Siderophore-mediated iron acquisition is essential for the virulence of Aspergillus fumigatus, a fungus causing life-threatening aspergillosis. Drugs targeting the siderophore biosynthetic pathway could help improve disease management. The transacetylases SidF and SidL generate intermediates for different siderophores in A. fumigatus. A. fumigatus has a yet unidentified transacetylase that complements SidL during iron deficiency in SidL-lacking mutants. We present the first X-ray structure of SidF, revealing a two-domain architecture with tetrameric assembly. The N-terminal domain contributes to protein solubility and oligomerization, while the C-terminal domain containing the GCN5-related N-acetyltransferase (GNAT) motif is crucial for the enzymatic activity and mediates oligomer formation. Notably, AlphaFold modelling demonstrates structural similarity between SidF and SidL. Enzymatic assays showed that SidF can utilize acetyl-CoA as a donor, previously thought to be a substrate of SidL but not SidF, and selectively uses N5-hydroxy-L-ornithine as an acceptor. This study elucidates the structure of SidF and reveals its role in siderophore biosynthesis. We propose SidF as the unknown transacetylase complementing SidL activity, highlighting its central role in A. fumigatus siderophore biosynthesis. Investigation of this uncharacterized GNAT protein enhances our understanding of fungal virulence and holds promise for its potential application in developing antifungal therapies.

• Klíčová slova
• GNATs, SAXS, SidF, Siderophore, X-ray crystallography,
• Publikační typ
• časopisecké články MeSH

Enteric bacteria have to adapt to environmental stresses in the human gastrointestinal tract such as acid and nutrient stress, oxygen limitation and exposure to antibiotics. Membrane lipid composition has recently emerged as a key factor for stress adaptation. The E. coli ravA-viaA operon is essential for aminoglycoside bactericidal activity under anaerobiosis but its mechanism of action is unclear. Here we characterise the VWA domain-protein ViaA and its interaction with the AAA+ ATPase RavA, and find that both proteins localise at the inner cell membrane. We demonstrate that RavA and ViaA target specific phospholipids and subsequently identify their lipid-binding sites. We further show that mutations abolishing interaction with lipids restore induced changes in cell membrane morphology and lipid composition. Finally we reveal that these mutations render E. coli gentamicin-resistant under fumarate respiration conditions. Our work thus uncovers a ravA-viaA-based pathway which is mobilised in response to aminoglycosides under anaerobiosis and engaged in cell membrane regulation.

• MeSH
• adenosintrifosfatasy * metabolismus   MeSH
• aminoglykosidy * farmakologie   MeSH
• antibakteriální látky farmakologie   MeSH
• ATPázy spojené s různými buněčnými aktivitami metabolismus   MeSH
• Escherichia coli * účinky léků   enzymologie   MeSH
• fosfolipidy MeSH
• fumaráty MeSH
• gentamiciny MeSH
• kyslík metabolismus   MeSH
• membránové lipidy MeSH
• proteiny z Escherichia coli * metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosintrifosfatasy * MeSH
• aminoglykosidy * MeSH
• antibakteriální látky MeSH
• ATPázy spojené s různými buněčnými aktivitami MeSH
• fosfolipidy MeSH
• fumaráty MeSH
• gentamiciny MeSH
• kyslík MeSH
• membránové lipidy MeSH
• proteiny z Escherichia coli * MeSH
• RavA protein, E coli MeSH Prohlížeč
• ViaA protein, E coli MeSH Prohlížeč

Signaling by the human C-type lectin-like receptor, natural killer (NK) cell inhibitory receptor NKR-P1, has a critical role in many immune-related diseases and cancer. C-type lectin-like receptors have weak affinities to their ligands; therefore, setting up a comprehensive model of NKR-P1-LLT1 interactions that considers the natural state of the receptor on the cell surface is necessary to understand its functions. Here we report the crystal structures of the NKR-P1 and NKR-P1:LLT1 complexes, which provides evidence that NKR-P1 forms homodimers in an unexpected arrangement to enable LLT1 binding in two modes, bridging two LLT1 molecules. These interaction clusters are suggestive of an inhibitory immune synapse. By observing the formation of these clusters in solution using SEC-SAXS analysis, by dSTORM super-resolution microscopy on the cell surface, and by following their role in receptor signaling with freshly isolated NK cells, we show that only the ligation of both LLT1 binding interfaces leads to effective NKR-P1 inhibitory signaling. In summary, our findings collectively support a model of NKR-P1:LLT1 clustering, which allows the interacting proteins to overcome weak ligand-receptor affinity and to trigger signal transduction upon cellular contact in the immune synapse.

