   metabolismus   MeSH
• bakteriální proteiny chemie   metabolismus   MeSH
• krystalografie rentgenová MeSH
• molekulární modely MeSH
• represorové proteiny chemie   metabolismus   MeSH
• terciární struktura proteinů MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• arabinosa MeSH
• bakteriální proteiny MeSH
• represorové proteiny MeSH

HotSpot Wizard is a web server for automatic identification of 'hot spots' for engineering of substrate specificity, activity or enantioselectivity of enzymes and for annotation of protein structures. The web server implements the protein engineering protocol, which targets evolutionarily variable amino acid positions located in the active site or lining the access tunnels. The 'hot spots' for mutagenesis are selected through the integration of structural, functional and evolutionary information obtained from: (i) the databases RCSB PDB, UniProt, PDBSWS, Catalytic Site Atlas and nr NCBI and (ii) the tools CASTp, CAVER, BLAST, CD-HIT, MUSCLE and Rate4Site. The protein structure and e-mail address are the only obligatory inputs for the calculation. In the output, HotSpot Wizard lists annotated residues ordered by estimated mutability. The results of the analysis are mapped on the enzyme structure and visualized in the web browser using Jmol. The HotSpot Wizard server should be useful for protein engineers interested in exploring the structure of their favourite protein and for the design of mutations in site-directed mutagenesis and focused directed evolution experiments. HotSpot Wizard is available at http://loschmidt.chemi.muni.cz/hotspotwizard/.

• MeSH
• beta-laktamasy chemie   MeSH
• glykosidhydrolasy chemie   MeSH
• hydrolasy triesterů kyseliny fosforečné chemie   MeSH
• hydrolasy chemie   MeSH
• internet MeSH
• proteinové inženýrství * MeSH
• reprodukovatelnost výsledků MeSH
• software * MeSH
• uživatelské rozhraní počítače MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• beta-laktamasy MeSH
• glykosidhydrolasy MeSH
• haloalkane dehalogenase MeSH Prohlížeč
• hydrolasy triesterů kyseliny fosforečné MeSH
• hydrolasy MeSH
• licheninase MeSH Prohlížeč