Flavivirus assembly is driven by the envelope glycoproteins pre-membrane (prM) and envelope (E) in the neutral pH environment of the endoplasmic reticulum. Newly budded, spiky particles are exported through the Golgi apparatus, where mildly acidic pH induces a major surface rearrangement. The glycoproteins reorganize into (prM/E)\₂ complexes at the surface of smooth particles, with prM trapped at the E dimer interface, thereby exposing a furin cleavage site (FCS) for proteolytic maturation into infectious virions. Here, we show that in the absence of furin, immature tick-borne flavivirus particles-tick-borne encephalitis virus, Langat virus, and Louping ill virus-remain fully infectious and pathogenic in female BALB/c mice, in contrast to mosquito-borne flaviviruses such as Usutu, West Nile, and Zika viruses. We further show that the FCS in tick-borne viruses remains exposed at neutral pH, allowing furin at the surface of target cells to activate viral fusogenicity, while mosquito-borne counterparts require acidic re-exposure. Mutations increasing the dynamic behavior of the E dimer mimic the mosquito-borne phenotype, with retracted FCS at neutral pH and loss of infectivity. Our multidisciplinary approach-combining virological assays, targeted mutagenesis, structural modeling, and molecular dynamics simulations-highlights the role of E dimer dynamics in regulating flavivirus maturation and infectivity.

• MeSH
• Flavivirus * patogenita   genetika   fyziologie   MeSH
• furin * metabolismus   genetika   MeSH
• koncentrace vodíkových iontů MeSH
• lidé MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• proteiny virového obalu * metabolismus   genetika   chemie   MeSH
• sestavení viru MeSH
• viry klíšťové encefalitidy * patogenita   genetika   fyziologie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• furin * MeSH
• proteiny virového obalu * MeSH

The quantitative characterization of residue contributions to protein-protein binding across extensive flexible interfaces poses a significant challenge for biophysical computations. It is attributable to the inherent imperfections in the experimental structures themselves, as well as to the lack of reliable computational tools for the evaluation of all types of noncovalent interactions. This study leverages recent advancements in semiempirical quantum-mechanical and implicit solvent approaches embodied in the PM6-D3H4S/COSMO2 method for the development of a hierarchical computational protocols encompassing molecular dynamics, fragmentation, and virtual glycine scan techniques for the investigation of flexible protein-protein interactions. As a model, the binding of insulin to its receptor is selected, a complex and dynamic process that has been extensively studied experimentally. The interaction energies calculated at the PM6-D3H4S/COSMO2 level in ten molecular dynamics snapshots did not correlate with molecular mechanics/generalized Born interaction energies because only the former method is able to describe nonadditive effects. This became evident by the examination of the energetics in small-model dimers featuring all the present types of noncovalent interactions with respect to DFT-D3 calculations. The virtual glycine scan has identified 15 hotspot residues on insulin and 15 on the insulin receptor, and their contributions have been quantified using PM6-D3H4S/COSMO2. The accuracy and credibility of the approach are further supported by the fact that all the insulin hotspots have previously been detected by biochemical and structural methods. The modular nature of the protocol has enabled the formulation of several variants, each tailored to specific accuracy and efficiency requirements. The developed computational strategy is firmly rooted in general biophysical chemistry and is thus offered as a general tool for the quantification of interactions across relevant flexible protein-protein interfaces.

• MeSH
• inzulin metabolismus   chemie   MeSH
• konformace proteinů MeSH
• lidé MeSH
• receptor inzulinu * chemie   metabolismus   MeSH
• simulace molekulární dynamiky * MeSH
• termodynamika MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• inzulin MeSH
• receptor inzulinu * MeSH

The main role of dimeric 14-3-3 proteins is to modulate the activity of several hundred binding partners by interacting with phosphorylated residues of the partner proteins, often located in disordered regions. The inherent flexibility or large size of 14-3-3 complexes hampers their structural characterization by X-ray crystallography, cryo-electron microscopy (EM) and traditional solution nuclear magnetic resonance (NMR) spectroscopy. Here, we employ solution 1D 19F-Trp NMR spectroscopy to characterize substrate binding and dimerization of 14-3-3 proteins, focusing on 14-3-3ζ - an abundant human isoform as an example. Both conserved Trp residues are located in distinct functionally important sites - the dimeric interface and the ligand-binding groove. We substituted them by 5F-Trp, thereby introducing a convenient NMR probe. Fluorination of the two Trp did not impact the stability and interaction properties of 14-3-3ζ in a substantive manner, permitting to carry out 19F NMR experiments to assess 14-3-3's structure and behavior. Importantly, 5F-Trp228 reports on binding of substrates in the amphipathic binding groove of 14-3-3ζ and permitted to distinguish distinct recognition modes. Thus, we established that 19F NMR is a powerful approach to evaluate the binding of partner proteins to 14-3-3 and to characterize the properties of the resulting complexes.