• MeSH
• antigeny povrchové MeSH
• buňky NK * MeSH
• difrakce rentgenového záření MeSH
• lektinové receptory NK-buněk - podrodina B MeSH
• lektiny typu C MeSH
• lidé MeSH
• ligandy MeSH
• maloúhlový rozptyl MeSH
• receptory buněčného povrchu * MeSH
• shluková analýza MeSH
• synapse MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny povrchové MeSH
• lektinové receptory NK-buněk - podrodina B MeSH
• lektiny typu C MeSH
• ligandy MeSH
• receptory buněčného povrchu * MeSH

Transcription elongation factor Spt6 associates with RNA polymerase II (Pol II) and acts as a histone chaperone, which promotes the reassembly of nucleosomes following the passage of Pol II. The precise mechanism of nucleosome reassembly mediated by Spt6 remains unclear. In this study, we used a hybrid approach combining cryo-electron microscopy and small-angle X-ray scattering to visualize the architecture of Spt6 from Saccharomyces cerevisiae. The reconstructed overall architecture of Spt6 reveals not only the core of Spt6, but also its flexible N- and C-termini, which are critical for Spt6's function. We found that the acidic N-terminal region of Spt6 prevents the binding of Spt6 not only to the Pol II CTD and Pol II CTD-linker, but also to pre-formed intact nucleosomes and nucleosomal DNA. The N-terminal region of Spt6 self-associates with the tSH2 domain and the core of Spt6 and thus controls binding to Pol II and nucleosomes. Furthermore, we found that Spt6 promotes the assembly of nucleosomes in vitro. These data indicate that the cooperation between the intrinsically disordered and structured regions of Spt6 regulates nucleosome and Pol II CTD binding, and also nucleosome assembly.

• MeSH
• elektronová kryomikroskopie MeSH
• genetická transkripce MeSH
• histonové chaperony genetika   metabolismus   MeSH
• nukleozomy * genetika   metabolismus   MeSH
• RNA-polymerasa II metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny * metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• transkripční elongační faktory metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• histonové chaperony MeSH
• nukleozomy * MeSH
• RNA-polymerasa II MeSH
• Saccharomyces cerevisiae - proteiny * MeSH
• SPT6 protein, S cerevisiae MeSH Prohlížeč
• transkripční elongační faktory MeSH

Expansion of the polyglutamine tract in the N terminus of Ataxin-1 is the main cause of the neurodegenerative disease, spinocerebellar ataxia type 1 (SCA1). However, the C-terminal part of the protein - including its AXH domain and a phosphorylation on residue serine 776 - also plays a crucial role in disease development. This phosphorylation event is known to be crucial for the interaction of Ataxin-1 with the 14-3-3 adaptor proteins and has been shown to indirectly contribute to Ataxin-1 stability. Here we show that 14-3-3 also has a direct anti-aggregation or "chaperone" effect on Ataxin-1. Furthermore, we provide structural and biophysical information revealing how phosphorylated S776 in the intrinsically disordered C terminus of Ataxin-1 mediates the cytoplasmic interaction with 14-3-3 proteins. Based on these findings, we propose that 14-3-3 exerts the observed chaperone effect by interfering with Ataxin-1 dimerization through its AXH domain, reducing further self-association. The chaperone effect is particularly important in the context of SCA1, as it was previously shown that a soluble form of mutant Ataxin-1 is the major driver of pathology.

