The heme-based oxygen sensor protein AfGcHK is a globin-coupled histidine kinase in the soil bacterium Anaeromyxobacter sp. Fw109-5. Its C-terminal functional domain exhibits autophosphorylation activity induced by oxygen binding to the heme-Fe(II) complex located in the oxygen-sensing N-terminal globin domain. A detailed understanding of the signal transduction mechanisms in heme-containing sensor proteins remains elusive. Here, we investigated the role of the globin domain's dimerization interface in signal transduction in AfGcHK. We present a crystal structure of a monomeric imidazole-bound AfGcHK globin domain at 1.8 Å resolution, revealing that the helices of the WT globin dimer are under tension and suggesting that Tyr-15 plays a role in both this tension and the globin domain's dimerization. Biophysical experiments revealed that whereas the isolated WT globin domain is dimeric in solution, the Y15A and Y15G variants in which Tyr-15 is replaced with Ala or Gly, respectively, are monomeric. Additionally, we found that although the dimerization of the full-length protein is preserved via the kinase domain dimerization interface in all variants, full-length AfGcHK variants bearing the Y15A or Y15G substitutions lack enzymatic activity. The combined structural and biophysical results presented here indicate that Tyr-15 plays a key role in the dimerization of the globin domain of AfGcHK and that globin domain dimerization is essential for internal signal transduction and autophosphorylation in this protein. These findings provide critical insights into the signal transduction mechanism of the histidine kinase AfGcHK from Anaeromyxobacter.

• Klíčová slova
• bacterial protein kinase, cell signaling, crystal structure, dimerization interface, globin, heme, heme-based oxygen sensor, histidine kinase, signal transduction, two component system,
• MeSH
• bakteriální proteiny chemie   metabolismus   MeSH
• fosforylace MeSH
• globiny chemie   metabolismus   MeSH
• histidinkinasa chemie   metabolismus   MeSH
• konformace proteinů, alfa-helix MeSH
• konformace proteinů MeSH
• krystalografie rentgenová MeSH
• molekulární modely MeSH
• multimerizace proteinu MeSH
• Myxococcales chemie   metabolismus   MeSH
• proteinové domény MeSH
• signální transdukce MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• globiny MeSH
• histidinkinasa MeSH

Among all species, caspase-2 (C2) is the most evolutionarily conserved caspase required for effective initiation of apoptosis following death stimuli. C2 is activated through dimerization and autoproteolytic cleavage and inhibited through phosphorylation at Ser139 and Ser164 , within the linker between the caspase recruitment and p19 domains of the zymogen, followed by association with the adaptor protein 14-3-3, which maintains C2 in its immature form procaspase (proC2). However, the mechanism of 14-3-3-dependent inhibition of C2 activation remains unclear. Here, we report the structural characterization of the complex between proC2 and 14-3-3 by hydrogen/deuterium mass spectrometry and protein crystallography to determine the molecular basis for 14-3-3-mediated inhibition of C2 activation. Our data reveal that the 14-3-3 dimer interacts with proC2 not only through ligand-binding grooves but also through other regions outside the central channel, thus explaining the isoform-dependent specificity of 14-3-3 protein binding to proC2 and the substantially higher binding affinity of 14-3-3 protein to proC2 than to the doubly phosphorylated peptide. The formation of the complex between 14-3-3 protein and proC2 does not induce any large conformational change in proC2. Furthermore, 14-3-3 protein interacts with and masks both the nuclear localization sequence and the C-terminal region of the p12 domain of proC2 through transient interactions in which both the p19 and p12 domains of proC2 are not firmly docked onto the surface of 14-3-3. This masked region of p12 domain is involved in C2 dimerization. Therefore, 14-3-3 protein likely inhibits proC2 activation by blocking its dimerization surface. DATABASES: Structural data are available in the Protein Data Bank under the accession numbers 6SAD and 6S9K.

