Signaling by the human C-type lectin-like receptor, natural killer (NK) cell inhibitory receptor NKR-P1, has a critical role in many immune-related diseases and cancer. C-type lectin-like receptors have weak affinities to their ligands; therefore, setting up a comprehensive model of NKR-P1-LLT1 interactions that considers the natural state of the receptor on the cell surface is necessary to understand its functions. Here we report the crystal structures of the NKR-P1 and NKR-P1:LLT1 complexes, which provides evidence that NKR-P1 forms homodimers in an unexpected arrangement to enable LLT1 binding in two modes, bridging two LLT1 molecules. These interaction clusters are suggestive of an inhibitory immune synapse. By observing the formation of these clusters in solution using SEC-SAXS analysis, by dSTORM super-resolution microscopy on the cell surface, and by following their role in receptor signaling with freshly isolated NK cells, we show that only the ligation of both LLT1 binding interfaces leads to effective NKR-P1 inhibitory signaling. In summary, our findings collectively support a model of NKR-P1:LLT1 clustering, which allows the interacting proteins to overcome weak ligand-receptor affinity and to trigger signal transduction upon cellular contact in the immune synapse.

• MeSH
• Antigens, Surface MeSH
• Killer Cells, Natural * MeSH
• X-Ray Diffraction MeSH
• NK Cell Lectin-Like Receptor Subfamily B MeSH
• Lectins, C-Type MeSH
• Humans MeSH
• Ligands MeSH
• Scattering, Small Angle MeSH
• Receptors, Cell Surface * MeSH
• Cluster Analysis MeSH
• Synapses MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antigens, Surface MeSH
• NK Cell Lectin-Like Receptor Subfamily B MeSH
• Lectins, C-Type MeSH
• Ligands MeSH
• Receptors, Cell Surface * MeSH

Targeted cancer immunotherapy is a promising tool for restoring immune surveillance and eradicating cancer cells. Hydrophilic polymers modified with coiled coil peptide tags can be used as universal carriers designed for cell-specific delivery of such biologically active proteins. Here, we describe the preparation of pHPMA-based copolymer conjugated with immunologically active protein B7-H6 via complementary coiled coil VAALEKE (peptide E) and VAALKEK (peptide K) sequences. Receptor B7-H6 was described as a binding partner of NKp30, and its expression has been proven for various tumor cell lines. The binding of B7-H6 to NKp30 activates NK cells and results in Fas ligand or granzyme-mediated apoptosis of target tumor cells. In this work, we optimized the expression of coiled coil tagged B7-H6, its ability to bind activating receptor NKp30 has been confirmed by isothermal titration calorimetry, and the binding stoichiometry of prepared chimeric biopolymer has been characterized by analytical ultracentrifugation. Furthermore, this coiled coil B7-H6-loaded polymer conjugate activates NK cells in vitro and, in combination with coiled coil scFv, enables their targeting towards a model tumor cell line. Prepared chimeric biopolymer represents a promising precursor for targeted cancer immunotherapy by activating the cytotoxic activity of natural killer cells.

• Keywords
• B7-H6, HPMA polymer, NK cell, NKp30, coiled coil, immunotherapy,
• 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

In this article, we present an enhanced sampling method based on a hybrid Hamiltonian which combines experimental distance restraints with a bias dependent from multiple path-dependent variables. This simulation method determines the bias-coordinates on the fly and does not require a priori knowledge about reaction coordinates. The hybrid Hamiltonian accelerates the sampling of proteins, and, combined with experimental distance information, the technique considers the restraints adaptively and in dependency of the system's intrinsic dynamics. We validate the methodology on the dipole relaxation of two water models and the conformational landscape of dialanine. Using experimental NMR-restraint data, we explore the folding landscape of the TrpCage mini-protein and in a second example apply distance restraints from chemical crosslinking/mass spectrometry experiments for the sampling of the conformation space of the Killer Cell Lectin-like Receptor Subfamily B Member 1A (NKR-P1A). The new methodology has the potential to adaptively introduce experimental restraints without affecting the conformational space of the system along an ergodic trajectory. Since only a limited number of input- and no-order parameters are required for the setup of the simulation, the method is broadly applicable and has the potential to be combined with coarse-graining methods.

