Fc receptors, which are expressed on the majority of hematopoietic cells, play important roles in antibody-mediated immune responses. [3][4][5]. In addition to variations in affinity, each receptor displays distinct IgG subtype specificities. Unlike the high affinity receptors that can bind monomeric antibodies, the low affinity receptors preferentially bind to and are activated by immune complexes.Human Fc␥RIII exists as two isoforms, Fc␥RIIIA and Fc␥RIIIB, that share 96% sequence identity in their extracellular immunoglobulin-binding regions. Fc␥RIIIA is expressed on macrophages, mast cells, and natural killer cells as a transmembrane receptor. In contrast, Fc␥RIIIB, present exclusively on neutrophils, is anchored by a glycosyl-phosphatidylinositol linker to the plasma membrane. Although Fc␥RIIIA associates with the immunoreceptor tyrosine-based activation motif containing Fc⑀RI ␥-chain or the T cell receptor -chain for its signaling, Fc␥RIIIB lacks a signaling component. Nevertheless, it plays an active role in triggering Ca 2ϩ mobilization and in neutrophil degranulation (6, 7). In addition, Fc␥RIIIB, in conjunction with Fc␥RIIA, activates phagocytosis, degranulation, and the oxidative burst that leads to the clearance of opsonized pathogens by neutrophils. A soluble form of Fc␥RIIIB was reported to activate the CR3 complement receptordependent inflammatory process (8).The Fc binding region on Fc␥RII and Fc␥RIII has been identified through the work of chimeric receptors with Fc⑀RI as primarily the membrane proximal domain, including both the BC and FG loops. Further site-directed mutations have revealed several residues of the receptor critical to Fc binding (9 -11). Similar regions on the ␣-chain of Fc⑀RI were also identified to be critical for IgE binding affinity (12). The receptor binding site on Fc has been located through the construction of chimeric IgG molecules and mutational analysis at the lower hinge region, residues located in the hinge region between the C H 1 and C H 2 domains and immediately adjacent to the N terminus of the C H 2 domain of IgG (13-15). In particular, residues 234 -238 (Leu-Leu-Gly-Gly-Pro) of the lower hinge of IgG1 have been implicated in the receptor binding. The corresponding region of IgE has also been implicated in the Fc⑀RI binding (16). Apart from the lower hinge region, a few residues on the C H 2 domain of an IgG2b were also suggested to interact * This work was supported by the intramural research funding of NIAID, National Institutes of Health and by INSERM, Institut Curie, France. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The recent crystal structures of Fc⑀RI␣, Fc␥RIIA, and Fc␥RIIB have each revealed a conserved Ig-like structure, with particularly the small hinge angle between the two Ig-like domains, which is unique to the Fc receptors (19 -21). We report here the crystal stru...

Transforming growth factor (TGF)-␤1, -␤2, and -␤3 are 25-kDa homodimeric polypeptides that play crucial nonoverlapping roles in embryogenesis, tissue development, carcinogenesis, and immune regulation. Here we report the 3.0-Å resolution crystal structure of the ternary complex between human TGF-␤1 and the extracellular domains of its type I and type II receptors, T␤RI and T␤RII. The TGF-␤1 ternary complex structure is similar to previously reported TGF-␤3 complex except with a 10°rotation in T␤RI docking orientation. Quantitative binding studies showed distinct kinetics between the receptors and the isoforms of TGF-␤. T␤RI showed significant binding to TGF-␤2 and TGF-␤3 but not TGF-␤1, and the binding to all three isoforms of TGF-␤ was enhanced considerably in the presence of T␤RII. The preference of TGF-␤2 to T␤RI suggests a variation in its receptor recruitment in vivo. Although TGF-␤1 and TGF-␤3 bind and assemble their ternary complexes in a similar manner, their structural differences together with differences in the affinities and kinetics of their receptor binding may underlie their unique biological activities. Structural comparisons revealed that the receptor-ligand pairing in the TGF-␤ superfamily is dictated by unique insertions, deletions, and disulfide bonds rather than amino acid conservation at the interface. The binding mode of T␤RII on TGF-␤ is unique to TGF-␤s, whereas that of type II receptor for bone morphogenetic protein on bone morphogenetic protein appears common to all other cytokines in the superfamily. Further, extensive hydrogen bonds and salt bridges are present at the high affinity cytokine-receptor interfaces, whereas hydrophobic interactions dominate the low affinity receptor-ligand interfaces.Transforming growth factor (TGF)-␤ 2 isoforms regulate the growth and differentiation of many cell types involved in normal development, immune function, and carcinogenesis (1-3). TGF-␤s are the founding members of a highly diversified family of signaling ligands and receptors, known as the TGF-␤ superfamily. To date the superfamily consists of more than 30 growth factors and cytokines, including TGF-␤s, bone morphogenetic proteins (BMPs), activins, inhibins, nodal, Müllerian inhibiting substance, growth differentiation factors, and others (4). TGF-␤s and related factors signal through two single-pass transmembrane receptors, known as the type I and type II receptors. These two receptor types have the same overall domain structure, including an extracellular ligand-binding domain displaying a three-finger toxin fold, a single transmembrane helix, and a cytosolic serine-threonine kinase domain. Signaling is initiated by the ligand, which binds the receptor extracellular domains, bringing them together and triggering a phosphorylation cascade, whereby the type II phosphorylates the type I, and the type I phosphorylates Smads, the cytoplasmic effectors of the pathway (3).Specificities have been determined based on cell-based affinity labeling studies with radiolabeled ligands and have enabled th...

