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Mitochondria play a key role in apoptosis due to their capacity to release potentially lethal proteins. One of these latent death factors is cytochrome c, which can stimulate the proteolytic activation of caspase zymogens. Another important protein is apoptosis-inducing factor (AIF), a flavoprotein that can stimulate a caspase-independent cell-death pathway required for early embryonic morphogenesis. Here, we report the crystal structure of mouse AIF at 2.0 Å. Its active site structure and redox properties suggest that AIF functions as an electron transferase with a mechanism similar to that of the bacterial ferredoxin reductases, its closest evolutionary homologs. However, AIF structurally differs from these proteins in some essential features, including a long insertion in a C-terminal β-hairpin loop, which may be related to its apoptogenic functions.

Sialidases are present on the surface of several trypanosomatid protozoan parasites. They are highly specific for sialic acid linked in α-(2,3) to a terminal β-galactose and include the strictly hydrolytic enzymes and trans-sialidases (sialyl-transferases). Based on the structural comparison of the sialidase from Trypanosoma rangeli and the trans-sialidase from T. cruzi (the agent of Chagas’ disease in humans), we have explored the role of specific amino acid residues sought to be important for substrate specificity. The substitution of a conserved tryptophanyl residue in the two enzymes, Trp312/313-Ala, changed substrate specificity, rendering the point mutants capable to hydrolyze both α-(2,3)- and α-(2,6)-linked sialoconjugates. The same mutation abolished sialyl-transferase activity, indicating that transfer (but not hydrolysis) requires a precise orientation of the bound substrate. The exchange substitution of another residue that modulates oligosaccharide binding, Gln284-Pro, was found to significantly increase the hydrolytic activity of sialidase, and residue Tyr119 was confirmed to be part of a second binding site for the acceptor substrate in trans-sialidase. Together with the structural information, these results provide a consistent framework to account for the unique enzymatic properties of trypanosome trans-sialidases.

ABSTRACTTrypanosoma cruzi, the agent of Chagas' disease, expresses trans-sialidase, a unique enzyme activity that enables the parasite to invade host cells by transferring sialyl residues from host glyconjugates to the parasite's surface acceptor molecules. The enzyme is also shed into the surrounding environment, causing apoptosis in cells from the immune system. During infections, an antibody response against the catalytic region of the trans-sialidase that is coincident with the control of the parasitemia and survival of the host is observed. This low-titer humoral response is characterized by its persistence for many years in benznidazole-treated patients. Here we analyzed the antigenic structure of the molecule by phage-displayed peptide combinatorial libraries and SPOT synthesis. Several epitopes were defined and located on the three-dimensional model of the enzyme. Unexpectedly, cross-reaction was found among several epitopes distributed in different locations displaying nonconsensus sequences. This finding was confirmed by the reactivity of three monoclonal antibodies able to recognize non-sequence-related peptides that together constitute the surface surrounding the catalytic site of the enzyme. The presence of cross-reacting epitopes within a single molecule suggests a mechanism developed to avoid a strong humoral response by displaying an undefined target to the immune system.

The three-dimensional structure of the major horse allergen Equ c 1 has been determined at 2.3 Å resolution by x-ray crystallography. Equ c 1 displays the typical fold of lipocalins, a β-barrel flanked by a C-terminal α-helix. The space between the two β-sheets of the barrel defines an internal cavity that could serve, as in other lipocalins, for the binding and transport of small hydrophobic ligands. Equ c 1 crystallizes in a novel dimeric form, which is distinct from that observed in other lipocalin dimers and corresponds to the functional form of the allergen. Binding studies of point mutants of the allergen with specific monoclonal antibodies raised in mouse and IgE serum from horse allergic patients allowed to identify putative B cell antigenic determinants. In addition, total inhibition of IgE serum recognition by a single specific monoclonal antibody revealed the restricted nature of the IgE binding target on the molecular surface of Equ c 1.