• Klíčová slova
• 14-3-3 protein, H/D exchange, caspase-2, crystallography, protein-protein interaction,
• MeSH
• fosforylace MeSH
• kaspasa 2 chemie   genetika   metabolismus   MeSH
• konformace proteinů * MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• molekulární modely * MeSH
• multimerizace proteinu * MeSH
• mutace MeSH
• protein - isoformy genetika   metabolismus   MeSH
• proteinové prekurzory chemie   genetika   metabolismus   MeSH
• proteiny 14-3-3 chemie   genetika   metabolismus   MeSH
• rekombinantní proteiny chemie   metabolismus   MeSH
• vazba proteinů MeSH
• vazebná místa genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kaspasa 2 MeSH
• protein - isoformy MeSH
• proteinové prekurzory MeSH
• proteiny 14-3-3 MeSH
• rekombinantní proteiny MeSH

Apart from its role in insulin receptor (IR) activation, the C terminus of the B-chain of insulin is also responsible for the formation of insulin dimers. The dimerization of insulin plays an important role in the endogenous delivery of the hormone and in the administration of insulin to patients. Here, we investigated insulin analogues with selective N-methylations of peptide bond amides at positions B24, B25, or B26 to delineate their structural and functional contribution to the dimer interface. All N-methylated analogues showed impaired binding affinities to IR, which suggests a direct IR-interacting role for the respective amide hydrogens. The dimerization capabilities of analogues were investigated by isothermal microcalorimetry. Selective N-methylations of B24, B25, or B26 amides resulted in reduced dimerization abilities compared with native insulin (K(d) = 8.8 μM). Interestingly, although the N-methylation in [NMeTyrB26]-insulin or [NMePheB24]-insulin resulted in K(d) values of 142 and 587 μM, respectively, the [NMePheB25]-insulin did not form dimers even at high concentrations. This effect may be attributed to the loss of intramolecular hydrogen bonding between NHB25 and COA19, which connects the B-chain β-strand to the core of the molecule. The release of the B-chain β-strand from this hydrogen bond lock may result in its higher mobility, thereby shifting solution equilibrium toward the monomeric state of the hormone. The study was complemented by analyses of two novel analogue crystal structures. All examined analogues crystallized only in the most stable R(6) form of insulin oligomers (even if the dimer interface was totally disrupted), confirming the role of R(6)-specific intra/intermolecular interactions for hexamer stability.

• MeSH
• inzulin prasečí chemie   MeSH
• krystalografie rentgenová MeSH
• kvarterní struktura proteinů MeSH
• metylace MeSH
• multimerizace proteinu * MeSH
• prasata MeSH
• sekundární struktura proteinů MeSH
• stabilita proteinů MeSH
• vodíková vazba MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• inzulin prasečí MeSH

BACKGROUND: Myristoylation of the matrix (MA) domain mediates the transport and binding of Gag polyproteins to the plasma membrane (PM) and is required for the assembly of most retroviruses. In betaretroviruses, which assemble immature particles in the cytoplasm, myristoylation is dispensable for assembly but is crucial for particle transport to the PM. Oligomerization of HIV-1 MA stimulates the transition of the myristoyl group from a sequestered to an exposed conformation, which is more accessible for membrane binding. However, for other retroviruses, the effect of MA oligomerization on myristoyl group exposure has not been thoroughly investigated. RESULTS: Here, we demonstrate that MA from the betaretrovirus mouse mammary tumor virus (MMTV) forms dimers in solution and that this process is stimulated by its myristoylation. The crystal structure of N-myristoylated MMTV MA, determined at 1.57 Å resolution, revealed that the myristoyl groups are buried in a hydrophobic pocket at the dimer interface and contribute to dimer formation. Interestingly, the myristoyl groups in the dimer are mutually swapped to achieve energetically stable binding, as documented by molecular dynamics modeling. Mutations within the myristoyl binding site resulted in reduced MA dimerization and extracellular particle release. CONCLUSIONS: Based on our experimental, structural, and computational data, we propose a model for dimerization of MMTV MA in which myristoyl groups stimulate the interaction between MA molecules. Moreover, dimer-forming MA molecules adopt a sequestered conformation with their myristoyl groups entirely buried within the interaction interface. Although this differs from the current model proposed for lentiviruses, in which oligomerization of MA triggers exposure of myristoyl group, it appears convenient for intracellular assembly, which involves no apparent membrane interaction and allows the myristoyl group to be sequestered during oligomerization.