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3-Deoxy-D-manno-octulosonate-8-phosphate synthase (KDO8PS) from the hyperthermophilic bacteriumAquifex aeolicus differs from its Escherichia coli counterpart in the requirement of a divalent metal for activity (Duewel, H. S., and Woodard, R. W. (2000) J. Biol. Chem. 275, 22824 -22831). Here we report the crystal structure of the A. aeolicus enzyme, which was determined by molecular replacement using E. coli KDO8PS as a model. The structures of the metal-free and Cd 2؉forms of the enzyme were determined in the uncomplexed state and in complex with various combinations of phosphoenolpyruvate (PEP), arabinose 5-phosphate (A5P), and erythrose 4-phosphate (E4P). Like the E. coli enzyme, A. aeolicus KDO8PS is a homotetramer containing four distinct active sites at the interface between subunits. The active site cavity is open in the substratefree enzyme or when either A5P alone or PEP alone binds, and becomes isolated from the aqueous phase when both PEP and A5P (or E4P) bind together. In the presence of metal, the enzyme is asymmetric and appears to alternate catalysis between the active sites located on one face of the tetramer and those located on the other face. In the absence of metal, the asymmetry is lost. Details of the active site that may be important for catalysis are visible at the high resolution achieved in these structures. Most notably, the shape of the PEPbinding pocket forces PEP to assume a distorted geometry at C-2, which might anticipate the conversion from sp 2 to sp 3 hybridization occurring during intermediate formation and which may modulate PEP reactivity toward A5P. Two water molecules are located in van der Waals contact with the si and re sides of C-2 PEP , respectively. Abstraction of a proton from either of these water molecules by a protein group is expected to elicit a nucleophilic attack of the resulting hydroxide ion on the nearby C-2 PEP , thus triggering the beginning of the catalytic cycle.3-Deoxy-D-manno-octulosonate-8-phosphate synthase (KDO8PS 1 ; phospho-2-dehydro-3-deoxyoctonate aldolase, EC 4.1.2.16) catalyzes the aldol-type condensation of phosphoenolpyruvate (PEP) with arabinose 5-phosphate (A5P) to form KDO8P and inorganic phosphate ( Fig. 1) (1). This reaction is the first step in the biosynthesis of 3-deoxy-Dmanno-octulosonate, an essential component of the lipopolysaccharide of all Gram-negative bacteria (2). A reaction very similar to KDO8P synthesis is the formation of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH7P) from erythrose 4-phosphate (E4P) and PEP catalyzed by DAH7P synthase (DAH7PS; phospho-2-dehydro-3-deoxyheptonate aldolase, EC 4.1.2.15), which constitutes the first step of the shikimate pathway (3). Earlier studies have established that KDO8PS and DAH7PS share several mechanistic characteristics. For example, in both enzymes, the condensation step of the reaction is stereospecific, involving the addition of the si face of C-3 PEP to the re face of the A5P/E4P carbonyl (4 -7). In particular, KDO8PS and DAH7PS are two of only four known PEP-utilizing e...