Interleukin (IL)-2 interacts with two types of functional receptors (IL-2Rαβγ and IL-2Rβγ) and acts on a broad range of target cells involved in inflammatory reactions and immune responses. For the first time, we show that a chemically synthesized fragment of the IL-2 sequence can fold into a molecule mimicking the quaternary structure of a hemopoietin. Indeed, peptide p1–30 (containing amino acids 1–30, covering the entire α helix A of IL-2) spontaneously folds into an α-helical homotetramer and stimulates the growth of T cell lines expressing human IL-2Rβ, whereas shorter versions of the peptide lack helical structure and are inactive. We also demonstrate that this neocytokine interacts with a previously undescribed dimeric form of IL-2Rβ. In agreement with its binding to IL-2Rβ, p1–30 activates Shc and p56lck but unlike IL-2, fails to activate Janus kinase (Jak)1, Jak3, and signal transducer and activator of transcription 5 (STAT5). Unexpectedly, we also show that p1–30 activates Tyk2, thus suggesting that IL-2Rβ may bind to different Jaks depending on its oligomerization. At the cellular level, p1–30 induces lymphokine-activated killer (LAK) cells and preferentially activates CD8low lymphocytes and natural killer cells, which constitutively express IL-2Rβ. A significant interferon γ production is also detected after p1–30 stimulation. A mutant form of p1–30 (Asp20→Lys), which is likely unable to induce vascular leak syndrome, remains capable of generating LAK cells, like the original p1–30 peptide. Altogether, our data suggest that p1–30 has therapeutic potential.

A series of mutants bearing single amino acid substitutions often encountered in the catalase/peroxidase, KatG, from isoniazid-resistant isolates of Mycobacterium tuberculosis has been produced by site-directed mutagenesis. The resultant enzymes were overexpressed, purified and characterized. Replacing Cys-20 by Ser abolished disulphide-bridge formation, but did not affect either dimerization of the enzyme or catalysis. The substitution of Thr-275, which is probably involved in electron transfer from the haem, by proline resulted in a highly unstable enzyme with insignificant enzyme activities. The most commonly occurring substitution in drug-resistant clinical isolates is the replacement of Ser-315 by Thr; this lowered catalase and peroxidase activities by 50% and caused a significant decrease in the KatG-mediated inhibition of the activity of the NADH-dependent enoyl-[acyl-carrier protein] reductase, InhA, in vitro. The ability of this enzyme to produce free radicals from isoniazid was severely impaired, as judged by its loss of NitroBlue Tetrazolium reduction activity. Replacement of Leu-587 by Pro resulted in marked instability of KatG, indicating that the C-terminal domain is also important for structural and functional integrity.

The barrier function of mitochondrial membranes is perturbed early during the apoptotic process. Here we show that the mitochondria contain a caspase-like enzymatic activity cleaving the caspase substrate Z-VAD.afc, in addition to three biological activities previously suggested to participate in the apoptotic process: (a) cytochrome c; (b) an apoptosis-inducing factor (AIF) which causes isolated nuclei to undergo apoptosis in vitro; and (c) a DNAse activity. All of these factors, which are biochemically distinct, are released upon opening of the permeability transition (PT) pore in a coordinate, Bcl-2–inhibitable fashion. Caspase inhibitors fully neutralize the Z-VAD.afc–cleaving activity, have a limited effect on the AIF activity, and have no effect at all on the DNase activities. Purification of proteins reacting with the biotinylated caspase substrate Z-VAD, immunodetection, and immunodepletion experiments reveal the presence of procaspase-2 and -9 in mitochondria. Upon induction of PT pore opening, these procaspases are released from purified mitochondria and become activated. Similarly, upon induction of apoptosis, both procaspases redistribute from the mitochondrion to the cytosol and are processed to generate enzymatically active caspases. This redistribution is inhibited by Bcl-2. Recombinant caspase-2 and -9 suffice to provoke full-blown apoptosis upon microinjection into cells. Altogether, these data suggest that caspase-2 and -9 zymogens are essentially localized in mitochondria and that the disruption of the outer mitochondrial membrane occurring early during apoptosis may be critical for their subcellular redistribution and activation.

