[Puffy hand syndrome: A little-known diagnosis]. / Le syndrome des grosses mains : un diagnostic méconnu.

INTRODUCTION: Puffy hand syndrome is a rare complication of intravenous drug addiction. Diagnosis is based on the patient's history and clinical examination. OBSERVATIONS: A woman and two men, aged 42, 39 and 36 years old, are described. All had a history of intravenous drug use of heroin and oral buprenorphine misuse. Puffy hand syndrome appeared during drug addiction (n = 2) or after its withdrawal (n = 1). It was associated with acrocyanosis (n = 1) or injection scars (n = 1). Upper limb ultrasonography showed sequelae of venous (n = 3) or arterial (n = 1) thrombosis. An upper limb lymphoscintigraphy in one patient showed decreased radionuclide uptake of axillary lymph node and subdermal reflux tracer in the forearm. Treatment was based on low-stretch bandages to reduce the volume and then elastic compression sleeve for long-term stabilization. CONCLUSION: Puffy hand syndrome seen in intravenous drug addicts is poorly understood. It is a chronic complication despite the cessation of drug use. This syndrome has to become more widely known because its management is mandatory, although symptomatic.

Structure of the histone-core octamer in KCl/phosphate crystals at 2.15 A resolution.

The structure of the native chicken histone octamer, crystallized in 2 M KCl, 1.35 M potassium phosphate pH 6.9, has been refined at 2.15 A resolution to a final R factor of 21.4% and an R(free) of 25.2%. Unique crystal-packing interactions between histone-core octamers are strong and one of them (area 4000 A(2)) involves two chloride ions and direct interactions between six acidic amino-acid residues on one octamer and the equivalent number of basic residues on the next. These interactions are on the structured part of the octamer (not involving tails). Five phosphate ions, 23 chloride ions and 437 water molecules have been identified in the structure. The phosphate and some chloride ions bind to basic amino-acid residues that interact with DNA in the nucleosome. The binding of most of the anions and the packing interactions are unique to these crystals. In other respects, and including the positions of four chloride ions, the octamer structure is very close to that of octamers in nucleosome-core particle crystals, particularly with respect to 'docking' sequences of the histone H2As and H4s. These sequences together with the H2B-H4 four-helix bundles stabilize the histone structure in the nucleosome and prevent the dissociation of the (H2A-H2B) dimers from the (H3-H4)(2) tetramer. Possible reasons why this happens at high salt in the absence of DNA are given.

The iota-carrageenase of Alteromonas fortis. A beta-helix fold-containing enzyme for the degradation of a highly polyanionic polysaccharide.

Carrageenans are gel-forming hydrocolloids extracted from the cell walls of marine red algae. They consist of d-galactose residues bound by alternate alpha(1-->3) and beta(1-->4) linkages and substituted by one (kappa-carrageenan), two (iota-carrageenan), or three (lambda-carrageenan) sulfate-ester groups per disaccharide repeating unit. Both the kappa- and iota-carrageenan chains adopt ordered conformations leading to the formation of highly ordered aggregates of double-stranded helices. Several kappa-carrageenases and iota-carrageenases have been cloned from marine bacteria. Kappa-carrageenases belong to family 16 of the glycoside hydrolases, which essentially encompasses polysaccharidases specialized in the hydrolysis of the neutral polysaccharides such as agarose, laminarin, lichenan, and xyloglucan. In contrast, iota-carrageenases constitute a novel glycoside hydrolase structural family. We report here the crystal structure of Alteromonas fortis iota-carrageenase at 1.6 A resolution. The enzyme folds into a right-handed parallel beta-helix of 10 complete turns with two additional C-terminal domains. Glu(245), Asp(247), or Glu(310), in the cleft of the enzyme, are proposed as candidate catalytic residues. The protein contains one sodium and one chloride binding site and three calcium binding sites shown to be involved in stabilizing the enzyme structure.

The kappa-carrageenase of P. carrageenovora features a tunnel-shaped active site: a novel insight in the evolution of Clan-B glycoside hydrolases.

BACKGROUND: kappa-carrageenans are gel-forming, sulfated 1,3-alpha-1,4-beta-galactans from the cell walls of marine red algae. The kappa-carrageenase from the marine, gram-negative bacterium Pseudoalteromonas carrageenovora degrades kappa-carrageenan both in solution and in solid state by an endoprocessive mechanism. This beta-galactanase belongs to the clan-B of glycoside hydrolases. RESULTS: The structure of P. carrageenovora kappa-carrageenase has been solved to 1.54 A resolution by the multiwavelength anomalous diffraction (MAD) method, using a seleno-methionine-substituted form of the enzyme. The enzyme folds into a curved beta sandwich, with a tunnel-like active site cavity. Another remarkable characteristic is the presence of an arginine residue at subsite -1. CONCLUSIONS: The crystal structure of P. carrageenovora kappa-carrageenase is the first three-dimensional structure of a carrageenase. Its tunnel-shaped active site, the first to be reported for enzymes other than cellulases, suggests that such tunnels are associated with the degradation of solid polysaccharides. Clan-B glycoside hydrolases fall into two subgroups, one with catalytic machinery held by an ancestral beta bulge, and the other in which it is held by a regular beta strand. At subsite -1, all of these hydrolases exhibit an aromatic amino acid that interacts with the hexopyranose ring of the monosaccharide undergoing catalysis. In addition, in kappa-carrageenases, an arginine residue recognizes the sulfate-ester substituents of the beta-linked kappa-carrageenan monomers. It also appears that, in addition to the nucleophile and acid/base catalysts, two other amino acids are involved with the catalytic cycle, accelerating the deglycosylation step.

