ORPs are lipid-transport proteins belonging to the oxysterol-binding protein family. They facilitate the transfer of lipids between different intracellular membranes, such as the ER and plasma membrane. We have solved the crystal structure of the ORP8 lipid transport domain (ORD8). The ORD8 exhibited a β-barrel fold composed of anti-parallel β-strands, with three α-helices replacing β-strands on one side. This mixed alpha-beta structure was consistent with previously solved structures of ORP2 and ORP3. A large cavity (≈1860 Å3) within the barrel was identified as the lipid-binding site. Although we were not able to obtain a lipid-bound structure, we used computer simulations based on our crystal structure to dock PS and PI4P molecules into the putative lipid-binding site of the ORD8. Comparative experiments between the short ORD8ΔLid (used for crystallography) and the full-length ORD8 (lid containing) revealed the lid's importance for stable lipid binding. Fluorescence assays revealed different transport efficiencies for PS and PI4P, with the lid slowing down transport and stabilizing cargo. Coarse-grained simulations highlighted surface-exposed regions and hydrophobic interactions facilitating lipid bilayer insertion. These findings enhance our comprehension of ORD8, its structure, and lipid transport mechanisms, as well as provide a structural basis for the design of potential inhibitors.

• Klíčová slova
• ER, ORD, ORP8, PI4P, PS, lipid transport, plasma membrane,
• MeSH
• biologický transport MeSH
• buněčná membrána metabolismus   MeSH
• lipidy * chemie   MeSH
• transportní proteiny * metabolismus   MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• lipidy * MeSH
• transportní proteiny * MeSH

The nucleocapsid protein of the SARS-CoV-2 virus comprises two RNA-binding domains and three regions that are intrinsically disordered. While the structures of the RNA-binding domains have been solved using protein crystallography and NMR, current knowledge of the conformations of the full-length nucleocapsid protein is rather limited. To fill in this knowledge gap, we combined coarse-grained molecular simulations with data from small-angle X-ray scattering (SAXS) experiments using the ensemble refinement of SAXS (EROS) method. Our results show that the dimer of the full-length nucleocapsid protein exhibits large conformational fluctuations with its radius of gyration ranging from about 4 to 8 nm. The RNA-binding domains do not make direct contacts. The disordered region that links these two domains comprises a hydrophobic α-helix which makes frequent and nonspecific contacts with the RNA-binding domains. Each of the intrinsically disordered regions adopts conformations that are locally compact, yet on average, much more extended than Gaussian chains of equivalent lengths. We offer a detailed picture of the conformational ensemble of the nucleocapsid protein dimer under near-physiological conditions, which will be important for understanding the nucleocapsid assembly process.

• Klíčová slova
• EROS, Nucleocapsid, SARS-CoV-2, SAXS,
• MeSH
• COVID-19 * MeSH
• difrakce rentgenového záření MeSH
• konformace proteinů MeSH
• lidé MeSH
• maloúhlový rozptyl MeSH
• nukleokapsida - proteiny chemie   MeSH
• nukleokapsida MeSH
• SARS-CoV-2 * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• nukleokapsida - proteiny MeSH

Phosphorylation by kinases governs many key cellular and extracellular processes, such as transcription, cell cycle progression, differentiation, secretion and apoptosis. Unsurprisingly, tight and precise kinase regulation is a prerequisite for normal cell functioning, whereas kinase dysregulation often leads to disease. Moreover, the functions of many kinases are regulated through protein-protein interactions, which in turn are mediated by phosphorylated motifs and often involve associations with the scaffolding and chaperon protein 14-3-3. Therefore, the aim of this review article is to provide an overview of the state of the art on 14-3-3-mediated kinase regulation, focusing on the most recent mechanistic insights into these important protein-protein interactions and discussing in detail both their structural aspects and functional consequences.

• Klíčová slova
• 14-3-3, ASK1, CaMKK2, LRRK2, PI4KB, PKC, RAF kinase, kinase, phosphorylation,
• MeSH
• alosterická regulace genetika   MeSH
• apoptóza genetika   MeSH
• fosforylace genetika   MeSH
• lidé MeSH
• mitogenem aktivované proteinkinasy p38 genetika   MeSH
• proteinkinasy genetika   MeSH
• proteiny 14-3-3 genetika   MeSH
• signální transdukce genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• mitogenem aktivované proteinkinasy p38 MeSH
• proteinkinasy MeSH
• proteiny 14-3-3 MeSH

Many picornaviruses hijack the Golgi resident Acyl-coenzyme A binding domain containing 3 (ACBD3) protein in order to recruit the phosphatidylinositol 4-kinase B (PI4KB) to viral replication organelles (ROs). PI4KB, once recruited and activated by ACBD3 protein, produces the lipid phosphatidylinositol 4-phosphate (PI4P), which is a key step in the biogenesis of viral ROs. To do so, picornaviruses use their small nonstructural protein 3A that binds the Golgi dynamics domain of the ACBD3 protein. Here, we present the analysis of the highly flexible ACBD3 proteins and the viral 3A protein in solution using small-angle X-ray scattering and computer simulations. Our analysis revealed that both the ACBD3 protein and the 3A:ACBD3 protein complex have an extended and flexible conformation in solution.

• Klíčová slova
• ACBD3, RNA virus, coarse-grained simulations, host factor, intrinsically disordered regions, picornavirus, small-angle X-ray scattering (SAXS),
• MeSH
• acylkoenzym A chemie   metabolismus   MeSH
• adaptorové proteiny signální transdukční chemie   metabolismus   MeSH
• lidé MeSH
• membránové proteiny chemie   metabolismus   MeSH
• Picornaviridae chemie   metabolismus   MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ACBD3 protein, human MeSH Prohlížeč
• acylkoenzym A MeSH
• adaptorové proteiny signální transdukční MeSH
• membránové proteiny MeSH