The biosynthesis of the lincosamide antibiotics lincomycin A and celesticetin involves the pyridoxal-5'-phosphate (PLP)-dependent enzymes LmbF and CcbF, which are responsible for bifurcation of the biosynthetic pathways. Despite recognizing the same S-glycosyl-L-cysteine structure of the substrates, LmbF catalyses thiol formation through β-elimination, whereas CcbF produces S-acetaldehyde through decarboxylation-coupled oxidative deamination. The structural basis for the diversification mechanism remains largely unexplored. Here we conduct structure-function analyses of LmbF and CcbF. X-ray crystal structures, docking and molecular dynamics simulations reveal that active-site aromatic residues play important roles in controlling the substrate binding mode and the reaction outcome. Furthermore, the reaction selectivity and oxygen-utilization of LmbF and CcbF were rationally engineered through structure- and calculation-based mutagenesis. Thus, the catalytic function of CcbF was switched to that of LmbF, and, remarkably, both LmbF and CcbF variants gained the oxidative-amidation activity to produce an unnatural S-acetamide derivative of lincosamide.

• MeSH
• katalytická doména MeSH
• krystalografie rentgenová MeSH
• linkosamidy * chemie   biosyntéza   metabolismus   MeSH
• pyridoxalfosfát * metabolismus   chemie   MeSH
• simulace molekulární dynamiky MeSH
• simulace molekulového dockingu MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• linkosamidy * MeSH
• pyridoxalfosfát * MeSH

Brassinosteroids (BRs) are steroidal phytohormones indispensable for plant growth, development, and responses to environmental stresses. The export of bioactive BRs to the apoplast is essential for BR signaling initiation, which requires binding of a BR molecule to the extracellular domains of the plasma membrane-localized receptor complex. We have previously shown that the Arabidopsis thaliana ATP-binding cassette (ABC) transporter ABCB19 functions as a BR exporter and, together with its close homolog ABCB1, positively regulates BR signaling. Here, we demonstrate that ABCB1 is another BR transporter. The ATP hydrolysis activity of ABCB1 can be stimulated by bioactive BRs, and its transport activity was confirmed in proteoliposomes and protoplasts. Structures of ABCB1 were determined in substrate-unbound (apo), brassinolide (BL)-bound, and ATP plus BL-bound states. In the BL-bound structure, BL is bound to the hydrophobic cavity formed by the transmembrane domain and triggers local conformational changes. Together, our data provide additional insights into ABC transporter-mediated BR export.

• Klíčová slova
• ABCB1, Arabidopsis, brassinosteroids, signaling, structure, transport,
• MeSH
• ABC transportéry * metabolismus   genetika   chemie   MeSH
• adenosintrifosfát metabolismus   MeSH
• Arabidopsis * metabolismus   genetika   MeSH
• biologický transport MeSH
• brassinosteroidy * metabolismus   MeSH
• proteiny huseníčku * metabolismus   genetika   chemie   MeSH
• steroidy heterocyklické MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• ABC transportéry * MeSH
• adenosintrifosfát MeSH
• brassinolide MeSH Prohlížeč
• brassinosteroidy * MeSH
• proteiny huseníčku * MeSH
• steroidy heterocyklické MeSH

Nucleases of the S1/P1 family have important applications in biotechnology and molecular biology. We have performed structural analyses of SmNuc1 nuclease from Stenotrophomonas maltophilia, including RNA cleavage product binding and mutagenesis in a newly discovered flexible Arg74-motif, involved in substrate binding and product release and likely contributing to the high catalytic rate. The Arg74Gln mutation shifts substrate preference towards RNA. Purine nucleotide binding differs compared to pyrimidines, confirming the plasticity of the active site. The enzyme-product interactions indicate a gradual, stepwise product release. The activity of SmNuc1 towards c-di-GMP in crystal resulted in a distinguished complex with the emerging product 5'-GMP. This enzyme from an opportunistic pathogen relies on specific architecture enabling high performance under broad conditions, attractive for biotechnologies.

• Klíčová slova
• Stenotrophomonas maltophilia, RNA, S1/P1 nuclease, X‐ray crystallography, c‐di‐GMP cleavage,
• MeSH
• bakteriální proteiny metabolismus   chemie   genetika   MeSH
• guanosinmonofosfát cyklický metabolismus   analogy a deriváty   chemie   MeSH
• katalytická doména * MeSH
• krystalografie rentgenová MeSH
• molekulární modely MeSH
• RNA metabolismus   chemie   genetika   MeSH
• Stenotrophomonas maltophilia * enzymologie   genetika   metabolismus   MeSH
• substrátová specifita MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bakteriální proteiny MeSH
• guanosinmonofosfát cyklický MeSH
• RNA MeSH