3-Deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase catalyzes the condensation of phosphoenolpyruvate (PEP) with arabinose 5-phosphate (A5P) to form KDO8P and inorganic phosphate. KDO8P is the phosphorylated precursor of 3-deoxy-D-manno-octulosonate, an essential sugar of the lipopolysaccharide of Gram-negative bacteria. The crystal structure of the Escherichia coli KDO8P synthase has been determined by multiple wavelength anomalous diffraction and the model has been refined to 2.4 Å (R-factor, 19.9%; R-free, 23.9%). KDO8P synthase is a homotetramer in which each monomer has the fold of a (␤/␣) 8 barrel. On the basis of the features of the active site, PEP and A5P are predicted to bind with their phosphate moieties 13 Å apart such that KDO8P synthesis would proceed via a linear intermediate. A reaction similar to KDO8P synthesis, the condensation of phosphoenolpyruvate, and erythrose 4-phosphate to form 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH7P), is catalyzed by DAH7P synthase. In the active site of DAH7P synthase the two substrates PEP and erythrose 4-phosphate appear to bind in a configuration similar to that proposed for PEP and A5P in the active site of KDO8P synthase. This observation suggests that KDO8P synthase and DAH7P synthase evolved from a common ancestor and that they adopt the same catalytic strategy. 3-Deoxy-D-manno-octulosonate (KDO)1 is an 8-carbon sugar present in the lipopolysaccharide (LPS) of all Gram-negative bacteria (1). KDO provides a link between lipid A, the membrane embedded moiety of LPS, and the O-antigen, an elongated polysaccharide chain that protrudes from the bacterial outer membrane into the surrounding environment and determines the antigenic specificity of the cell. Although the composition of the O-antigen varies between species and also between strains, the inner core region containing KDO is fairly constant among all Gram-negative bacteria (2).3-Deoxy-D-manno-octulosonate 8-phosphate synthase (KDO8P synthase, EC 4.1.2.16) plays a key role in the biosynthesis of KDO. This enzyme catalyzes the aldol-type condensation of phosphoenolpyruvate (PEP) with arabinose 5-phosphate (A5P) to form KDO8P (precursor to KDO) and inorganic phosphate ( Fig. 1) (3). Dephosphorylation of KDO8P to KDO and synthesis of CMP-KDO (from CTP and KDO) occur prior to insertion of the sugar into LPS (2). Strains of Salmonella have been isolated with mutations in KDO8P synthase that confer temperature-sensitive growth (4, 5). Such strains fail to synthesize KDO at the nonpermissive temperature, which leads to the inhibition of LPS biosynthesis and, as a consequence, to the arrest of cell growth. These studies indicate that KDO8P synthase provides an essential function for bacterial homeostasis.Earlier studies have determined that the reaction of KDO8P synthesis is a sequential process in which the binding of PEP precedes the binding of A5P and the release of inorganic phosphate precedes the release of KDO8P (6). The condensation step of the reaction is stereospecific, involving the additio...

NKG2D is known to trigger the natural killer (NK) cell lysis of various tumor and virally infected cells. In the NKG2D/ULBP3 complex, the structure of ULBP3 resembles the alpha1 and alpha2 domains of classical MHC molecules without a bound peptide. The lack of alpha3 and beta2m domains is compensated by replacing two hydrophobic patches at the underside of the class I MHC-like beta sheet floor with a group of hydrophilic and charged residues in ULBP3. NKG2D binds diagonally across the ULBP3 alpha 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.

Hepatic stellate cells (HSCs) store 75% of the body's supply of vitamin A (retinol) and play a key role in liver fibrogenesis. During liver injury, HSCs become activated and susceptible to natural killer (NK) cell killing due to increased expression of the NK cell activating ligand retinoic acid early inducible gene 1 (RAE-1). To study the mechanism by which RAE-1 is upregulated in HSCs during activation, an in vitro model of cultured mouse HSCs was employed. RAE-1 was detected at low levels in quiescent HSCs but upregulated in 4- and 7-day cultured HSCs (early activated HSCs), whereas 21-day cultured HSCs (fully activated HSCs) lost RAE-1 expression. High levels of RAE-1 in 4- and 7-day cultured HSCs correlated with their susceptibility to NK cell killing, which was diminished by treatment with RAE-1 neutralizing antibody. Furthermore, retinoic acid (RA) and retinal dehydrogenase (Raldh) levels were upregulated in early activated HSCs compared with quiescent or fully activated HSCs. Blocking RA synthesis by the Raldh inhibitor or blocking RA signaling by the retinoic acid receptor antagonist abolished upregulation of RAE-1 whereas treatment with RA induced RAE-1 expression in HSCs. In conclusion, during activation, HSCs lose retinol, which is either secreted out or oxidized into RA; the latter stimulates RAE-1 expression and sensitizes early activated HSCs to NK cell killing. In contrast, fully activated HSCs become resistant to NK cell killing because of lack of RAE1 expression, leading to chronic liver fibrosis and disease.

Serine hydroxymethyltransferase (SHMT) catalyzes the reversible cleavage of serine to form glycine and single carbon groups that are essential for many biosynthetic pathways. SHMT requires both pyridoxal phosphate (PLP) and tetrahydropteroylpolyglutamate (H4PteGlun) as cofactors, the latter as a carrier of the single carbon group. We describe here the crystal structure at 2.8 A resolution of rabbit cytosolic SHMT (rcSHMT) in two forms: one with the PLP covalently bound as an aldimine to the Nepsilon-amino group of the active site lysine and the other with the aldimine reduced to a secondary amine. The rcSHMT structure closely resembles the structure of human SHMT, confirming its similarity to the alpha-class of PLP enzymes. The structures reported here further permit identification of changes in the PLP group that accompany formation of the geminal diamine complex, the first intermediate in the reaction pathway. On the basis of the current mechanism derived from solution studies and the properties of site mutants, we are able to model the binding of both the serine substrate and the H4PteGlun cofactor. This model explains the properties of several site mutants of SHMT and offers testable hypotheses for a more detailed mechanism of this enzyme.