Interferon-γ receptor 2 is a cell-surface receptor that is required for interferon-γ signalling and therefore plays a critical immunoregulatory role in innate and adaptive immunity against viral and also bacterial and protozoal infections. A crystal structure of the extracellular part of human interferon-γ receptor 2 (IFNγR2) was solved by molecular replacement at 1.8 Å resolution. Similar to other class 2 receptors, IFNγR2 has two fibronectin type III domains. The characteristic structural features of IFNγR2 are concentrated in its N-terminal domain: an extensive π-cation motif of stacked residues KWRWRH, a NAG-W-NAG sandwich (where NAG stands for N-acetyl-D-glucosamine) and finally a helix formed by residues 78-85, which is unique among class 2 receptors. Mass spectrometry and mutational analyses showed the importance of N-linked glycosylation to the stability of the protein and confirmed the presence of two disulfide bonds. Structure-based bioinformatic analysis revealed independent evolutionary behaviour of both receptor domains and, together with multiple sequence alignment, identified putative binding sites for interferon-γ and receptor 1, the ligands of IFNγR2.

• Klíčová slova
• class 2 cytokine receptors, fibronectin type III domain, interferon-γ receptor 2,
• MeSH
• aminokyselinové motivy MeSH
• disulfidy chemie   MeSH
• glykosylace MeSH
• konformace proteinů MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• molekulární modely MeSH
• proteinové domény MeSH
• receptory interferonů chemie   MeSH
• sbalování proteinů MeSH
• stabilita proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• disulfidy MeSH
• IFNGR2 protein, human MeSH Prohlížeč
• receptory interferonů MeSH

BACKGROUND: β-N-Acetylhexosaminidase (GH20) from the filamentous fungus Talaromyces flavus, previously identified as a prominent enzyme in the biosynthesis of modified glycosides, lacks a high resolution three-dimensional structure so far. Despite of high sequence identity to previously reported Aspergillus oryzae and Penicilluim oxalicum β-N-acetylhexosaminidases, this enzyme tolerates significantly better substrate modification. Understanding of key structural features, prediction of effective mutants and potential substrate characteristics prior to their synthesis are of general interest. RESULTS: Computational methods including homology modeling and molecular dynamics simulations were applied to shad light on the structure-activity relationship in the enzyme. Primary sequence analysis revealed some variable regions able to influence difference in substrate affinity of hexosaminidases. Moreover, docking in combination with consequent molecular dynamics simulations of C-6 modified glycosides enabled us to identify the structural features required for accommodation and processing of these bulky substrates in the active site of hexosaminidase from T. flavus. To access the reliability of predictions on basis of the reported model, all results were confronted with available experimental data that demonstrated the principal correctness of the predictions as well as the model. CONCLUSIONS: The main variable regions in β-N-acetylhexosaminidases determining difference in modified substrate affinity are located close to the active site entrance and engage two loops. Differences in primary sequence and the spatial arrangement of these loops and their interplay with active site amino acids, reflected by interaction energies and dynamics, account for the different catalytic activity and substrate specificity of the various fungal and bacterial β-N-acetylhexosaminidases.

• MeSH
• beta-N-acetylhexosaminidasy chemie   metabolismus   MeSH
• fylogeneze MeSH
• glykosylace MeSH
• katalytická doména MeSH
• kinetika MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• reprodukovatelnost výsledků MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• simulace molekulární dynamiky MeSH
• substrátová specifita MeSH
• Talaromyces enzymologie   MeSH
• výpočetní biologie * MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• beta-N-acetylhexosaminidasy MeSH

BACKGROUND: Fungal beta-N-acetylhexosaminidases catalyze the hydrolysis of chitobiose into its constituent monosaccharides. These enzymes are physiologically important during the life cycle of the fungus for the formation of septa, germ tubes and fruit-bodies. Crystal structures are known for two monomeric bacterial enzymes and the dimeric human lysosomal beta-N-acetylhexosaminidase. The fungal beta-N-acetylhexosaminidases are robust enzymes commonly used in chemoenzymatic syntheses of oligosaccharides. The enzyme from Aspergillus oryzae was purified and its sequence was determined. RESULTS: The complete primary structure of the fungal beta-N-acetylhexosaminidase from Aspergillus oryzae CCF1066 was used to construct molecular models of the catalytic subunit of the enzyme, the enzyme dimer, and the N-glycosylated dimer. Experimental data were obtained from infrared and Raman spectroscopy, and biochemical studies of the native and deglycosylated enzyme, and are in good agreement with the models. Enzyme deglycosylated under native conditions displays identical kinetic parameters but is significantly less stable in acidic conditions, consistent with model predictions. The molecular model of the deglycosylated enzyme was solvated and a molecular dynamics simulation was run over 20 ns. The molecular model is able to bind the natural substrate - chitobiose with a stable value of binding energy during the molecular dynamics simulation. CONCLUSION: Whereas the intracellular bacterial beta-N-acetylhexosaminidases are monomeric, the extracellular secreted enzymes of fungi and humans occur as dimers. Dimerization of the fungal beta-N-acetylhexosaminidase appears to be a reversible process that is strictly pH dependent. Oligosaccharide moieties may also participate in the dimerization process that might represent a unique feature of the exclusively extracellular enzymes. Deglycosylation had only limited effect on enzyme activity, but it significantly affected enzyme stability in acidic conditions. Dimerization and N-glycosylation are the enzyme's strategy for catalytic subunit stabilization. The disulfide bridge that connects Cys448 with Cys483 stabilizes a hinge region in a flexible loop close to the active site, which is an exclusive feature of the fungal enzymes, neither present in bacterial nor mammalian structures. This loop may play the role of a substrate binding site lid, anchored by a disulphide bridge that prevents the substrate binding site from being influenced by the flexible motion of the loop.

• MeSH
• Aspergillus oryzae enzymologie   MeSH
• beta-N-acetylhexosaminidasy chemie   izolace a purifikace   metabolismus   MeSH
• dimerizace MeSH
• glykosylace MeSH
• koncentrace vodíkových iontů MeSH
• konformace proteinů MeSH
• molekulární modely MeSH
• počítačová simulace * MeSH
• Ramanova spektroskopie metody   MeSH
• spektroskopie infračervená s Fourierovou transformací metody   MeSH
• stabilita enzymů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• beta-N-acetylhexosaminidasy MeSH