Fibroblast growth factor 2 (FGF2) is a signaling protein that plays a significant role in tissue development and repair. FGF2 binds to fibroblast growth factor receptors (FGFRs) alongside its co-factor heparin, which protects FGF2 from degradation. The binding between FGF2 and FGFRs induces intracellular signaling pathways such as RAS-MAPK, PI3K-AKT, and STAT. FGF2 has strong potential for application in cell culturing, wound healing, and cosmetics but the potential is severely limited by its low protein stability. The thermostable variant FGF2-STAB was constructed by computer-assisted protein engineering to overcome the natural limitation of FGF2. Previously reported characterization of FGF2-STAB revealed an enhanced ability to induce MAP/ERK signaling while having a lower dependence on heparin when compared with FGF2-wt. Here we report the crystal structure of FGF2-STAB solved at 1.3 Å resolution. Protein stabilization is achieved by newly formed hydrophobic interactions, polar contacts, and one additional hydrogen bond. The overall structure of FGF2-STAB is similar to FGF2-wt and does not reveal information on the experimentally observed lower dependence on heparin. A noticeable difference in flexibility in the receptor binding region can explain the differences in signaling between FGF2-STAB and its wild-type counterpart. Our structural analysis provided molecular insights into the stabilization and unique biological properties of FGF2-STAB.

• Klíčová slova
• Protein flexibility, Stabilized fibroblast growth factor 2, X-ray structural analysis,
• Publikační typ
• časopisecké články MeSH

Membrane penetration by non-enveloped viruses is diverse and generally not well understood. Enteroviruses, one of the largest groups of non-enveloped viruses, cause diseases ranging from the common cold to life-threatening encephalitis. Enteroviruses enter cells by receptor-mediated endocytosis. However, how enterovirus particles or RNA genomes cross the endosome membrane into the cytoplasm remains unknown. Here we used cryo-electron tomography of infected cells to show that endosomes containing enteroviruses deform, rupture, and release the virus particles into the cytoplasm. Blocking endosome acidification with bafilomycin A1 reduced the number of particles that released their genomes, but did not prevent them from reaching the cytoplasm. Inhibiting post-endocytic membrane remodeling with wiskostatin promoted abortive enterovirus genome release in endosomes. The rupture of endosomes also occurs in control cells and after the endocytosis of very low-density lipoprotein. In summary, our results show that cellular membrane remodeling disrupts enterovirus-containing endosomes and thus releases the virus particles into the cytoplasm to initiate infection. Since the studied enteroviruses employ different receptors for cell entry but are delivered into the cytoplasm by cell-mediated endosome disruption, it is likely that most if not all enteroviruses, and probably numerous other viruses from the family Picornaviridae, can utilize endosome rupture to infect cells.

• MeSH
• buněčná membrána ultrastruktura   virologie   MeSH
• Cercopithecus aethiops MeSH
• COS buňky MeSH
• cytoplazma virologie   MeSH
• elektronová kryomikroskopie MeSH
• endocytóza * MeSH
• endozomy * patologie   virologie   MeSH
• HeLa buňky MeSH
• lidé MeSH
• makrolidy farmakologie   MeSH
• pikornavirové infekce * virologie   MeSH
• Rhinovirus * genetika   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bafilomycin A1 MeSH Prohlížeč
• makrolidy MeSH

Phosphofructokinase-1 (PFK1) catalyzes the rate-limiting step of glycolysis, committing glucose to conversion into cellular energy. PFK1 is highly regulated to respond to the changing energy needs of the cell. In bacteria, the structural basis of PFK1 regulation is a textbook example of allostery; molecular signals of low and high cellular energy promote transition between an active R-state and inactive T-state conformation, respectively. Little is known, however, about the structural basis for regulation of eukaryotic PFK1. Here, we determine structures of the human liver isoform of PFK1 (PFKL) in the R- and T-state by cryoEM, providing insight into eukaryotic PFK1 allosteric regulatory mechanisms. The T-state structure reveals conformational differences between the bacterial and eukaryotic enzyme, the mechanisms of allosteric inhibition by ATP binding at multiple sites, and an autoinhibitory role of the C-terminus in stabilizing the T-state. We also determine structures of PFKL filaments that define the mechanism of higher-order assembly and demonstrate that these structures are necessary for higher-order assembly of PFKL in cells.

• MeSH
• adenosintrifosfát * metabolismus   MeSH
• alosterická regulace MeSH
• elektronová kryomikroskopie MeSH
• fosfofruktokinasa-1 * metabolismus   chemie   genetika   MeSH
• glykolýza MeSH
• játra enzymologie   metabolismus   MeSH
• konformace proteinů MeSH
• lidé MeSH
• molekulární modely MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adenosintrifosfát * MeSH
• fosfofruktokinasa-1 * MeSH
• PFKL protein, human MeSH Prohlížeč
• Pfkl protein, mouse MeSH Prohlížeč

Monogenic diabetes is a gateway to precision medicine through molecular mechanistic insight. Hepatocyte nuclear factor 1A (HNF-1A) and HNF-4A are transcription factors that engage in crossregulatory gene transcription networks to maintain glucose-stimulated insulin secretion in pancreatic β cells. Variants in the HNF1A and HNF4A genes are associated with maturity-onset diabetes of the young (MODY). Here, we explored 4 variants in the P2-HNF4A promoter region: 3 in the HNF-1A binding site and 1 close to the site, which were identified in 63 individuals from 21 families of different MODY disease registries across Europe. Our goal was to study the disease causality for these variants and to investigate diabetes mechanisms on the molecular level. We solved a crystal structure of HNF-1A bound to the P2-HNF4A promoter and established a set of techniques to probe HNF-1A binding and transcriptional activity toward different promoter variants. We used isothermal titration calorimetry, biolayer interferometry, x-ray crystallography, and transactivation assays, which revealed changes in HNF-1A binding or transcriptional activities for all 4 P2-HNF4A variants. Our results suggest distinct disease mechanisms of the promoter variants, which can be correlated with clinical phenotype, such as age of diagnosis of diabetes, and be important tools for clinical utility in precision medicine.

