1. Natural Killer Cell Inhibitory Receptors CD94/NKG2, with its cytoplasmic immuno-tyrosine inhibitory motif (ITIM), defines a family of MHC class I molecules mediated negative/positive signaling receptors on the surface of natural killer cells. Both chains are members of type II transmembrane C-lectin family of receptors. Recently, 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 by themselves. The crystallization of CD94 yielded crystals of a hexagonal space group P6522 with a=b=91.7 ? and c=84.2 ?. We have subsequently determined the structure of CD94 using multiple isomorphous replacement method to 2.6 ? resolution. Another family of inhibitory receptors on NK cells, termed Killer Inhibitory Receptors have been identified recently on human NK cells to have immunoglobulin-like extracellular ligand binding domains. Members of this family are type I transmembrane glycoproteins that have either two (p58 molecules) or three (p70 molecules) Ig-like domains. To understand the function of these receptors, we have expressed and reconstituted a truncated p58 molecule, KIR 2DL2 using a bacteria expression system and in vitro refolding. Crystallization of 2DL2 have resulted an orthorhombic crystal form P212121 with a= 40.8, b=72.0, c=88.5 ?, and a trigonal crystal form P3221 with a=b=91.5, c=61.5 ?. Both crystals diffract to 2.9 ? resolution and their structures have been solved by molecular replacement method. 2. Natural Killer Cell Activating receptor CD16 CD16, an immunoglobulin Fc receptor, functions as the primary activating receptor on NK cells and mediates positive signals. Activation of CD16 upon antibody cross-linking results lysis of target cells. CD16 is also known to play a role in T cell development and function. In human, certain auto-reactive IgG immune complexes often results in symptoms of inflammatory response and autoimmune diseases, such as lupus and rheumatoid arthritis. Their extracellular ligand binding domain consists of two immunoglobulin modules and their cytoplasmic domain interacts with ITAM containing co-receptors. To date, there has not been a crystal structure available for the family of Fc receptors. As a result, the molecular mechanism governing the activation of Fc receptors remains largely unknown. In pursuing the crystallization of the ligand binding domain of a murine CD32, a homologous Fcg receptor, and its complex with the Fc portion of IgG, we have developed a method particularly effective in removing the carbohydrate moiety on the receptor under non-denaturing conditions. This method results in partial deglycosylation of the receptor and a molecular mass shifting from 45 kD to about 20 kD. Preliminary crystallization experiments have yielded small crystals of the receptor. We have constructed an E.coli expression system to express the extracellular ligand binding domain of human CD16 molecule. The initial results of in vitro refolding indicate that the soluble receptor can be successfully refolded into a monomeric form. Our goal is to form complexes between Fc fragments and this soluble CD16 receptor molecule and to determine the three dimensional structure of the complexes. In doing so, we hope to map the corresponding interaction regions and to define important residues on the receptor and the Fc domain that are responsible for the receptor activation. 3. Transcription regulator C/EBP The CCAAT-enhancer binding protein (C/EBP) is a transcription factor which regulates cell proliferation and differentiation, particularly in cell growth arrest and terminal differentiation. It belongs to a family of leucine zipper DNA-binding proteins with a basic region responsible for DNA sequence recognition. We have crystallized the DNA binding domain of C/EBP in complex with its cognate DNA. The crystals diffract to approximate 3.0 angstrom resolution and they belong to an orthorhombic space group P212121 with cell dimensions a=51.9, b=66.9 and c=140.2 ?.
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