• Klíčová slova
• (19)F NMR, 14-3-3 monomer, 14-3-3 proteins, Quaternary structure, Substrate binding,
• MeSH
• lidé MeSH
• ligandy MeSH
• magnetická rezonanční spektroskopie MeSH
• molekulární modely MeSH
• multimerizace proteinu * MeSH
• nukleární magnetická rezonance biomolekulární * MeSH
• proteiny 14-3-3 * chemie   metabolismus   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• ligandy MeSH
• proteiny 14-3-3 * MeSH

P2Y12 receptors on the platelet plasma membrane are targeted by several antiplatelets drugs. Although oligomerization and functioning of P2Y12 receptors depend on the membrane environment, little is known about their preferred membrane localization and the role of surrounding lipid composition, especially the arachidonic acids (ARA), which are abundant in platelets. Coarse-grained molecular dynamics simulations of platelet plasma membrane based on the lipidomics data were used to investigate the P2Y12 lipid environment and the involvement of ARA in its oligomerization in platelet plasma membranes. The platelet plasma membrane contains two types of lipids nanodomains: ordered, enriched in SM and cholesterol, and disordered, enriched in ARA-containing lipids. P2Y12 receptors prefer to localize in these ARA-rich domains and induce the sorting of the ARA-containing lipids in their vicinity. This ARA-rich environment promotes the oligomerization of P2Y12 receptors and stabilizes the protein-protein interfaces of oligomers. As summary, oligomerization of P2Y12 receptors is promoted in ARA-rich nano-domains of the platelet plasma membrane.

• Klíčová slova
• Arachidonic acid, Blood platelets, Molecular dynamics simulation, Protein Multimerization, Protein–lipid interaction, Purinergic receptor P2Y12,
• MeSH
• buněčná membrána * metabolismus   chemie   MeSH
• cholesterol metabolismus   chemie   MeSH
• kyselina arachidonová * metabolismus   chemie   MeSH
• lidé MeSH
• multimerizace proteinu MeSH
• purinergní receptory P2Y12 * metabolismus   chemie   MeSH
• simulace molekulární dynamiky * MeSH
• trombocyty * metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cholesterol MeSH
• kyselina arachidonová * MeSH
• P2RY12 protein, human MeSH Prohlížeč
• purinergní receptory P2Y12 * MeSH

Aging workers of the termite Neocapritermes taracua can defend their colony by sacrificing themselves by body rupture, mixing the externally stored blue laccase BP76 with hydroquinones to produce a sticky liquid rich in toxic benzoquinones. Here, we describe the crystal structure of BP76 isolated from N. taracua in its native form. The structure reveals several stabilization strategies, including compact folding, glycosylation, and flexible loops with disulfide bridges and tight dimer interface. The remarkable stability of BP76 maintains its catalytic activity in solid state during the lifespan of N. taracua workers, providing old workers with an efficient defensive weapon to protect their colony.

• Klíčová slova
• Neocapritermes taracua, X-ray crystallography, autothysis, benzoquinone, colony defence, insect glycosylation, laccase, protein stabilization, single-wavelength anomalous diffraction,
• MeSH
• glykosylace MeSH
• hmyzí proteiny chemie   metabolismus   MeSH
• Isoptera * MeSH
• katalytická doména MeSH
• krystalografie rentgenová MeSH
• lakasa * chemie   metabolismus   MeSH
• molekulární modely * MeSH
• multimerizace proteinu MeSH
• sbalování proteinů MeSH
• sekvence aminokyselin MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• hmyzí proteiny MeSH
• lakasa * MeSH