• MeSH
• biologické modely MeSH
• buněčné linie MeSH
• krysa rodu Rattus MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• molekulární modely MeSH
• multimerizace proteinu * MeSH
• posttranslační úpravy proteinů * MeSH
• proteiny virové matrix chemie   metabolismus   MeSH
• simulace molekulární dynamiky MeSH
• virus myšího tumoru prsní žlázy chemie   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteiny virové matrix 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

The adsorption, mobility, and self-association of naphthalene (NPH) and 1-methylnaphthalene (1MN), two of the simplest polycyclic aromatic hydrocarbons (PAHs), at the surface of liquid water at 289 K were investigated using classical molecular dynamics (MD) simulations and free energy profile calculations across the water-vapor interface. Both NPH and 1MN, which exhibit a strong preference to be adsorbed at the water-vapor interface, are found to readily self-associate at the water surface, adopting mostly configurations with distinctly nonparallel arrangement of the two monomers. The additional methyl group of 1MN represents only a minor perturbation in terms of the hydration properties, interfacial orientation, and self-association with respect to NPH. Implications of the observed self-association behavior for fluorescence spectroscopy of NPH and 1MN in aqueous interfacial environment are discussed.

• MeSH
• adsorpce MeSH
• dimerizace MeSH
• molekulární konformace MeSH
• naftaleny chemie   MeSH
• pára * MeSH
• pohyb těles * MeSH
• simulace molekulární dynamiky * MeSH
• termodynamika MeSH
• vakuum MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• 1-methylnaphthalene MeSH Prohlížeč
• naftaleny MeSH
• pára * 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

SARS-CoV-2 encodes eight accessory proteins, one of which, ORF8, has a poorly conserved sequence with SARS-CoV and its role in viral pathogenicity has recently been identified. ORF8 in SARS-CoV-2 has a unique functional feature that allows it to form a dimer structure linked by a disulfide bridge between Cys20 and Cys20 (S-S). This study provides structural characterization of natural mutant variants as well as the identification of potential drug candidates capable of binding directly to the interchain disulfide bridge. The lead compounds reported in this work have a tendency to settle in the dimeric interfaces by direct interaction with the disulfide bridge. These molecules may disturb the dimer formation and may have an inhibition impact on its potential functional role in host immune evasion and virulence pathogenicity. This work provides detailed insights on the sequence and structural variability through computational mutational studies, as well as potent drug candidates with the ability to interrupt the intermolecular disulfide bridge formed between Cys20 and Cys20. Furthermore, the interactions of ORF8 peptides complexed with MHC-1 is studied, and the binding mode reveals that certain ORF8 peptides bind to MHC-1 in a manner similar to other viral peptides. Overall, this study is a narrative of various computational approaches used to provide detailed structural insights into SARS-CoV-2 ORF8 interchain disulfide bond disruptors.

• Klíčová slova
• COVID-19, MHC-1, ORF8, SARS-CoV, SARS-CoV-2, dimer, disulfide bond, immune evasion, molecular dynamics, protein–protein interactions, viral peptides,
• MeSH
• COVID-19 * MeSH
• dimerizace MeSH
• lidé MeSH
• SARS-CoV-2 * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ORF8 protein, SARS-CoV-2 MeSH Prohlížeč

