The phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis exhibits several types of interfacial activation. In the crystal structure of the closely related Bacillus cereus PI-PLC, the rim of the active site is flanked by a short helix B and a loop that show an unusual clustering of hydrophobic amino acids. Two of the seven tryptophans in PI-PLC are among the exposed residues. To test the importance of these residues in substrate and activator binding, we prepared several mutants of Trp-47 (in helix B) and Trp-242 (in the loop). Two other tryptophans, Trp-178 and Trp-280, which are not near the rim, were mutated as controls. Kinetic (both phosphotransferase and cyclic phosphodiesterase activities), fluorescence, and vesicle binding analyses showed that both Trp-47 and Trp-242 residues are important for the enzyme to bind to interfaces, both activating zwitterionic and substrate anionic surfaces. Partitioning of the enzyme to vesicles is decreased more than 10-fold for either W47A or W242A, and removal of both tryptophans (W47A/W242A) yields enzyme with virtually no affinity for phospholipid surfaces. Replacement of either tryptophan with phenylalanine or isoleucine has moderate effects on enzyme affinity for surfaces but yields a fully active enzyme. These results are used to describe how the enzyme is activated by interfaces. Phosphatidylinositol-specific phospholipase C (PI-PLC)1 catalyzes the hydrolysis of PI in two steps: (i) an intramolecular phosphotransferase reaction to form inositol 1,2-cyclic-phosphate (cIP), followed by (ii) a cyclic phosphodiesterase activity that converts cIP to inositol 1-phosphate (I1P). Although the enzymes in eukaryotes play key roles in generating membraneassociated second messengers and in some case water-soluble second messengers (1, 2), PI-PLC enzymes in bacteria are secreted and play critical roles in cell infectivity (3, 4). The PI-PLC from Bacillus thuringiensis exhibits several types of kinetic interfacial activation by interfaces. Micellar PI is a better substrate than monomeric PI (5, 6), and interfaces of phosphatidylcholine, a nonsubstrate that does not bind at the active site, activate the enzyme for both PI cleavage (7) and cIP hydrolysis (8). In the available crystal structure (9) of the closely related Bacillus cereus PI-PLC, bound myo-inositol localized the active site inside the ␤␣ barrel. However, the orientation of PI substrate side chains and any other sites for interfacial PC were not defined.The rim of the active site of bacterial PI-PLC (9) has a short helix B and one particular loop (residues 237-243) that show an unusual clustering of hydrophobic amino acids that are fully exposed to solvent (Fig. 1). This structural characteristic could contribute to the binding of substrate fatty acyl chains (for PI) but also to the binding of the PC activator. Tryptophan residues are often elements inserted into bilayers when a peripheral protein binds (e.g. annexin V (10) or phospholipase A 2 (11)). Two of the seven tryptophans (Trp-47 an...

The activating NKG2D receptor plays a critical role in innate and adaptive immune responses by natural killer cells and subpopulations of T cells. The human receptor assembles with the DAP10 signaling dimer, and it is thought that one NKG2D homodimer pairs with a single DAP10 dimer by formation of two salt bridges between charged transmembrane (TM) residues. However, direct stoichiometry measurements demonstrated that one NKG2D homodimer assembles with four DAP10 chains. Selective mutation of one of the basic TM residues of NKG2D resulted in loss of two DAP10 chains, indicating that each TM arginine serves as an interaction site for a DAP10 dimer. Assembly of the hexameric structure was cooperative because this mutation also significantly reduced NKG2D dimerization. A monomeric NKG2D TM peptide was sufficient for assembly with a DAP10 dimer, indicating that the interaction between these proteins occurs in the membrane environment. Formation of a three-helix interface among the TM domains involved ionizable residues from all three chains, the TM arginine of NKG2D and both TM aspartic acids of the DAP10 dimer. The organization of the TM domains thus shows similarities to the T cell antigen receptor-CD3 complex, in particular to the six-chain assembly intermediate between T cell antigen receptor and the CD3␦ and CD3␥ dimers. Binding of a single ligand can thus result in phosphorylation of four DAP10 chains, which may be relevant for the sensitivity of NKG2D receptor signaling, in particular in situations of low ligand density.natural killer cells ͉ stoichiometry ͉ membrane biochemistry T he NKG2D receptor recognizes a group of ligands that represent distant relatives of MHC class I molecules, and expression of these ligands is induced or up-regulated by infected and transformed cells (1)(2)(3)(4)(5)(6)(7)(8). In innate immune responses mediated by natural killer (NK) cells, NKG2D serves as a primary activating receptor and ligand binding triggers cytotoxicity and cytokine production. The receptor is also expressed by CD8 T cells and ␥␦ T cells and provides important costimulatory signals in T cell-mediated adaptive immune responses by amplifying T cell cytokine production and proliferation (2, 3, 9, 10). The human NKG2D ligands MICA and MICB have a domain organization similar to MHC class I molecules (␣1, ␣2, and ␣3 domains) but do not associate with ␤ 2 -microglobulin or peptide (1). The NKG2D homodimer binds in a diagonal orientation across the surface of the ␣1 and ␣2 domains, and each receptor monomer contacts one of the long ␣-helices of the ␣1-␣2 platform, reminiscent of the binding of T cell antigen receptor (TCR) ␣␤ heterodimers to MHC͞peptide complexes (11). The human NKG2D ligands UL binding proteins 1, 2, 3, and 4 and the murine ligands RAE-1␣-, H60, and murine UL binding protein-like transcript 1 lack the membrane-proximal ␣3 domain and are quite dissimilar in sequence (4, 5, 12), but NKG2D nevertheless binds in a similar diagonal orientation to the ␣1-␣2 domains of these proteins (13, 14).The NKG2D rec...

Activating receptors in cells of hematopoetic origin include members of two unrelated protein families, the immunoglobulin (Ig) and C type lectins, which differ even in the orientation of the transmembrane (TM) domains. We examined assembly of four receptors with diverse function: the NK receptors KIR2DS and NKG2C/CD94, the Fc receptor for IgA, and the GPVI collagen receptor. For each of the four different receptors studied here, assembly results in the formation of a three-helix interface in the membrane involving two acidic TM residues from the signaling dimer and a basic TM residue from the ligand recognition module, an arrangement remarkably similar to the T cell receptor (TCR)-CD3 complex. The fact that the TM domains of Ig family and C type lectins adopt opposite orientations proves that these receptor families independently evolved toward the same structural arrangement of the interacting TM helices. This assembly mechanism is thus widely utilized by receptors in cells of hematopoetic origin.