Primary and secondary functions of HLA-E are determined by stability and conformation of the peptide-bound complexes.

MHC-E regulates NK cells by displaying MHC class Ia signal peptides (VL9) to NKG2A:CD94 receptors. MHC-E can also present sequence-diverse, lower-affinity, pathogen-derived peptides to T cell receptors (TCRs) on CD8+ T cells. To understand these affinity differences, human MHC-E (HLA-E)-VL9 versus pathogen-derived peptide structures are compared. Small-angle X-ray scatter (SAXS) measures biophysical parameters in solution, allowing comparison with crystal structures. For HLA-E-VL9, there is concordance between SAXS and crystal parameters. In contrast, HLA-E-bound pathogen-derived peptides produce larger SAXS dimensions that reduce to their crystallographic dimensions only when excess peptide is supplied. Further crystallographic analysis demonstrates three amino acids, exclusive to MHC-E, that not only position VL9 close to the α2 helix, but also allow non-VL9 peptide binding with re-configuration of a key TCR-interacting α2 region. Thus, non-VL9-bound peptides introduce an alternative peptide-binding motif and surface recognition landscape, providing a likely basis for VL9- and non-VL9-HLA-E immune discrimination.

Mouse and human antibodies bind HLA-E-leader peptide complexes and enhance NK cell cytotoxicity.

The non-classical class Ib molecule human leukocyte antigen E (HLA-E) has limited polymorphism and can bind HLA class Ia leader peptides (VL9). HLA-E-VL9 complexes interact with the natural killer (NK) cell receptors NKG2A-C/CD94 and regulate NK cell-mediated cytotoxicity. Here we report the isolation of 3H4, a murine HLA-E-VL9-specific IgM antibody that enhances killing of HLA-E-VL9-expressing cells by an NKG2A+ NK cell line. Structural analysis reveal that 3H4 acts by preventing CD94/NKG2A docking on HLA-E-VL9. Upon in vitro maturation, an affinity-optimized IgG form of 3H4 showes enhanced NK killing of HLA-E-VL9-expressing cells. HLA-E-VL9-specific IgM antibodies similar in function to 3H4 are also isolated from naïve B cells of cytomegalovirus (CMV)-negative, healthy humans. Thus, HLA-E-VL9-targeting mouse and human antibodies isolated from the naïve B cell antibody pool have the capacity to enhance NK cell cytotoxicity.

Oligomeric Architecture of Mouse Activating Nkrp1 Receptors on Living Cells.

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.

Pathogen-derived HLA-E bound epitopes reveal broad primary anchor pocket tolerability and conformationally malleable peptide binding.

Through major histocompatibility complex class Ia leader sequence-derived (VL9) peptide binding and CD94/NKG2 receptor engagement, human leucocyte antigen E (HLA-E) reports cellular health to NK cells. Previous studies demonstrated a strong bias for VL9 binding by HLA-E, a preference subsequently supported by structural analyses. However, Mycobacteria tuberculosis (Mtb) infection and Rhesus cytomegalovirus-vectored SIV vaccinations revealed contexts where HLA-E and the rhesus homologue, Mamu-E, presented diverse pathogen-derived peptides to CD8+ T cells, respectively. Here we present crystal structures of HLA-E in complex with HIV and Mtb-derived peptides. We show that despite the presence of preferred primary anchor residues, HLA-E-bound peptides can adopt alternative conformations within the peptide binding groove. Furthermore, combined structural and mutagenesis analyses illustrate a greater tolerance for hydrophobic and polar residues in the primary pockets than previously appreciated. Finally, biochemical studies reveal HLA-E peptide binding and exchange characteristics with potential relevance to its alternative antigen presenting function in vivo.

Solution structure of the lymphocyte receptor Nkrp1a reveals a distinct conformation of the long loop region as compared to in the crystal structure.

Mouse Nkrp1a receptor is a C-type lectin-like receptor expressed on the surface of natural killer cells that play an important role against virally infected and tumor cells. The recently solved crystal structure of Nkrp1a raises questions about a long loop region which was uniquely extended from the central region in the crystal. To understand the functional significance of the loop, the solution structure of Nkrp1a using nuclear magnetic resonance (NMR) spectroscopy was determined. A notable difference between the crystal and NMR structure of Nkrp1a appears in the conformation of the long loop region. While the extended loop points away from the central core and mediates formation of a domain swapped dimer in the crystal, the solution structure is monomeric with the loop tightly anchored to the central region. The findings described the first solution structure in the Nkrp1 family and revealed intriguing similarities and differences to the crystal structure. Proteins 2016; 84:1304-1311. © 2016 Wiley Periodicals, Inc.

Nkrp1 family, from lectins to protein interacting molecules.

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.

Re-evaluation of binding properties of recombinant lymphocyte receptors NKR-P1A and CD69 to chemically synthesized glycans and peptides.

The binding of monosaccharides and short peptides to lymphocyte receptors (human CD69 and rat NKR-P1A) was first reported in 1994 and then in a number of subsequent publications. Based on this observation, numerous potentially high-affinity saccharide ligands have been synthesized over the last two decades in order to utilize their potential in antitumor therapy. Due to significant inconsistencies in their reported binding properties, we decided to re-examine the interaction between multiple ligands and CD69 or NKR-P1A. Using NMR titration and isothermal titration calorimetry we were unable to detect the binding of the tested ligands such as N-acetyl-D-hexosamines and oligopeptides to both receptors, which contradicts the previous observations published in more than twenty papers over the last fifteen years.