Isoforms of microtubule-associated protein 2 (MAP2) differ from their homolog Tau in the sequence and interactions of the N-terminal region. Binding of the N-terminal region of MAP2c (N-MAP2c) to the dimerization/docking domains of the regulatory subunit RIIα of cAMP-dependent protein kinase (RIIDD2) and to the Src-homology domain 2 (SH2) of growth factor receptor-bound protein 2 (Grb2) have been described long time ago. However, the structural features of the complexes remained unknown due to the disordered nature of MAP2. Here, we provide structural description of the complexes. We have solved solution structure of N-MAP2c in complex with RIIDD2, confirming formation of an amphiphilic α-helix of MAP2c upon binding, defining orientation of the α-helix in the complex and showing that its binding register differs from previous predictions. Using chemical shift mapping, we characterized the binding interface of SH2-Grb2 and rat MAP2c phosphorylated by the tyrosine kinase Fyn in their complex and proposed a model explaining differences between SH2-Grb2 complexes with rat MAP2c and phosphopeptides with a Grb2-specific sequence. The results provide the structural basis of a potential role of MAP2 in regulating cAMP-dependent phosphorylation cascade via interactions with RIIDD2 and Ras signaling pathway via interactions with SH2-Grb2.

• Klíčová slova
• A-kinase anchoring protein (AKAP), growth factor receptor-bound protein 2 (GRB2), microtubule associated protein (MAP) 2, nuclear magnetic resonance (NMR), protein kinase A (PKA),
• MeSH
• adaptorový protein Grb2 * metabolismus   chemie   MeSH
• lidé MeSH
• proteinové domény MeSH
• proteiny asociované s mikrotubuly * metabolismus   chemie   genetika   MeSH
• protoonkogenní proteiny c-fyn metabolismus   chemie   genetika   MeSH
• signální transdukce MeSH
• src homologní domény MeSH
• vazba proteinů * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adaptorový protein Grb2 * MeSH
• GRB2 protein, human MeSH Prohlížeč
• proteiny asociované s mikrotubuly * MeSH
• protoonkogenní proteiny c-fyn MeSH

Tau protein is an intrinsically disordered protein that plays a key role in Alzheimer's disease (AD). In brains of AD patients, Tau occurs abnormally phosphorylated and aggregated in neurofibrillary tangles (NFTs). Together with Tau, 14-3-3 proteins - abundant cytosolic dimeric proteins - were found colocalized in the NFTs. However, so far, the molecular mechanism of the process leading to pathological changes in Tau structure as well as the direct involvement of 14-3-3 proteins are not well understood. Here, we aimed to reveal the effects of phosphorylation by protein kinase A (PKA) on Tau structural preferences and provide better insight into the interaction between Tau and 14-3-3 proteins. We also addressed the impact of monomerization-inducing phosphorylation of 14-3-3 at S58 on the binding to Tau protein. Using multidimensional nuclear magnetic resonance spectroscopy (NMR), chemical cross-linking analyzed by mass spectrometry (MS) and PAGE, we unveiled differences in their binding affinity, stoichiometry, and interfaces with single-residue resolution. We revealed that the interaction between 14-3-3 and Tau proteins is mediated not only via the 14-3-3 amphipathic binding grooves, but also via less specific interactions with 14-3-3 protein surface and, in the case of monomeric 14-3-3, also partially via the exposed dimeric interface. In addition, the hyperphosphorylation of Tau changes its affinity to 14-3-3 proteins. In conclusion, we propose quite complex interaction mode between the Tau and 14-3-3 proteins.

• Klíčová slova
• 14-3-3ζ protein, Cross-linking, Interaction, NMR, Phosphorylation, Tau protein,
• MeSH
• Alzheimerova nemoc metabolismus   MeSH
• fosforylace MeSH
• lidé MeSH
• molekulární modely MeSH
• multimerizace proteinu MeSH
• proteinkinasy závislé na cyklickém AMP metabolismus   MeSH
• proteiny 14-3-3 * metabolismus   chemie   MeSH
• proteiny tau * metabolismus   chemie   MeSH
• vazba proteinů * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• proteinkinasy závislé na cyklickém AMP MeSH
• proteiny 14-3-3 * MeSH
• proteiny tau * MeSH

The redox behavior and chemisorption of cysteamine (CA) at a charged mercury surface are described, with an emphasis on its acid-base properties supported by molecular dynamics and quantum mechanical calculations. It was found that CA forms chemisorbed layers on the surface of the mercury electrode. The formation of Hg-CA complexes is connected to mercury disproportionation, as reflected in peaks SII and SI at potentials higher than the electrode potential of zero charge (p.z.c.). Both the process of chemisorption of CA and its consequent redox transformation are proton-dependent. Also, depending on the protonation of CA, the formation of typical populations of chemisorbed conformers can be observed. In addition, cystamine (CA disulfide dimer) can be reduced on the mercury surface. Between the potentials of this reduction and peak SI, the p.z.c. of the electrode used can be found. Furthermore, CA can serve as an LMW catalyst for hydrogen evolution. The mechanistic insights presented here can be used for follow-up research on CA chemisorption and targeted modification of other metallic surfaces.

