Eukaryotic-like gephyrin and cognate membrane receptor coordinate corynebacterial cell division and polar elongation.

The order Corynebacteriales includes major industrial and pathogenic actinobacteria such as Corynebacterium glutamicum or Mycobacterium tuberculosis . Their elaborate multi-layered cell wall, composed primarily of the mycolyl-arabinogalactan-peptidoglycan complex, and their polar growth mode impose a stringent coordination between the septal divisome, organized around the tubulin-like protein FtsZ, and the polar elongasome, assembled around the tropomyosin-like protein Wag31. Here, we report the identification of two new divisome members, a gephyrin-like repurposed molybdotransferase (GLP) and its membrane receptor (GLPR). We show that the interplay between the GLPR/GLP module, FtsZ and Wag31 is crucial for orchestrating cell cycle progression. Our results provide a detailed molecular understanding of the crosstalk between two essential machineries, the divisome and elongasome, and reveal that Corynebacteriales have evolved a protein scaffold to control cell division and morphogenesis similar to the gephyrin/GlyR system that in higher eukaryotes mediates synaptic signaling through network organization of membrane receptors and the microtubule cytoskeleton.

The first alpha helix of interleukin (IL)-2 folds as a homotetramer, acts as an agonist of the IL-2 receptor beta chain, and induces lymphokine-activated killer cells.

Interleukin (IL)-2 interacts with two types of functional receptors (IL-2Ralphabetagamma and IL-2Rbetagamma) 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 alpha helix A of IL-2) spontaneously folds into an alpha-helical homotetramer and stimulates the growth of T cell lines expressing human IL-2Rbeta, 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-2Rbeta. In agreement with its binding to IL-2Rbeta, p1-30 activates Shc and p56(lck) 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-2Rbeta may bind to different Jaks depending on its oligomerization. At the cellular level, p1-30 induces lymphokine-activated killer (LAK) cells and preferentially activates CD8(low) lymphocytes and natural killer cells, which constitutively express IL-2Rbeta. A significant interferon gamma 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.

Mitochondrial release of caspase-2 and -9 during the apoptotic process.

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.

Proteomic identification of M. tuberculosis protein kinase substrates: PknB recruits GarA, a FHA domain-containing protein, through activation loop-mediated interactions.

Genes for functional Ser/Thr protein kinases (STPKs) are ubiquitous in prokaryotic genomes, but little is known about their physiological substrates and their actual involvement in bacterial signal transduction pathways. We report here the identification of GarA (Rv1827), a Forkhead-associated (FHA) domain-containing protein, as a putative physiological substrate of PknB, an essential Ser/Thr protein kinase from Mycobacterium tuberculosis. Using a global proteomic approach, GarA was found to be the best detectable substrate of the PknB catalytic domain in non-denatured whole-cell protein extracts from M. tuberculosis and the saprophyte Mycobacterium smegmatis. Enzymological and binding studies of the recombinant proteins demonstrate that docking interactions between the activation loop of PknB and the C-terminal FHA domain of GarA are required to enable efficient phosphorylation at a single N-terminal threonine residue, Thr22, of the substrate. The predicted amino acid sequence of the garA gene, including both the N-terminal phosphorylation motif and the FHA domain, is strongly conserved in mycobacteria and other related actinomycetes, suggesting a functional role of GarA in putative STPK-mediated signal transduction pathways. The ensuing model of PknB-GarA interactions suggests a substrate recruitment mechanism that might apply to other mycobacterial kinases bearing multiple phosphorylation sites in their activation loops.

The crystal structure of endoglucanase CelA, a family 8 glycosyl hydrolase from Clostridium thermocellum.

BACKGROUND: Cellulases, which catalyze the hydrolysis of glycosidic bonds in cellulose, can be classified into several different protein families. Endoglucanase CelA is a member of glycosyl hydrolase family 8, a family for which no structural information was previously available. RESULTS: The crystal structure of CelA was determined by multiple isomorphous replacement and refined to 1.65 A resolution. The protein folds into a regular (alpha/alpha)6 barrel formed by six inner and six outer alpha helices. Cello-oligosaccharides bind to an acidic cleft containing at least five D-glucosyl-binding subsites (A-E) such that the scissile glycosidic linkage lies between subsites C and D. The strictly conserved residue Glu95, which occupies the center of the substrate-binding cleft and is hydrogen bonded to the glycosidic oxygen, has been assigned the catalytic role of proton donor. CONCLUSIONS: The present analysis provides a basis for modeling homologous family 8 cellulases. The architecture of the active-site cleft, presenting at least five glucosyl-binding subsites, explains why family 8 cellulases cleave cello-oligosaccharide polymers that are at least five D-glycosyl subunits long. Furthermore, the structure of CelA allows comparison with (alpha/alpha)6 barrel glycosidases that are not related in sequence, suggesting a possible, albeit distant, evolutionary relationship between different families of glycosyl hydrolases.