We have investigated photoinduced hole hopping in a Pseudomonas aeruginosa azurin mutant Re126WWCuI, where two adjacent tryptophan residues (W124 and W122) are inserted between the CuI center and a Re photosensitizer coordinated to a H126 imidazole (Re = ReI(H126)(CO)3(dmp)+, dmp = 4,7-dimethyl-1,10-phenanthroline). Optical excitation of this mutant in aqueous media (≤40 μM) triggers 70 ns electron transport over 23 Å, yielding a long-lived (120 μs) ReI(H126)(CO)3(dmp•-)WWCuII product. The Re126FWCuI mutant (F124, W122) is not redox-active under these conditions. Upon increasing the concentration to 0.2-2 mM, {Re126WWCuI}2 and {Re126FWCuI}2 are formed with the dmp ligand of the Re photooxidant of one molecule in close contact (3.8 Å) with the W122' indole on the neighboring chain. In addition, {Re126WWCuI}2 contains an interfacial tryptophan quadruplex of four indoles (3.3-3.7 Å apart). In both mutants, dimerization opens an intermolecular W122' → //*Re ET channel (// denotes the protein interface, *Re is the optically excited sensitizer). Excited-state relaxation and ET occur together in two steps (time constants of ∼600 ps and ∼8 ns) that lead to a charge-separated state containing a Re(H126)(CO)3(dmp•-)//(W122•+)' unit; then (CuI)' is oxidized intramolecularly (60-90 ns) by (W122•+)', forming ReI(H126)(CO)3(dmp•-)WWCuI//(CuII)'. The photocycle is closed by ∼1.6 μs ReI(H126)(CO)3(dmp•-) → //(CuII)' back ET that occurs over 12 Å, in contrast to the 23 Å, 120 μs step in Re126WWCuI. Importantly, dimerization makes Re126FWCuI photoreactive and, as in the case of {Re126WWCuI}2, channels the photoproduced "hole" to the molecule that was not initially photoexcited, thereby shortening the lifetime of ReI(H126)(CO)3(dmp•-)//CuII. Although two adjacent W124 and W122 indoles dramatically enhance CuI → *Re intramolecular multistep ET, the tryptophan quadruplex in {Re126WWCuI}2 does not accelerate intermolecular electron transport; instead, it acts as a hole storage and crossover unit between inter- and intramolecular ET pathways. Irradiation of {Re126WWCuII}2 or {Re126FWCuII}2 also triggers intermolecular W122' → //*Re ET, and the Re(H126)(CO)3(dmp•-)//(W122•+)' charge-separated state decays to the ground state by ∼50 ns ReI(H126)(CO)3(dmp•-)+ → //(W122•+)' intermolecular charge recombination. Our findings shed light on the factors that control interfacial hole/electron hopping in protein complexes and on the role of aromatic amino acids in accelerating long-range electron transport.

• MeSH
• azurin chemie   genetika   metabolismus   MeSH
• elektrony MeSH
• imidazoly chemie   MeSH
• kvantová teorie MeSH
• měď chemie   MeSH
• molekulární modely MeSH
• mutageneze MeSH
• oxidace-redukce MeSH
• Pseudomonas aeruginosa metabolismus   MeSH
• světlo MeSH
• terciární struktura proteinů MeSH
• transport elektronů MeSH
• tryptofan chemie   MeSH
• voda chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• azurin MeSH
• imidazole MeSH Prohlížeč
• imidazoly MeSH
• měď MeSH
• tryptofan MeSH
• voda MeSH

The effect of non-denaturing concentrations of three different organic solvents, formamide, acetone and isopropanol, on the structure of haloalkane dehalogenases DhaA, LinB, and DbjA at the protein-solvent interface was studied using molecular dynamics simulations. Analysis of B-factors revealed that the presence of a given organic solvent mainly affects the dynamical behavior of the specificity-determining cap domain, with the exception of DbjA in acetone. Orientation of organic solvent molecules on the protein surface during the simulations was clearly dependent on their interaction with hydrophobic or hydrophilic surface patches, and the simulations suggest that the behavior of studied organic solvents in the vicinity of hyrophobic patches on the surface is similar to the air/water interface. DbjA was the only dimeric enzyme among studied haloalkane dehalogenases and provided an opportunity to explore effects of organic solvents on the quaternary structure. Penetration and trapping of organic solvents in the network of interactions between both monomers depends on the physico-chemical properties of the organic solvents. Consequently, both monomers of this enzyme oscillate differently in different organic solvents. With the exception of LinB in acetone, the structures of studied enzymes were stabilized in water-miscible organic solvents.

• MeSH
• 2-propanol chemie   farmakologie   MeSH
• aceton chemie   farmakologie   MeSH
• formamidy chemie   farmakologie   MeSH
• hydrofobní a hydrofilní interakce MeSH
• hydrolasy chemie   MeSH
• krystalografie rentgenová MeSH
• kvarterní struktura proteinů účinky léků   MeSH
• molekulární modely MeSH
• rozpouštědla chemie   MeSH
• simulace molekulární dynamiky MeSH
• terciární struktura proteinů účinky léků   MeSH
• voda chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 2-propanol MeSH
• aceton MeSH
• formamide MeSH Prohlížeč
• formamidy MeSH
• haloalkane dehalogenase MeSH Prohlížeč
• hydrolasy MeSH
• rozpouštědla MeSH
• voda MeSH