The structure of the peroxisomal catalase A from the budding yeast Saccharomyces cerevisiae, with 515 residues per subunit, has been determined and refined to 2.4 A˚ resolution. The crystallographic agreement factors R and Rfree are 15.4 % and 19.8 %, respectively. A tetramer with accurate 222-molecular symmetry is located in the asymmetric unit of the crystal. The conformation of the central core of catalase A, about 300 residues, remains similar to the structure of catalases from distantly related organisms. In contrast, catalase A lacks a carboxy-terminal domain equivalent to that found in catalase from Penicillium vitalae, the only other fungal catalase structure available. Structural peculiarities related with the heme and NADP(H) binding pockets can be correlated with biochemical characteristics of the catalase A enzyme. The network of molecular cavities and channels, filled with solvent molecules, supports the existence of one major substrate entry and at least two possible alternative pathways to the heme active site. The structure of the variant protein Val111Ala, also determined by X-ray crystallography at 2.8 A˚ resolution, shows a few, well-localized, differences with respect to the wild-type enzyme. These differences, that include the widening of the entry channel in its narrowest point, provide an explanation for both the increased peroxidatic activity and the reduced catalatic activity of this mutant.

The crystal structure ofClostridium thermocellumendoglucanase CelD revealed an extended NH₂-terminal segment (involving residues from the putative leader peptide) sticking out from the enzyme core to interact with a symmetry related molecule through an intermolecular salt bridge (Lys38-Asp201). Enzymatic digestion of CelD with various proteases emphasized the flexibility of the NH₂-segment in solution. Proteolytic removal of Lys38 or the substitution of bridge-forming residues by site-directed mutagenesis promoted crystal packing arrangements that differ from that of wild type CelD. Crystals of wild-type CelD (a=99.3 Åc=191.8 Å) are trigonal, space groupP3₁21, with one molecule in the asymmetric unit (form A), whereas crystals of papain-treated CelD (a=100.4 Å,c=248.7 Å), of CelD_K38M(a=100.1 Å,c=248.4 Å) and of papain-treated CelD_D201A(a=99.9 Å,c=250.0 Å) are trigonal, space groupP3₁21, with two crystallographically independent molecules (form B), and crystals of chymotrypsin-treated CelD (a=100.0 Å,c=254.3 Å) and of CelD_D201A(a=99.8 Å,c=254.7 Å) are hexagonal, space groupP6₁22, with one molecule in the asymmetric unit (form C). Only chymotrypsin-treated CelD (which preserves both Lys38 and Asp201) can grow in crystal form A upon macroseeding, indicating that formation of the intermolecular salt bridge is critical for stability of this crystal form. Flexible NH₂- and COOH-terminal peptide extensions were found to influence crystal nucleation, but not crystal growth. The crystal structures of papain-treated CelD and chymotrypsin-treated CelD, determined at 3.5 Å resolution by molecular replacement techniques, demonstrate that a small change in molecular orientation promoted by Lys38 account for the differences between crystal forms B and C.

The partial amino acid sequence of the tetrameric isolectin B4 from Vicia villosaseeds has been determined by peptide analysis, and its three‐dimensional structure solved by molecular replacement techniques and refined at 2.9 Å resolution to a crystallographic R‐factor of 21%. Each subunit displays the thirteen‐stranded β‐barrel topology characteristic of legume lectins. The amino acid residues involved in metal‐ and sugar‐binding are similar to those of other GalNAc‐specific lectins, indicating that residues outside the carbohydrate‐binding pocket modulate the affinity for the Tn glycopeptide. Isolectin B4 displays an unusual quaternary structure, probably due to protein glycosylation.