• Keywords
• enhanced molecular dynamics simulations, protein folding,
• MeSH
• Time Factors MeSH
• Magnetic Resonance Spectroscopy MeSH
• Peptides chemistry   MeSH
• Molecular Dynamics Simulation * MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Peptides MeSH

Human LLT1 is a C-type lectin-like ligand of NKR-P1 (CD161, gene KLRB1), a C-type lectin-like receptor of natural killer cells. Using X-ray diffraction, the first experimental structures of human LLT1 were determined. Four structures of LLT1 under various conditions were determined: monomeric, dimeric deglycosylated after the first N-acetylglucosamine unit in two forms and hexameric with homogeneous GlcNAc2Man5 glycosylation. The dimeric form follows the classical dimerization mode of human CD69. The monomeric form keeps the same fold with the exception of the position of an outer part of the long loop region. The hexamer of glycosylated LLT1 consists of three classical dimers. The hexameric packing may indicate a possible mode of interaction of C-type lectin-like proteins in the glycosylated form.

• Keywords
• C-type lectin-like ligand, LLT1,
• MeSH
• Glycosylation MeSH
• Protein Structure, Quaternary MeSH
• NK Cell Lectin-Like Receptor Subfamily B chemistry   genetics   metabolism   MeSH
• Lectins, C-Type chemistry   genetics   metabolism   MeSH
• Humans MeSH
• Protein Multimerization * MeSH
• Receptors, Cell Surface chemistry   genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• CLEC2D protein, human MeSH Browser
• KLRB1 protein, human MeSH Browser
• NK Cell Lectin-Like Receptor Subfamily B MeSH
• Lectins, C-Type MeSH
• Receptors, Cell Surface MeSH

The C-type lectin-like receptors include the Nkrp1 protein family that regulates the activity of natural killer (NK) cells. Rat Nkrp1a was reported to bind monosaccharide moieties in a Ca2+-dependent manner in preference order of GalNac > GlcNAc >> Fuc >> Gal > Man. These findings established for rat Nkrp1a have been extrapolated to all additional Nkrp1 receptors and have been supported by numerous studies over the past two decades. However, since 1996 there has been controversy and another article showed lack of interactions with saccharides in 1999. Nevertheless, several high affinity saccharide ligands were synthesized in order to utilize their potential in antitumor therapy. Subsequently, protein ligands were introduced as specific binders for Nkrp1 proteins and three dimensional models of receptor/protein ligand interaction were derived from crystallographic data. Finally, for at least some members of the NK cell C-type lectin-like proteins, the "sweet story" was impaired by two reports in recent years. It has been shown that the rat Nkrp1a and CD69 do not bind saccharide ligands such as GlcNAc, GalNAc, chitotetraose and saccharide derivatives (GlcNAc-PAMAM) do not directly and specifically influence cytotoxic activity of NK cells as it was previously described.

• MeSH
• Killer Cells, Natural * chemistry   immunology   metabolism   MeSH
• Antigens, CD * chemistry   immunology   metabolism   MeSH
• Antigens, Differentiation, T-Lymphocyte * chemistry   immunology   metabolism   MeSH
• Rats MeSH
• NK Cell Lectin-Like Receptor Subfamily B * chemistry   immunology   metabolism   MeSH
• Lectins, C-Type * chemistry   immunology   metabolism   MeSH
• Humans MeSH
• Oligosaccharides * chemistry   immunology   metabolism   MeSH
• Protein Structure, Tertiary MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antigens, CD * MeSH
• CD69 antigen MeSH Browser
• Antigens, Differentiation, T-Lymphocyte * MeSH
• KLRB1 protein, human MeSH Browser
• NK Cell Lectin-Like Receptor Subfamily B * MeSH
• Lectins, C-Type * MeSH
• Oligosaccharides * MeSH