NKp30 is one of the main human natural killer (NK) cell activating receptors used in directed immunotherapy. The oligomerization of the NKp30 ligand binding domain depends on the length of the C-terminal stalk region, but our structural knowledge of NKp30 oligomerization and its role in signal transduction remains limited. Moreover, ligand binding of NKp30 is affected by the presence and type of N-glycosylation. In this study, we assessed whether NKp30 oligomerization depends on its N-glycosylation. Our results show that NKp30 forms oligomers when expressed in HEK293S GnTI- cell lines with simple N-glycans. However, NKp30 was detected only as monomers after enzymatic deglycosylation. Furthermore, we characterized the interaction between NKp30 and its best-studied cognate ligand, B7-H6, with respect to glycosylation and oligomerization, and we solved the crystal structure of this complex with glycosylated NKp30, revealing a new glycosylation-induced mode of NKp30 dimerization. Overall, this study provides new insights into the structural basis of NKp30 oligomerization and explains how the stalk region and glycosylation of NKp30 affect its ligand affinity. This furthers our understanding of the molecular mechanisms involved in NK cell activation, which is crucial for the successful design of novel NK cell-based targeted immunotherapeutics.

• Keywords
• B7-H6, NK cell, NKp30, glycosylation, oligomerization,
• Publication type
• Journal Article MeSH

Working at the border between innate and adaptive immunity, natural killer (NK) cells play a key role in the immune system by protecting healthy cells and by eliminating malignantly transformed, stressed or virally infected cells. NK cell recognition of a target cell is mediated by a receptor "zipper" consisting of various activating and inhibitory receptors, including C-type lectin-like receptors. Among this major group of receptors, two of the largest rodent receptor families are the NKR-P1 and the Clr receptor families. Although these families have been shown to encode receptor-ligand pairs involved in MHC-independent self-nonself discrimination and are a target for immune evasion by tumour cells and viruses, structural mechanisms of their mutual recognition remain less well characterized. Therefore, we developed a non-viral eukaryotic expression system based on transient transfection of suspension-adapted human embryonic kidney 293 cells to produce soluble native disulphide dimers of NK cell C-type lectin-like receptor ectodomains. The expression system was optimized using green fluorescent protein and secreted alkaline phosphatase, easily quantifiable markers of recombinant protein production. We describe an application of this approach to the recombinant protein production and characterization of native rat NKR-P1B and Clr-11 proteins suitable for further structural and functional studies.

• MeSH
• HEK293 Cells MeSH
• Rats MeSH
• NK Cell Lectin-Like Receptor Subfamily B chemistry   genetics   metabolism   MeSH
• Humans MeSH
• Protein Multimerization MeSH
• Calcitonin Receptor-Like Protein chemistry   genetics   metabolism   MeSH
• Protein Domains MeSH
• Protein Engineering methods   MeSH
• Recombinant Proteins chemistry   genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Calcrl protein, rat MeSH Browser
• NK Cell Lectin-Like Receptor Subfamily B MeSH
• Calcitonin Receptor-Like Protein MeSH
• Recombinant Proteins MeSH

Mouse activating Nkrp1 proteins are commonly described as type II transmembrane receptors with disulfide-linked homodimeric structure. Their function and the manner in which Nkrp1 proteins of mouse strain (C57BL/6) oligomerize are still poorly understood. To assess the oligomerization state of Nkrp1 proteins, mouse activating EGFP-Nkrp1s were expressed in mammalian lymphoid cells and their oligomerization evaluated by Förster resonance energy transfer (FRET). Alternatively, Nkrp1s oligomers were detected by Western blotting to specify the ratio between monomeric and dimeric forms. We also performed structural characterization of recombinant ectodomains of activating Nkrp1 receptors. Nkrp1 isoforms c1, c2 and f were expressed prevalently as homodimers, whereas the Nkrp1a displays larger proportion of monomers on the cell surface. Cysteine-to-serine mutants revealed the importance of all stalk cysteines for protein dimerization in living cells with a major influence of cysteine at position 74 in two Nkrp1 protein isoforms. Our results represent a new insight into the oligomerization of Nkrp1 receptors on lymphoid cells, which will help to determine their function.