The gene encoding the major horse allergen, designated Equus caballusallergen 1 (Equ c1), was cloned from total cDNA of sublingual salivary glands by reverse transcription-polymerase chain reaction using synthetic degenerate oligonucleotides deduced from N-terminal and internal peptide sequences of the glycosylated hair dandruff protein. A recombinant form of the protein, with a polyhistidine tail, was expressed in Escherichia coliand purified by immobilized metal affinity chromatography. The recombinant protein is able to induce a passive cutaneous anaphylaxis reaction in rat, and it behaves similarly to the native Equ c1 in several immunological tests with allergic patients' IgE antibodies, mouse monoclonal antibodies, or rabbit polyclonal IgG antibodies. Amino acid sequence identity of 49-51% with rodent urinary proteins from mice and rats suggests that Equ c1 is a new member of the lipocalin superfamily of hydrophobic ligand-binding proteins that includes several other major allergens. An RNA blot analysis demonstrates the expression of mRNA Equ c1 in liver and in sublingual and submaxillary salivary glands.

CD45 is a receptor-like protein tyrosine phosphatase critically involved in the regulation of initial effector functions in B- and T-cells. The protein comprises two phosphatase (PTP) domains in its cytoplasmic region. However, whether each PTP domain has enzyme activity by itself or whether both domains are required to build up a functional enzyme is unclear. We have studied different constructions of human CD45 comprising the two PTP domains, both separately and as a single protein, fused to maltose-binding protein (MBP). In apparent contrast with previous studies, we show that the first PTP domain of CD45 (when fused to MBP) may be a viable phosphatase in the absence of the second domain. Phosphatase activity resides in the monomeric form of the protein and is lost after proteolytic cleavage of the fusion partner, indicating that MBP specifically activates the first PTP domain. Furthermore, changes in the optimal pH for activity with respect to wild-type CD45 suggest that protein–protein interactions involving residues in the neighbourhood of the catalytic site mediate enzyme activation.

The structures of the Glu140→Gln mutant ofClostridium thermocellumendoglucanase CelC in unliganded form (CelC_E140Q) and in complex with cellohexaose (CelC_E140Q– Glc₆) have been refined to crystallographicR-factors of 19.4% at 1.9 Å and 17.8% at 2.3 Å resolution, respectively. The structure of CelC_E140Q– Glc₆complex shows two

The pivaloyloxy methyl 6,6-dihalo penicillanates 1a, 1b, and 1chave been stereoselectively prepared from the reaction of pivaloyloxy methyl 6-diazo penicillanate 2with either N-halosuccinimide/halide or the interhalogens Xl (X = Cl, Br). The crystal structures of 3S,5R,6Rpivaloyloxy methyl 6-bromo, 6-chloro penicillanate1a; 3S,5R,6Rpivaloyloxy methyl 6-iodo, 6-bromo penicillanate 1b, and 3S, 5R, 6R pivaloyloxymethyl 6-iodo, 6-chloro penicillanate 1chave been determined by X-ray single crystal analysis. The stereochemistry of the displacement reaction is discussed.

The quaternary organization of the cellulosome, a multi-enzymatic extracellular complex produced by cellulolytic bacteria, depends on specific interactions between dockerin domains, double EF-hand subunits carried by the catalytic components, and cohesin domains, individual receptor subunits linearly arranged within a non-catalytic scaffolding polypeptide. Cohesin-dockerin complexes with distinct specificities are also thought to mediate the attachment of cellulosomes to the cell membrane.

The structure of TEM-1 β-lactamase complexed with the inhibitor BLIP has been determined at 1.7 Å resolution. The two tandemly repeated domains of BLIP form a polar, concave surface that docks onto a predominantly polar, convex protrusion on the enzyme. The ability of BLIP to adapt to a variety of class A β-lactamases is most likely due to an observed flexibility between the two domains of the inhibitor and to an extensive layer of water molecules entrapped between the enzyme and inhibitor. A β-hairpin loop from domain 1 of BLIP is inserted into the active site of the β-lactamase. The carboxylate of Asp 49 forms hydrogen bonds to four conserved, catalytic residues in the β-lactamase, thereby mimicking the position of the penicillin G carboxylate observed in the acyl–enzyme complex of TEM-1 with substrate. This β-hairpin may serve as a template with which to create a new family of peptide-analogue β-lactamase inhibitors.