Preliminary crystallographic study of a complex between an heteroclitic anti-hen egg-white lysozyme antibody and the heterologous antigen pheasant egg-white lysozyme.

We report the preparation, crystallization and preliminary X-ray crystallographic study of the Fab fragment from a heteroclitic murine (BALB/c) monoclonal anti-hen egg-white lysozyme antibody complexed with a heterologous antigen, pheasant lysozyme. The complex between the heterologous antigen and the antibody has been crystallized from polyethylene glycol 8000 solutions in a form suitable for X-ray crystallographic studies. The crystals are monoclinic, space group C2 with a = 158.2 A, b = 49.1 A, c = 177.6 A, beta = 92.0 degrees (1 A = 0.1 nm).

Preliminary crystallographic study of the Fab fragment of a monoclonal antibody directed against a cobra cardiotoxin.

We report on the preparation, crystallization and preliminary X-ray crystallographic study of the Fab fragments from a murine monoclonal anti-cardiotoxin antibody M gamma 2-3 directed against a cobra cardiotoxin. The Fab fragment has been crystallized from polyethylene glycol 8000 solutions in a form suitable for high-resolution, X-ray crystallographic studies. The crystals are monoclinic, space group C2, with a = 161.2 A, b = 40.4 A, c = 96.5 A, beta = 118.3 degrees.

The crystal structure of a type I cohesin domain at 1.7 A resolution.

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. We report here the crystal structure of a single cohesin domain from the scaffolding protein of Clostridium thermocellum. The cohesin domain folds into a nine-stranded beta-sandwich with an overall "jelly roll" topology, similar to that observed in bacterial cellulose-binding domains. Surface-exposed patches of conserved residues promote extensive intermolecular contacts in the crystal, and suggest a possible binding target for the EF-hand pair of the cognate dockerin domain. Comparative studies of cohesin domains indicate that, in spite of low sequence similarities and different functional roles, all cohesin domains share a common nine-stranded beta-barrel fold stabilized by a conserved hydrophobic core. The formation of stable cohesin-dockerin complexes requires the presence of Ca2+. However, the structure of the cohesin domain reported here reveals no obvious Ca2+-binding site, and previous experiments have failed to detect high affinity binding of Ca2+ to the unliganded dockerin domain of endoglucanase CelD. Based on structural and biochemical evidence, we propose a model of the cohesin-dockerin complex in which the dockerin domain requires complexation with its cohesin partner for protein stability and high-affinity Ca2+ binding.

Crystallization and preliminary diffraction analysis of the catalytic domain of xylanase Z from Clostridium thermocellum.

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 A, b = 50.8 A, c = 70.3 A, alpha = 100.7 degrees, beta = 83.8 degrees, gamma = 101.6 degrees, and two molecules in the unit cell. These crystals diffract X-rays to at least 1.8 A resolution and are suitable for high-resolution X-ray analysis.

The crystal structure of a family 5 endoglucanase mutant in complexed and uncomplexed forms reveals an induced fit activation mechanism.

The structures of the Glu140-->Gln mutant of the Clostridium thermocellum endoglucanase CelC in unliganded form (CelC(E140Q)) and in complex with cellohexaose (CelC(E140Q)-Gl(C6)) have been refined to crystallographic R-factors of 19.4% at 1.9 A and 17.8% at 2.3 A resolution, respectively. The structure of CelC(E140Q)-Gl(C6) complex shows two D-glucosyl residues bound to the non-reducing end of the substrate-binding cleft. Comparison of the unliganded and complexes structures reveals conformational changes due to substrate binding, including a significant reorientation of the loop 138-141 which carries the general acid/base catalyst Glu140 in wild-type CelC. Endoglucanase CelC, a family 5 glycohydrolase, exhibits a (beta/alpha)8-fold with an additional subdomain of 54 amino acids inserted between beta-strand 6 and alpha-helix 6. Seven amino acid residues (Arg46, His90, Asn139, Glu140, His198, Tyr200, and Glu280) located close to the catalytic reaction center are strictly conserved in family 5 cellulases. Only three of these residues (His90, Gln140 and Glu280) make direct contacts with the substrate, but all participate in a network of hydrogen bonds which contribute to the stability of the active site architecture and may influence the protonation state of the two catalytic residues. Residue Trp313, which interacts with the nucleophile Glu280 and is within hydrogen bonding distance of the substrate, is involved in a non-proline cis-peptide bond. An aromatic residue occurs at an equivalent position in many other (beta/alpha)8-barrel glycosidases; the presence of a cis-peptide bond at this position in the structures of family 1 beta-glucosidases, family 2 beta-galactosidases, family 5 cellulases, family 17 beta-glucanases, and family 18 chitinases provides further evidence of an evolutionary relationship between glycosyl hydrolases with a (beta/alpha)8- architecture.

