Abstract2B4, a transmembrane receptor expressed primarily on natural killer (NK) cells and on a subset of CD8 + T cells, plays an important role in activating NK-mediated cytotoxicity through its interaction with CD48 on target cells. We report here the atomic-resolution structure of the ligand-binding (D1) domain of 2B4 in solution determined by nuclear magnetic resonance (NMR) spectroscopy. The overall main chain structure resembles an immunoglobulin variable (V) domain fold, very similar to that seen previously for domain 1 of CD2 and CD4. The structure contains nine β-strands assembled into two β-sheets conventionally labeled DEB and AGFCC'C". The six-stranded sheet (AGFCC'C") contains structural features that may have implications for ligand recognition and receptor function. A noncanonical disulfide bridge between Cys2 and Cys99 stabilizes a long and parallel β-structure between strand A (residues 3-12) and strand G (residues 100-108). A β-bulge at residues Glu45 and Ile46 places a bend in the middle of strand C' that orients two conserved and adjacent hydrophobic residues (Ile46 and Leu47) inside the β-sandwich as seen in other V domains. Finally, the FG-loop (implicated in ligand recognition in the CD2-CD58 complex) is dynamically disordered in 2B4 in the absence of a ligand. We propose that ligand binding to 2B4 might stabilize the structure of the FG-loop in the ligand complex.Natural killer (NK) 1 cells are a fundamental component of the innate immune system that play a vital role in the detection and destruction of virally infected and tumor cells (1,2). The cytolytic activity of NK cells is regulated by a dynamic interplay between activating and inhibitory signals transmitted by distinct classes of receptors found on their surface. The dominant signal received by an NK cell through its interaction with normal levels of major histocompatibility complex (MHC) class I on target cells is inhibitory. When expression of MHC class I is reduced through infectious or tumorigenic processes, this inhibitory signal is attenuated, and the NK cell is activated. In this way, cells with abnormal MHC class I expression become targets of NK lytic activity that results from loss of inhibition of NK cell activation. Inhibitory receptors specific for MHC class I include the killer immunoglobulinlike receptors (KIRs), members of the Ly49 family, and the CD94/NKG2D family (3). Activating receptors include 2B4, CD16, CD44, CD69, NKR-P1, NKp30, NKp44, and NKp46 (1,2). However, only for some of these stimulatory receptors are the physiological ligands known: CD48 for 2B4 (4), IgG Fc for CD16 (5), and Clr for NKR-P1 (6). The balance between positive signaling receptors (resulting in target cell lysis) and negative signaling receptors (preventing lysis) ultimately determines the outcome of NK cell-target cell encounters (1,2
EXPERIMENTAL PROCEDURES Protein Expression and PurificationA DNA fragment encoding the ligand-binding domain of rat 2B4 (residues 1-112, referred to throughout the text as simply 2B4) was generated by PCR...