Metabotropic glutamate receptors play major roles in the activation of excitatory synapses in the central nerve system. We determined the crystal structure of the entire extracellular region of the group II receptor and that of the ligand-binding region of the group III receptor. A comparison among groups I, II, and III provides the structural basis that could account for the discrimination of groupspecific agonists. Furthermore, the structure of group II includes the cysteine-rich domain, which is tightly linked to the ligand-binding domain by a disulfide bridge, suggesting a potential role in transmitting a ligand-induced conformational change into the downstream transmembrane region. The structure also reveals the lateral interaction between the two cysteine-rich domains, which could stimulate clustering of the dimeric receptors on the cell surface. We propose a general activation mechanism of the dimeric receptor coupled with both ligand-binding and interprotomer rearrangements.crystallization ͉ cysteine-rich region ͉ ligand binding ͉ G protein-coupled receptor

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Superantigens (SAGs) are a class of immunostimulatory proteins of bacterial or viral origin that activate T cells by binding to the V beta domain of the T cell antigen receptor (TCR). The three-dimensional structure of the complex between a TCR beta chain (mouse V beta8.2) and the SAG staphylococcal enterotoxin B (SEB) at 2.4 A resolution reveals why SEB recognizes only certain V beta families, as well as why only certain SAGs bind mouse V beta8.2. Models of the TCR-SEB-peptide/MHC class II complex indicate that V alpha interacts with the MHC beta chain in the TCR-SAG-MHC complex. The extent of the interaction is variable and is largely determined by the geometry of V alpha/V beta domain association. This variability can account for the preferential expression of certain V alpha regions among T cells reactive with SEB.

Lectin-like, oxidized low-density lipoprotein (LDL) receptor 1, LOX-1, is the major receptor for oxidized LDL (OxLDL) in endothelial cells. We have determined the crystal structure of the ligand binding domain of LOX-1, with a short stalk region connecting the domain to the membrane-spanning region, as a homodimer linked by an interchain disulfide bond. In vivo assays with LOX-1 mutants revealed that the "basic spine," consisting of linearly aligned arginine residues spanning over the dimer surface, is responsible for ligand binding. Single amino acid substitution in the dimer interface caused a severe reduction in LOX-1 binding activity, suggesting that the correct dimer arrangement is crucial for binding to OxLDL. Based on the LDL model structure, possible binding modes of LOX-1 to OxLDL are proposed.

Alanine scanning mutagenesis, double mutant cycles, and X-ray crystallography were used to characterize the interface between the anti-hen egg white lysozyme (HEL) antibody D1.3 and HEL. Twelve out of the 13 nonglycine contact residues on HEL, as determined by the high-resolution crystal structure of the D1.3-HEL complex, were individually truncated to alanine. Only four positions showed a DeltaDeltaG (DeltaGmutant - DeltaGwild-type) of greater than 1.0 kcal/mol, with HEL residue Gln121 proving the most critical for binding (DeltaDeltaG = 2.9 kcal/mol). These residues form a contiguous patch at the periphery of the epitope recognized by D1.3. To understand how potentially disruptive mutations in the antigen are accommodated in the D1.3-HEL interface, we determined the crystal structure to 1.5 A resolution of the complex between D1.3 and HEL mutant Asp18 --> Ala. This mutation results in a DeltaDeltaG of only 0.3 kcal/mol, despite the loss of a hydrogen bond and seven van der Waals contacts to the Asp18 side chain. The crystal structure reveals that three additional water molecules are stably incorporated in the antigen-antibody interface at the site of the mutation. These waters help fill the cavity created by the mutation and form part of a rearranged solvent network linking the two proteins. To further dissect the energetics of specific interactions in the D1.3-HEL interface, double mutant cycles were carried out to measure the coupling of 14 amino acid pairs, 10 of which are in direct contact in the crystal structure. The highest coupling energies, 2.7 and 2.0 kcal/mol, were measured between HEL residue Gln121 and D1.3 residues VLTrp92 and VLTyr32, respectively. The interaction between Gln121 and VLTrp92 consists of three van der Waals contacts, while the interaction of Gln121 with VLTyr32 is mediated by a hydrogen bond. Surprisingly, however, most cycles between interface residues in direct contact in the crystal structure showed no significant coupling. In particular, a number of hydrogen-bonded residue pairs were found to make no net contribution to complex stabilization. We attribute these results to accessibility of the mutation sites to water, such that the mutated residues exchange their interaction with each other to interact with water. This implies that the strength of the protein-protein hydrogen bonds in these particular cases is comparable to that of the protein-water hydrogen bonds they replace. Thus, the simple fact that two residues are in direct contact in a protein-protein interface cannot be taken as evidence that there necessarily exists a productive interaction between them. Rather, the majority of such contacts may be energetically neutral, as in the D1.3-HEL complex.

An ATP-dependent protease, FtsH, digests misassembled membrane proteins in order to maintain membrane integrity and digests short-lived soluble proteins in order to control their cellular regulation. This enzyme has an N-terminal transmembrane segment and a C-terminal cytosolic region consisting of an AAA+ ATPase domain and a protease domain. Here we present two crystal structures: the protease domain and the whole cytosolic region. The cytosolic region fully retains an ATP-dependent protease activity and adopts a three-fold-symmetric hexameric structure. The protease domains displayed a six-fold symmetry, while the AAA+ domains, each containing ADP, alternate two orientations relative to the protease domain, making "open" and "closed" interdomain contacts. Apparently, ATPase is active only in the closed form, and protease operates in the open form. The protease catalytic sites are accessible only through a tunnel following from the AAA+ domain of the adjacent subunit, raising a possibility of translocation of polypeptide substrate to the protease sites through this tunnel.

The atomic view of the active site coupling termed channelling is a major subject in molecular biology. We have determined two distinct crystal structures of the bacterial multienzyme complex that catalyzes the last three sequential reactions in the fatty acid b-oxidation cycle. The a 2 b 2 heterotetrameric structure shows the uneven ring architecture, where all the catalytic centers of 2-enoyl-CoA hydratase (ECH), L-3-hydroxyacyl-CoA dehydrogenase (HACD) and 3-ketoacyl-CoA thiolase (KACT) face a large inner solvent region. The substrate, anchored through the 3 0 -phosphate ADP moiety, allows the fatty acid tail to pivot from the ECH to HACD active sites, and finally to the KACT active site. Coupling with striking domain rearrangements, the incorporation of the tail into the KACT cavity and the relocation of 3 0 -phosphate ADP bring the reactive C2-C3 bond to the correct position for cleavage. The ahelical linker specific for the multienzyme contributes to the pivoting center formation and the substrate transfer through its deformation. This channelling mechanism could be applied to other b-oxidation multienzymes, as revealed from the homology model of the human mitochondrial trifunctional enzyme complex.

FtsH is a cytoplasmic membrane-integrated, ATP-dependent metalloprotease, which processively degrades both cytoplasmic and membrane proteins in concert with unfolding. The FtsH protein is divided into the N-terminal transmembrane region and the larger C-terminal cytoplasmic region, which consists of an ATPase domain and a protease domain. We have determined the crystal structures of the Thermus thermophilus FtsH ATPase domain in the nucleotide-free and AMP-PNP- and ADP-bound states, in addition to the domain with the extra preceding segment. Combined with the mapping of the putative substrate binding region, these structures suggest that FtsH internally forms a hexameric ring structure, in which ATP binding could cause a conformational change to facilitate transport of substrates into the protease domain through the central pore.