• Publikační typ
• časopisecké články MeSH

Fibroblast growth factor 2 (FGF2) exits cells by direct translocation across the plasma membrane, a type I pathway of unconventional protein secretion. This process is initiated by phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2)-dependent formation of highly dynamic FGF2 oligomers at the inner plasma membrane leaflet, inducing the formation of lipidic membrane pores. Cell surface heparan sulfate chains linked to glypican-1 (GPC1) capture FGF2 at the outer plasma membrane leaflet, completing FGF2 membrane translocation into the extracellular space. While the basic steps of this pathway are well understood, the molecular mechanism by which FGF2 oligomerizes on membrane surfaces remains unclear. In the current study, we demonstrate the initial step of this process to depend on C95-C95 disulfide-bridge-mediated FGF2 dimerization on membrane surfaces, producing the building blocks for higher FGF2 oligomers that drive the formation of membrane pores. We find FGF2 with a C95A substitution to be defective in oligomerization, pore formation, and membrane translocation. Consistently, we demonstrate a C95A variant of FGF2 to be characterized by a severe secretion phenotype. By contrast, while also important for efficient FGF2 secretion from cells, a second cysteine residue on the molecular surface of FGF2 (C77) is not involved in FGF2 oligomerization. Rather, we find C77 to be part of the interaction interface through which FGF2 binds to the α1 subunit of the Na,K-ATPase, the landing platform for FGF2 at the inner plasma membrane leaflet. Using cross-linking mass spectrometry, atomistic molecular dynamics simulations combined with a machine learning analysis and cryo-electron tomography, we propose a mechanism by which disulfide-bridged FGF2 dimers bind with high avidity to PI(4,5)P2 on membrane surfaces. We further propose a tight coupling between FGF2 secretion and the formation of ternary signaling complexes on cell surfaces, hypothesizing that C95-C95-bridged FGF2 dimers are functioning as the molecular units triggering autocrine and paracrine FGF2 signaling.

• Klíčová slova
• E. coli, FGF2, Protein trafficking, Protein-lipid interaction, Protein-protein interaction, Unconventional protein secretion, biochemistry, chemical biology, cho, cho k1, hela s3,
• MeSH
• dimerizace MeSH
• disulfidy MeSH
• extracelulární prostor * MeSH
• fibroblastový růstový faktor 2 * MeSH
• sodíko-draslíková ATPasa MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• disulfidy MeSH
• fibroblastový růstový faktor 2 * MeSH
• sodíko-draslíková ATPasa MeSH

Mitochondria are double-membrane-bounded organelles that depend critically on phospholipids supplied by the endoplasmic reticulum. These lipids must cross the outer membrane to support mitochondrial function, but how they do this is unclear. We identify the Voltage Dependent Anion Channel (VDAC), an abundant outer membrane protein, as a scramblase-type lipid transporter that catalyzes lipid entry. On reconstitution into membrane vesicles, dimers of human VDAC1 and VDAC2 catalyze rapid transbilayer translocation of phospholipids by a mechanism that is unrelated to their channel activity. Coarse-grained molecular dynamics simulations of VDAC1 reveal that lipid scrambling occurs at a specific dimer interface where polar residues induce large water defects and bilayer thinning. The rate of phospholipid import into yeast mitochondria is an order of magnitude lower in the absence of VDAC homologs, indicating that VDACs provide the main pathway for lipid entry. Thus, VDAC isoforms, members of a superfamily of beta barrel proteins, moonlight as a class of phospholipid scramblases - distinct from alpha-helical scramblase proteins - that act to import lipids into mitochondria.

• MeSH
• fosfolipidy * metabolismus   MeSH
• lidé MeSH
• mitochondrie metabolismus   MeSH
• napětím ovládané aniontové kanály metabolismus   MeSH
• napětím ovládaný aniontový kanál 1 * metabolismus   MeSH
• Saccharomyces cerevisiae metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• fosfolipidy * MeSH
• napětím ovládané aniontové kanály MeSH
• napětím ovládaný aniontový kanál 1 * MeSH