• Klíčová slova
• HDX-MS, SAXS, crystal structure, neurodegeneration, protein aggregation,
• MeSH
• ataxin-1 chemie   metabolismus   MeSH
• buněčné linie MeSH
• cytoplazma metabolismus   MeSH
• fosforylace MeSH
• HEK293 buňky MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• multimerizace proteinu MeSH
• proteinové domény MeSH
• proteiny 14-3-3 metabolismus   MeSH
• stabilita proteinů MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ataxin-1 MeSH
• ATXN1 protein, human MeSH Prohlížeč
• proteiny 14-3-3 MeSH

Death-associated protein kinase 2 (DAPK2) is a CaM-regulated Ser/Thr protein kinase, involved in apoptosis, autophagy, granulocyte differentiation and motility regulation, whose activity is controlled by autoinhibition, autophosphorylation, dimerization and interaction with scaffolding proteins 14-3-3. However, the structural basis of 14-3-3-mediated DAPK2 regulation remains unclear. Here, we structurally and biochemically characterize the full-length human DAPK2:14-3-3 complex by combining several biophysical techniques. The results from our X-ray crystallographic analysis revealed that Thr369 phosphorylation at the DAPK2 C terminus creates a high-affinity canonical mode III 14-3-3-binding motif, further enhanced by the diterpene glycoside Fusicoccin A. Moreover, concentration-dependent DAPK2 dimerization is disrupted by Ca2+/CaM binding and stabilized by 14-3-3 binding in solution, thereby protecting the DAPK2 inhibitory autophosphorylation site Ser318 against dephosphorylation and preventing Ca2+/CaM binding. Overall, our findings provide mechanistic insights into 14-3-3-mediated DAPK2 inhibition and highlight the potential of the DAPK2:14-3-3 complex as a target for anti-inflammatory therapies.

• MeSH
• dimerizace MeSH
• fosforylace MeSH
• lidé MeSH
• proteinkinasy asociované se smrtí genetika   metabolismus   MeSH
• proteiny 14-3-3 genetika   metabolismus   MeSH
• regulace genové exprese MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DAPK2 protein, human MeSH Prohlížeč
• proteinkinasy asociované se smrtí MeSH
• proteiny 14-3-3 MeSH
• YWHAG protein, human MeSH Prohlížeč

Neural precursor cell expressed developmentally down-regulated 4 ligase (Nedd4-2) is an E3 ubiquitin ligase that targets proteins for ubiquitination and endocytosis, thereby regulating numerous ion channels, membrane receptors and tumor suppressors. Nedd4-2 activity is regulated by autoinhibition, calcium binding, oxidative stress, substrate binding, phosphorylation and 14-3-3 protein binding. However, the structural basis of 14-3-3-mediated Nedd4-2 regulation remains poorly understood. Here, we combined several techniques of integrative structural biology to characterize Nedd4-2 and its complex with 14-3-3. We demonstrate that phosphorylated Ser342 and Ser448 are the key residues that facilitate 14-3-3 protein binding to Nedd4-2 and that 14-3-3 protein binding induces a structural rearrangement of Nedd4-2 by inhibiting interactions between its structured domains. Overall, our findings provide the structural glimpse into the 14-3-3-mediated Nedd4-2 regulation and highlight the potential of the Nedd4-2:14-3-3 complex as a pharmacological target for Nedd4-2-associated diseases such as hypertension, epilepsy, kidney disease and cancer.

• MeSH
• down regulace MeSH
• fosforylace MeSH
• myši genetika   metabolismus   MeSH
• proteiny 14-3-3 genetika   metabolismus   MeSH
• ubikvitinace MeSH
• ubikvitinligasy Nedd4 genetika   metabolismus   MeSH
• vazba proteinů MeSH
• WW domény * MeSH
• zvířata MeSH
• Check Tag
• myši genetika   metabolismus   MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• Nedd4l protein, mouse MeSH Prohlížeč
• proteiny 14-3-3 MeSH
• Sfn protein, mouse MeSH Prohlížeč
• ubikvitinligasy Nedd4 MeSH

Biomolecular force fields optimized for globular proteins fail to properly reproduce properties of intrinsically disordered proteins. In particular, parameters of the water model need to be modified to improve applicability of the force fields to both ordered and disordered proteins. Here, we compared performance of force fields recommended for intrinsically disordered proteins in molecular dynamics simulations of three proteins differing in the content of ordered and disordered regions (two proteins consisting of a well-structured domain and of a disordered region with and without a transient helical motif and one disordered protein containing a region of increased helical propensity). The obtained molecular dynamics trajectories were used to predict measurable parameters, including radii of gyration of the proteins and chemical shifts, residual dipolar couplings, paramagnetic relaxation enhancement, and NMR relaxation data of their individual residues. The predicted quantities were compared with experimental data obtained within this study or published previously. The results showed that the NMR relaxation parameters, rarely used for benchmarking, are particularly sensitive to the choice of force-field parameters, especially those defining the water model. Interestingly, the TIP3P water model, leading to an artificial structural collapse, also resulted in unrealistic relaxation properties. The TIP4P-D water model, combined with three biomolecular force-field parameters for the protein part, significantly improved reliability of the simulations. Additional analysis revealed only one particular force field capable of retaining the transient helical motif observed in NMR experiments. The benchmarking protocol used in our study, being more sensitive to imperfections than the commonly used tests, is well suited to evaluate the performance of newly developed force fields.