Re-evaluation of the involvement of NK cells and C-type lectin-like NK receptors in modulation of immune responses by multivalent GlcNAc-terminated oligosaccharides.

Recognition of glycosylation patterns is one of the basic features of innate immunity. Ability of C-type lectin-like receptors such as NKR-P1 to bind saccharide moieties has become recently a controversial issue. In the present study, binding assay with soluble fluorescently labeled recombinant rat NKR-P1A and mouse NKR-P1C proteins revealed apparently no affinity to the various neoglycoproteins. Lack of functional linkage between NKR-P1 and previously described saccharide binder was supported by the fact, that synthetic N-acetyl-D-glucosamine octabranched dendrimer on polyamidoamine scaffold (GN8P) did not change gene expression of NKR-P1 isoforms in C57BL/6 and BALB/c mice divergent in the NK gene complex (both in vitro and in vivo). Surprisingly, N-acetyl-D-glucosamine-coated tetrabranched polyamido-amine dendrimer specifically binds to NKT cells and macrophages but not to NK cells (consistently with changes in cytokine patterns). Despite the fact that GN8P has been tested as an immunomodulator in anti-cancer treatment animal models for many years, surprisingly no changes in cytokine profiles in serum relevant to anti-cancer responses using B16F10 and CT26 harboring mouse strains C57BL/6 and BALB/c are observed. Our results indicate possible indirect involvement of NK cells in GN8P mediated immune responses.

Molecular architecture of mouse activating NKR-P1 receptors.

Receptors belonging to NKR-P1 family and their specific Clr ligands form an alternative missing self recognition system critical in immunity against tumors and viruses, elimination of tumor cells subjected to genotoxic stress, activation of T cell dependent immune response, and hypertension. The three-dimensional structure of the extracellular domain of the mouse natural killer (NK) cell receptor mNKR-P1Aex has been determined by X-ray diffraction. The core of the C-type lectin domain (CTLD) is homologous to the other CTLD receptors whereas one quarter of the domain forms an extended loop interacting tightly with a neighboring loop in the crystal. This domain swapping mechanism results in a compact interaction interface. A second dimerization interface resembles the known arrangement of other CTLD NK receptors. A functional dimeric form of the receptor is suggested, with the loop, evolutionarily conserved within this family, proposed to participate in interactions with ligands.

Synthetic N-acetyl-D-glucosamine based fully branched tetrasaccharide, a mimetic of the endogenous ligand for CD69, activates CD69+ killer lymphocytes upon dimerization via a hydrophilic flexible linker.

On the basis of the highly branched ovomucoid-type undecasaccharide that had been shown previously to be an endogenous ligand for CD69 leukocyte receptor, a systematic investigation of smaller oligosaccharide mimetics was performed based on linear and branched N-acetyl-d-hexosamine homooligomers prepared synthetically using hitherto unexplored reaction schemes. The systematic structure-activity studies revealed the tetrasaccharide GlcNAcbeta1-3(GlcNAcbeta1-4)(GlcNAcbeta1-6)GlcNAc (compound 52) and its alpha-benzyl derivative 49 as the best ligand for CD69 with IC(50) as high as 10(-9) M. This compound thus approaches the affinity of the classical high-affinity neoglycoprotein ligand GlcNAc(23)BSA. Compound 68, GlcNAc tetrasaccharide 52 dimerized through a hydrophilic flexible linker, turned out to be effective in activating CD69(+) lymphocytes. It also proved efficient in enhancing natural killing in vitro, decreasing the growth of tumors in vivo, and activating the CD69(+) tumor infiltrating lymphocytes examined ex vivo. This compound is thus a candidate for carbohydrate-based immunomodulators with promising antitumor potential.

Cooperation between subunits is essential for high-affinity binding of N-acetyl-D-hexosamines to dimeric soluble and dimeric cellular forms of human CD69.

CD69 is an earliest lymphocyte activation antigen and a universal leukocyte triggering molecule expressed at sites of active immune response. The binding of GlcNAc to the dimeric human CD69 was followed by equilibrium dialysis, fluorescence titration, and NMR. Clear cooperation was observed in the high-affinity binding (K(d) = 4.0 x 10(-7) M) of the carbohydrate to two subunits of the dimeric CD69 (Hill coefficient 1.94). A control monosaccharide ManNAc was not bound by human CD69, and both monosaccharides had no effects on the structure of the receptor. However, a monomeric CD69 obtained by mutating Q93 and R134 at the dimer interface exhibited a much lower affinity for GlcNAc (K(d) = 1.3 x 10(-5) M) and no cooperativity (Hill coefficient 1.07). Perturbation of the dimer interface resulted in a severe impairment of the signaling ability of cellular CD69 when cross-linked with an antibody or with a bivalent high-affinity N-acetylhexosamine dimer-based ligand. The availability of stable preparations of soluble CD69 receptor with well-documented ligand binding properties will be beneficial for immunological experiments evaluating the role of this antigen in the complex environment of the immune system. Moreover, such preparations in combination with efficient ligand mimetics able to both activate CD69(+) lymphocytes and to block undesired hyperactivation caused by other cellular ligands will also become indispensable tools in explaining the exact role of the CD69 antigen in the interaction between the tumor cell and the effector natural killer lymphocyte.