Crystallization and preliminary crystallographic analysis of a tetrameric isolectin from Vicia villosa, specific for the Tn antigen.

Isolectin B4 isolated from Vicia villosa seeds 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 P4(1) (or P4(3), with a = 91.3 A, c = 151.7 A and one tetramer in the asymmetric unit. The crystals diffract X-rays to 2.8 A resolution and are suitable for high-resolution structural analysis.

Multiple crystal forms of endoglucanase CelD: signal peptide residues modulate lattice formation.

The crystal structure of Clostridium thermocellum endoglucanase 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 A c = 191.8 A) are trigonal, space group P3(1)21, with one molecule in the asymmetric unit (form A), whereas crystals of papain-treated CelD (a = 100.4 A, c = 248.7 A), of CelDK38M (a = 100.1 A, c = 248.4 A) and of papain-treated CelDD201A (a = 99.9 A, c = 250.0 A) are trigonal, space group P3(1)21, with two crystallographically independent molecules (form B), and crystals of chymotrypsin-treated CelD (a = 100.0 A, c = 254.3 A) and of CelDD201A (a = 99.8 A, c = 254.7 A) are hexagonal, space group P6(1)22, 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 A 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.

Structure of catalase-A from Saccharomyces cerevisiae.

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.

Crystal structures of pheasant and guinea fowl egg-white lysozymes.

The crystal structures of pheasant and guinea fowl lysozymes have been determined by X-ray diffraction methods. Guinea fowl lysozyme crystallizes in space group P6(1)22 with cell dimensions a = 89.2 A and c = 61.7 A. The structure was refined to a final crystallographic R-factor of 17.0% for 8,854 observed reflections in the resolution range 6-1.9 A. Crystals of pheasant lysozyme are tetragonal, space group P4(3)2(1)2, with a = 98.9 A, c = 69.3 A and 2 molecules in the asymmetric unit. The final R-factor is 17.8% to 2.1 A resolution. The RMS deviation from ideality is 0.010 A for bond lengths and 2.5 degrees for bond angles in both models. Three amino acid positions beneath the active site are occupied by Thr 40, Ile 55, and Ser 91 in hen, pheasant, and other avian lysozymes, and by Ser 40, Val 55, and Thr 91 in guinea fowl and American quail lysozymes. In spite of their internal location, the structural changes associated with these substitutions are small. The pheasant enzyme has an additional N-terminal glycine residue, probably resulting from an evolutionary shift in the site of cleavage of prelysozyme. In the 3-dimensional structure, this amino acid partially fills a cleft on the surface of the molecule, close to the C alpha atom of Gly 41 and absent in lysozymes from other species (which have a large side-chain residue at position 41: Gln, His, Arg, or Lys). The overall structures are similar to those of other c-type lysozymes, with the largest deviations occurring in surface loops. Comparison of the unliganded and antibody-bound models of pheasant lysozyme suggests that surface complementarity of contacting surfaces in the antigen-antibody complex is the result of local, small rearrangements in the epitope. Structural evidence based upon this and other complexes supports the notion that antigenic variation in c-type lysozymes is primarily the result of amino acid substitutions, not of gross structural changes.

Conservative substitutions in the hydrophobic core of Rhodobacter sphaeroides thioredoxin produce distinct functional effects.

The internal residue Phe 25 in Rhodobacter sphaeroides thioredoxin was changed to five amino acids (Ala, Val, Leu, Ile, Tyr) by site-directed mutagenesis, and the mutant proteins were characterized in vitro and in vivo using the mutant trxA genes in an Escherichia coli TrxA- background. The substitution F25A severely impaired the functional properties of the enzyme. Strains expressing all other mutations can grow on methionine sulfoxide with growth efficiencies of 45-60% that of the wild type at 37 degrees, and essentially identical at 42 degrees. At both temperatures, however, strains harboring the substitutions F25V and F25Y had lower growth rates and formed smaller colonies. In another in vivo assay, only the wild type and the F25I substitution allowed growth of phage T3/7 at 37 degrees, demonstrating that subtle modifications of the protein interior at position 25 Ile/Leu or Phe/Tyr) can produce significant biological effects. All F25 mutants were good substrates for E. coli thioredoxin reductase. Although turnover rates and apparent Km values were significantly lower for all mutants compared to the wild type, catalytic efficiency of thioredoxin reductase was similar for all substrates. Determination of the free energy of unfolding showed that the aliphatic substitutions (Val, Leu, Ile) significantly destabilized the protein, whereas the F25Y substitution did not affect protein stability. Thus, thermodynamic stability of R. sphaeroides thioredoxin variants is not correlated with the distinct functional effects observed both in vivo and in vitro.