• Keywords
• Förster resonance energy transfer, Nkrp1, cysteine, dimerization, disulfide bond arrangement,
• MeSH
• Antigens, Ly analysis   MeSH
• Chlorocebus aethiops MeSH
• COS Cells MeSH
• Jurkat Cells MeSH
• NK Cell Lectin-Like Receptor Subfamily B analysis   chemistry   MeSH
• Humans MeSH
• Protein Multimerization MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Receptors, Immunologic analysis   MeSH
• Protein Refolding MeSH
• Fluorescence Resonance Energy Transfer MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Antigens, Ly MeSH
• Klrb1a protein, mouse MeSH Browser
• Klrb1c protein, mouse MeSH Browser
• NK Cell Lectin-Like Receptor Subfamily B MeSH
• Nkrp1f protein, mouse MeSH Browser
• Receptors, Immunologic MeSH

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.

• Keywords
• class 2 cytokine receptors, fibronectin type III domain, interferon-γ receptor 2,
• MeSH
• Amino Acid Motifs MeSH
• Disulfides chemistry   MeSH
• Glycosylation MeSH
• Protein Conformation MeSH
• Crystallography, X-Ray MeSH
• Humans MeSH
• Models, Molecular MeSH
• Protein Domains MeSH
• Receptors, Interferon chemistry   MeSH
• Protein Folding MeSH
• Protein Stability MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Disulfides MeSH
• IFNGR2 protein, human MeSH Browser
• Receptors, Interferon MeSH

Receptor proteins at the cell surface regulate the ability of natural killer cells to recognize and kill a variety of aberrant target cells. The structural features determining the function of natural killer receptor proteins 1 (NKR-P1s) are largely unknown. In the present work, refined homology models are generated for the C-type lectin-like extracellular domains of rat NKR-P1A and NKR-P1B, mouse NKR-P1A, NKR-P1C, NKR-P1F, and NKR-P1G, and human NKR-P1 receptors. Experimental data on secondary structure, tertiary interactions, and thermal transitions are acquired for four of the proteins using Raman and infrared spectroscopy. The experimental and modeling results are in agreement with respect to the overall structures of the NKR-P1 receptor domains, while suggesting functionally significant local differences among species and isoforms. Two sequence regions that are conserved in all analyzed NKR-P1 receptors do not correspond to conserved structural elements as might be expected, but are represented by loop regions, one of which is arranged differently in the constructed models. This region displays high flexibility but is anchored by conserved sequences, suggesting that its position relative to the rest of the domain might be variable. This loop may contribute to ligand-binding specificity via a coupled conformational transition.

• MeSH
• Amino Acid Motifs MeSH
• Phylogeny MeSH
• Conserved Sequence * MeSH
• Rats MeSH
• NK Cell Lectin-Like Receptor Subfamily B chemistry   classification   MeSH
• Humans MeSH
• Molecular Sequence Data MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Spectrum Analysis, Raman MeSH
• Amino Acid Sequence MeSH
• Sequence Alignment MeSH
• Molecular Dynamics Simulation MeSH
• Spectroscopy, Fourier Transform Infrared MeSH
• Structural Homology, Protein MeSH
• Protein Structure, Tertiary MeSH
• Thermodynamics MeSH
• Binding Sites MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• NK Cell Lectin-Like Receptor Subfamily B MeSH

Fungal β-N-acetylhexosaminidases are enzymes that are used in the chemoenzymatic synthesis of biologically interesting oligosaccharides. The enzyme from Aspergillus oryzae was produced and purified from its natural source and crystallized using the hanging-drop vapour-diffusion method. Diffraction data from two crystal forms (primitive monoclinic and primitive tetragonal) were collected to resolutions of 3.2 and 2.4 Å, respectively. Electrophoretic and quantitative N-terminal protein-sequencing analyses confirmed that the crystals are formed by a complete biologically active enzyme consisting of a glycosylated catalytic unit and a noncovalently attached propeptide.

• MeSH
• Aspergillus oryzae enzymology   MeSH
• beta-N-Acetylhexosaminidases chemistry   metabolism   MeSH
• Glycosylation MeSH
• Catalytic Domain MeSH
• Crystallization MeSH
• Crystallography, X-Ray MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Names of Substances
• beta-N-Acetylhexosaminidases MeSH