• Klíčová slova
• Diabetes, Metabolism, Structural biology, Transcription,
• MeSH
• diabetes mellitus 2. typu * genetika   metabolismus   MeSH
• hepatocytární jaderný faktor 1-alfa * genetika   metabolismus   MeSH
• hepatocytární jaderný faktor 4 * genetika   metabolismus   MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• promotorové oblasti (genetika) * genetika   MeSH
• regulace genové exprese MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• hepatocytární jaderný faktor 1-alfa * MeSH
• hepatocytární jaderný faktor 4 * MeSH
• HNF1A protein, human MeSH Prohlížeč
• HNF4A protein, human MeSH Prohlížeč

Inhibition of hypoxanthine-guanine-xanthine phosphoribosyltransferase activity decreases the pool of 6-oxo and 6-amino purine nucleoside monophosphates required for DNA and RNA synthesis, resulting in a reduction in cell growth. Therefore, inhibitors of this enzyme have potential to control infections, caused by Plasmodium falciparum and Plasmodium vivax, Trypanosoma brucei, Mycobacterium tuberculosis, and Helicobacter pylori. Five compounds synthesized here that contain a purine base covalently linked by a prolinol group to one or two phosphonate groups have Ki values ranging from 3 nM to >10 μM, depending on the structure of the inhibitor and the biological origin of the enzyme. X-ray crystal structures show that, on binding, these prolinol-containing inhibitors stimulated the movement of active site loops in the enzyme. Against TBr in cell culture, a prodrug exhibited an EC50 of 10 μM. Thus, these compounds are excellent candidates for further development as drug leads against infectious diseases as well as being potential anticancer agents.

• MeSH
• inhibitory enzymů * farmakologie   chemie   chemická syntéza   MeSH
• katalytická doména MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• molekulární modely MeSH
• molekulární struktura MeSH
• pentosyltransferasy * antagonisté a inhibitory   metabolismus   MeSH
• racionální návrh léčiv * MeSH
• Trypanosoma brucei brucei účinky léků   enzymologie   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• hypoxanthine-guanine-xanthine phosphoribosyltransferase MeSH Prohlížeč
• inhibitory enzymů * MeSH
• pentosyltransferasy * MeSH

Phosphofructokinase-1 (PFK1) catalyzes the rate-limiting step of glycolysis, committing glucose to conversion into cellular energy. PFK1 is highly regulated to respond to the changing energy needs of the cell. In bacteria, the structural basis of PFK1 regulation is a textbook example of allostery; molecular signals of low and high cellular energy promote transition between an active R-state and inactive T-state conformation, respectively Little is known, however, about the structural basis for regulation of eukaryotic PFK1. Here, we determine structures of the human liver isoform of PFK1 (PFKL) in the R- and T-state by cryoEM, providing insight into eukaryotic PFK1 allosteric regulatory mechanisms. The T-state structure reveals conformational differences between the bacterial and eukaryotic enzyme, the mechanisms of allosteric inhibition by ATP binding at multiple sites, and an autoinhibitory role of the C-terminus in stabilizing the T-state. We also determine structures of PFKL filaments that define the mechanism of higher-order assembly and demonstrate that these structures are necessary for higher-order assembly of PFKL in cells.

• Publikační typ
• časopisecké články MeSH
• preprinty MeSH

Chloride Intracellular Channel (CLIC) family members uniquely transition between soluble and membrane-associated conformations. Despite decades of extensive functional and structural studies, CLICs' function as ion channels remains debated, rendering our understanding of their physiological role incomplete. Here, we expose the function of CLIC5 as a fusogen. We demonstrate that purified CLIC5 directly interacts with the membrane and induces fusion, as reflected by increased liposomal diameter and lipid and content mixing between liposomes. Moreover, we show that this activity is facilitated by acidic pH, a known trigger for CLICs' transition to a membrane-associated conformation, and that increased exposure of the hydrophobic inter-domain interface is crucial for this process. Finally, mutation of a conserved hydrophobic interfacial residue diminishes the fusogenic activity of CLIC5 in vitro and impairs excretory canal extension in C. elegans in vivo. Together, our results unravel the long-sought physiological role of these enigmatic proteins.

• MeSH
• Caenorhabditis elegans * genetika   metabolismus   MeSH
• chloridové kanály metabolismus   MeSH
• chloridy * metabolismus   MeSH
• liposomy MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• chloridové kanály MeSH
• chloridy * MeSH
• liposomy MeSH