Subcloning of a DNA fragment encoding a single cohesin domain of the Clostridium thermocellum cellulosome-integrating protein CipA: purification, crystallization, and preliminary diffraction analysis of the encoded polypeptide.

An Escherichia coli clone encoding a single cohesin domain of the cellulosome-integrating protein CipA from Clostridium thermocellum was constructed, and the corresponding polypeptide was purified, treated with papain, and crystallized from a PEG 8000 solution. Crystals exhibit orthorhombic symmetry, space group P2(1)2(1)2(1), with cell dimensions a = 37.7 A, b = 80.7 A, c = 93.3 A, and four or eight molecules in the unit cell. The crystals diffract X-rays to beyond 2 A resolution and are suitable for further crystallographic studies.

Carbamate kinase: New structural machinery for making carbamoyl phosphate, the common precursor of pyrimidines and arginine.

The enzymes carbamoyl phosphate synthetase (CPS) and carbamate kinase (CK) make carbamoyl phosphate in the same way: by ATP-phosphorylation of carbamate. The carbamate used by CK is made chemically, whereas CPS itself synthesizes its own carbamate in a process involving the phosphorylation of bicarbonate. Bicarbonate and carbamate are analogs and the phosphorylations are carried out by homologous 40 kDa regions of the 120 kDa CPS polypeptide. CK can also phosphorylate bicarbonate and is a homodimer of a 33 kDa subunit that was believed to resemble the 40 kDa regions of CPS. Such belief is disproven now by the CK structure reported here. The structure does not conform to the biotin carboxylase fold found in the 40 kDa regions of CPS, and presents a new type of fold possibly shared by homologous acylphosphate-making enzymes. A molecular 16-stranded open beta-sheet surrounded by alpha-helices is the hallmark of the CK dimer. Each subunit also contains two smaller sheets and a large crevice found at the location expected for the active center. Intersubunit interactions are very large and involve a central hydrophobic patch and more hydrophilic peripheral contacts. The crevice holds a sulfate that may occupy the site of an ATP phosphate, and is lined by conserved residues. Site-directed mutations tested at two of these residues inactivate the enzyme. These findings support active site location in the crevice. The orientation of the crevices in the dimer precludes their physical cooperation in the catalytic process. Such cooperation is not needed in the CK reaction but is a requirement of the mechanism of CPSs.

Crystallization and preliminary structural analysis of catalase A from Saccharomyces cerevisiae.

Yeast peroxisomal catalase A, obtained at high yields by over expression of the C-terminally modified gene from a 2 mu-plasmid, has been crystallized in a form suitable for high resolution X-ray diffraction studies. Brownish crystals with bipyrimidal morphology and reaching ca. 0.8 mm in size were produced by the hanging drop method using ammonium sulphate as precipitant. These crystals diffract better than 2.0 A resolution and belong to the hexagonal space group P6(1)22 with unit cell parameters a = b = 184.3 A and c = 305.5 A. An X-ray data set with 76% completeness at 3.2 A resolution was collected in a rotating anode generator using mirrors to improve the collimation of the beam. An initial solution was obtained by molecular replacement only when using a beef liver catalase tetramer model in which fragments with no sequence homology had been omitted, about 150 residues per subunit. In the structure found a single molecule of catalase A (a tetramer with accurate 222 molecular symmetry) is located in the asymmetric unit of the crystal with an estimated solvent content of about 61%. The preliminary analysis of the structure confirms the absence of a carboxy terminal domain as the one found in the catalase from Penicillium vitalae, the only other fungal catalase structure available. The NADPH binding site appears to be involved in crystal contacts, suggesting that heterogeneity in the occupancy of the nucleotide can be a major difficulty during crystallization.

The MBP fusion protein restores the activity of the first phosphatase domain of CD45.

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.

Crystallization and preliminary X-ray diffraction data of the Fab fragment of a monoclonal antibody against apamin, a bee venom neurotoxin.

Fab fragments of anti-apamin monoclonal antibodies have been purified to homogeneity and crystallized. The crystals belong to the monoclinic space group P21 with cell dimensions a = 99.0 +/- 0.3 A, b = 137.1 +/- 0.4 A, c = 76.0 +/- 0.2 A and beta = 92.9 +/- 0.9 degrees. They most likely contain four molecules in the asymmetric unit (Vm = 2.39 A3/Da). The possibility of the existence of non-crystallographic symmetry is discussed.