• MeSH
• konformace proteinů MeSH
• reprodukovatelnost výsledků MeSH
• simulace molekulární dynamiky MeSH
• vnitřně neuspořádané proteiny * MeSH
• voda MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• vnitřně neuspořádané proteiny * MeSH
• voda MeSH

The integration of complementary molecular methods (including X-ray crystallography, NMR spectroscopy, small angle X-ray/neutron scattering, and computational techniques) is frequently required to obtain a comprehensive understanding of dynamic macromolecular complexes. In particular, these techniques are critical for studying intrinsically disordered protein regions (IDRs) or intrinsically disordered proteins (IDPs) that are part of large protein:protein complexes. Here, we explain how to prepare IDP samples suitable for study using NMR spectroscopy, and describe a novel SAXS modeling method (ensemble refinement of SAXS; EROS) that integrates the results from complementary methods, including crystal structures and NMR chemical shift perturbations, among others, to accurately model SAXS data and describe ensemble structures of dynamic macromolecular complexes.

• Klíčová slova
• EROS, Ensemble, Intrinsically disordered proteins (IDP), NMR spectroscopy, SAXS,
• MeSH
• endozomální třídící komplexy pro transport chemie   metabolismus   MeSH
• konformace proteinů MeSH
• krystalografie rentgenová metody   MeSH
• lidé MeSH
• magnetická rezonanční spektroskopie metody   MeSH
• mitogenem aktivované proteinkinasy chemie   metabolismus   MeSH
• molekulární modely MeSH
• radiační rozptyl * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• endozomální třídící komplexy pro transport MeSH
• mitogenem aktivované proteinkinasy MeSH

To maintain genome integrity, segmented double-stranded RNA viruses of the Reoviridae family must accurately select and package a complete set of up to a dozen distinct genomic RNAs. It is thought that the high fidelity segmented genome assembly involves multiple sequence-specific RNA-RNA interactions between single-stranded RNA segment precursors. These are mediated by virus-encoded non-structural proteins with RNA chaperone-like activities, such as rotavirus (RV) NSP2 and avian reovirus σNS. Here, we compared the abilities of NSP2 and σNS to mediate sequence-specific interactions between RV genomic segment precursors. Despite their similar activities, NSP2 successfully promotes inter-segment association, while σNS fails to do so. To understand the mechanisms underlying such selectivity in promoting inter-molecular duplex formation, we compared RNA-binding and helix-unwinding activities of both proteins. We demonstrate that octameric NSP2 binds structured RNAs with high affinity, resulting in efficient intramolecular RNA helix disruption. Hexameric σNS oligomerizes into an octamer that binds two RNAs, yet it exhibits only limited RNA-unwinding activity compared to NSP2. Thus, the formation of intersegment RNA-RNA interactions is governed by both helix-unwinding capacity of the chaperones and stability of RNA structure. We propose that this protein-mediated RNA selection mechanism may underpin the high fidelity assembly of multi-segmented RNA genomes in Reoviridae.

• MeSH
• genom virový genetika   MeSH
• konformace nukleové kyseliny MeSH
• molekulární chaperony chemie   genetika   metabolismus   MeSH
• molekulární modely MeSH
• proteiny vázající RNA chemie   genetika   metabolismus   MeSH
• ptačí orthoreovirus genetika   metabolismus   MeSH
• RNA virová chemie   genetika   metabolismus   MeSH
• sekundární struktura proteinů MeSH
• sekvence nukleotidů MeSH
• vazba proteinů MeSH
• virové nestrukturální proteiny chemie   genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• molekulární chaperony MeSH
• NS35 protein, rotavirus MeSH Prohlížeč
• proteiny vázající RNA MeSH
• RNA virová MeSH
• virové nestrukturální proteiny MeSH