Interleukin 2 (IL-2) interacts with a receptor (IL-2R) composed of three subunits (IL-2Rα, IL-2Rβ and IL-2Rγ). IL-2Rβ plays a critical role in signal transduction. An anti-human IL-2 mAb (H2-8) produced after immunization with peptide 1-30 of IL-2 was found to recognize the region occupied by Asp20, at the exposed interface between α-helices A and C. Muteins at position 17 and 20 are not recognized by mAb H2-8. mAb H2-8 specifically inhibits the IL-2 proliferation of TS1β cells which are dependent on the expression of human Il-2Rβ chain for IL-2 proliferation. Substitution at internal position Leu17 demonstrated that this position is essential for IL-2 binding and IL-2 bioactivity. New IL-2 mutants at position Asp20 have been analysed. Substitutions Asp→Asn, Asp→Lys, Asp→Leu, show a correlation between diminished affinity for IL-2 receptor and reduced bioactivity measured on TS1β cells. Mutein Asp→Arg lose affinity for IL-2R and bioactivity simultaneously. Furthermore, during the course of the study we have found that mutein Asp20→Leu is an IL-2 antagonist. The biological effects of mAb H2-8 and the properties of new mutants at positions 17 and 20 demonstrate that this region of α helix-A is involved in IL-2–IL-2Rβ interactions.

The secreted protein Equ c 1 is the major component responsible for the induction of specific IgE antibodies in patients sensitized to horse allergens. Equ c 1 belongs to the lipocalin superfamily of hydrophobic ligand‐binding proteins, which also includes other known allergens. Equilibrium sedimentation and gel‐filtration studies demonstrate that both the glycosylated form of Equ c 1 purified from horse salivary glands and the non‐glycosylated recombinant form expressed in bacteria exist predominantly as dimers in solution. As observed for other dimeric lipocalins, acidic pH and low protein concentration favour dimer dissociation. The recombinant form of Equ c 1 has been crystallized using ammonium sulfate as a precipitant. The crystals belong to the tetragonal space group P41212 with cell parameters a= b= 84.0, c= 56.1 Å, and contain a single molecule in the asymmetric unit. A complete data set from native crystals was collected at the synchrotron source in Hamburg to 2.9 Å resolution using a frozen crystal, and structure determination is in progress.

The catalytic domain of a thermostable xylanase from Clostridium thermocellum has been expressed in Escherichia coli and crystallized from a polyethylene glycol 2000 solution by the hanging drop method. Crystals belong to the triclinic space group P1 with cell dimensions a = 46·8 Å, b = 50·8 Å, c = 70·3 Å, α = 100·7°, β = 83·8° γ = 101·6°, and two molecules in the unit cell. These crystals diffract X-rays to at least 1·8 Å resolution and are suitable for high-resolution X-ray analysis. Copyright 1994, 1999 Academic Press

Isolectin B4isolated from Vicia villosaseeds is specific for the Tn antigen, a carcinoma-associated molecular marker. Crystals of the isolectin grown in the presence of carbohydrate are tetragonal, space group P41(or P43), with a= 91·3 Å, c= 151·7 Å and one tetramer in the asymmetric unit. The crystals diffract X-rays to 2·8 Å resolution and are suitable for high-resolution structural analysis.

The FHA domain of mycobacterial GarA mediates interactions with both phosphorylated and nonphosphorylated partners.

Carbapenems are “last resort” β-lactam antibiotics used to treat serious and life-threatening health care-associated infections caused by multidrug-resistant Gram-negative bacteria. Unfortunately, the worldwide spread of genes coding for carbapenemases among these bacteria is threatening these life-saving drugs.

In this issue of Structure, we describe the crystal structure of the Ser/Thr protein phosphatase PstP from Mycobacterium tuberculosis, opening new perspectives to understand the putative roles of "eukaryotic-like" signaling elements in bacteria.