The function of natural killer (NK) and myeloid cells depends on the balance of a number of activating and inhibitory receptors. In the past few years, much of the effort has been focused on characterizing the structure and the function of the inhibitory NK cell receptors specific for MHC class I molecules. These receptors inhibit NK cell-mediated lysis of target cells expressing class I MHC molecules, while allowing lysis of class I negative cells. Natural Killer Cell Inhibitory Receptors CD94/NKG2, with its cytoplasmic immuno-tyrosine inhibitory motif (ITIM), defines a family of MHC class I molecule mediated negative/positive signaling receptors on the surface of natural killer (NK) cells. Both chains are members of the type II transmembrane C-type lectin family of receptors (CTLR). The ligand of this receptor family has been identified to be HLA-E, which presents the signal peptides of other class I MHC molecules. We have expressed and reconstituted several forms of CD94 and NKG2A extracellular ligand binding domains and determined the crystal structure of a CD94 homodimer. Another family of inhibitory receptors, termed Killer Immunoglobulin-like Receptors (KIR), recognize the classical class I MHC molecules. We have expressed a truncated form of a KIR receptor (2DL2)that recognizes HLA-Cw3 and determined the crystal structure of KIR2DL2 and HLA-Cw3 complex. KIR binds in a nearly orthogonal orientation across the alpha1 and alpha2 helices of HLA-Cw3, directly contacting positions 7 and 8 of the peptide. No significant conformational changes in KIR are observed upon complex formation. The receptor footprint on HLA overlaps with, but is distinct from that of the T-cell receptor. Charge complementarity dominates the KIR/HLA interface and mutations disrupting interface salt bridges seriously diminished binding. While most KIR contacts are to conserved HLA-C residues, a hydrogen bond between Lys 44 of KIR2DL2 and Asn 80 of Cw3 confers the allotype specificity. KIR contact requires that P8 of the peptide to residues smaller than Val. A second KIR/HLA interface produced an ordered receptor- ligand aggregation in the crystal which may resemble receptor clustering during immune synapse formation. NKG2D, a member of CTLR superfamily and distantly related to NKG2A, B, C, and E, is found on NK cells to trigger cytotoxicity against certain tumor cells and on CD8+ T cells to provide a co-stimulatory signal against virally infected cells. More recently, a group of human cytomegalovirus glycoproteins UL16 binding proteins, named as ULBPs, have been identified as ligands to human NKG2D. We have expressed both NKG2D and its ligands ULBP2, and ULBP3. We also completed the structure of NKG2D and ULBP3 complex at 2.6 angstrom resolution. In the NKG2D/ULBP3 complex, the structure of ULBP3 resembles that of the a1 and a2 domains of classical MHC molecules without a bound peptide. The lack of a3 and b2m domains is compensated by replacing two hydrophobic patches at the underside of class I MHC beta-sheet floor with a group of hydrophilic and charged residues in ULBP3. NKG2D binds diagonally across the ULBP3 helices, creating a complementary interface, an asymmetrical subunit orientation and local conformational adjustments in the receptor. The interface is stabilized primarily by hydrogen bonds and hydrophobic interactions. Unlike the KIR receptors that recognize a conserved HLA region by a lock-and-key mechanism, NKG2D recognizes diverse ligands by an induced-fit mechanism.
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