Crystal structure of UDP-N-acetylmuramoyl-L-alanyl-D-glutamate: meso-diaminopimelate ligase from Escherichia coli.

UDP-N-acetylmuramoyl-l-alanyl-d-glutamate:meso-diaminopimelate ligase is a cytoplasmic enzyme that catalyzes the addition of meso-diaminopimelic acid to nucleotide precursor UDP-N-acetylmuramoyl-l-alanyl-d-glutamate in the biosynthesis of bacterial cell-wall peptidoglycan. The crystal structure of the Escherichia coli enzyme in the presence of the final product of the enzymatic reaction, UDP-MurNAc-l-Ala-gamma-d-Glu-meso-A(2)pm, has been solved to 2.0 A resolution. Phase information was obtained by multiwavelength anomalous dispersion using the K shell edge of selenium. The protein consists of three domains, two of which have a topology reminiscent of the equivalent domain found in the already established three-dimensional structure of the UDP-N-acetylmuramoyl-l-alanine: D-glutamate-ligase (MurD) ligase, which catalyzes the immediate previous step of incorporation of d-glutamic acid in the biosynthesis of the peptidoglycan precursor. The refined model reveals the binding site for UDP-MurNAc-l-Ala-gamma-d-Glu-meso-A(2)pm, and comparison with the six known MurD structures allowed the identification of residues involved in the enzymatic mechanism. Interestingly, during refinement, an excess of electron density was observed, leading to the conclusion that, as in MurD, a carbamylated lysine residue is present in the active site. In addition, the structural determinant responsible for the selection of the amino acid to be added to the nucleotide precursor was identified.

"Open" structures of MurD: domain movements and structural similarities with folylpolyglutamate synthetase.

UDP-N-acetylmuramoyl-l-alanine:d-glutamate (MurD) ligase catalyses the addition of d-glutamate to the nucleotide precursor UDP-N-acetylmuramoyl-l-alanine (UMA). The crystal structures of Escherichia coli in the substrate-free form and MurD complexed with UMA have been determined at 2.4 A and 1.88 A resolution, respectively. The MurD structure comprises three domains each of a topology reminiscent of nucleotide-binding folds. In the two structures the C-terminal domain undergoes a large rigid-body rotation away from the N-terminal and central domains. These two "open" structures were compared with the four published "closed" structures of MurD. In addition the comparison reveals which regions are affected by the binding of UMA, ATP and d-Glu. Also we compare and discuss two structurally characterized enzymes which belong to the same ligase superfamily: MurD and folylpolyglutamate synthetase (FGS). The analysis allows the identification of key residues involved in the reaction mechanism of FGS. The determination of the two "open" conformation structures represents a new step towards the complete elucidation of the enzymatic mechanism of the MurD ligase.

Crystal structure of human survivin reveals a bow tie-shaped dimer with two unusual alpha-helical extensions.

Survivin is a mitotic spindle-associated protein involved in linking mitotic spindle function to activation of apoptosis in mammalian cells. The structure of the full-length human survivin has been determined by X-ray crystallography to 2.7 A. Strikingly, the structure forms a very unusual bow tie-shaped dimer. It does not dimerize through a C-terminal coiled-coil, contrary to sequence analysis prediction. The C-terminal helices contain hydrophobic clusters with the potential for protein-protein interactions. The unusual shape and dimensions of survivin suggest it serves an adaptor function through its alpha-helical extensions.

Structural effects of the active site mutation cysteine to serine in Bacillus cereus zinc-beta-lactamase.

Beta-lactamases are involved in bacterial resistance. Members of the metallo-enzyme class are now found in many pathogenic bacteria and are becoming thus of major clinical importance. Despite the availability of Zn-beta-lactamase X-ray structures their mechanism of action is still unclear. One puzzling observation is the presence of one or two zincs in the active site. To aid in assessing the role of zinc content in beta-lactam hydrolysis, the replacement by Ser of the zinc-liganding residue Cys168 in the Zn-beta-lactamase from Bacillus cereus strain 569/H/9 was carried out: the mutant enzyme (C168S) is inactive in the mono-Zn form, but active in the di-Zn form. The structure of the mono-Zn form of the C168S mutant has been determined at 1.85 A resolution. Ser168 occupies the same position as Cys168 in the wild-type enzyme. The protein residues mostly affected by the mutation are Asp90-Arg91 and His210. A critical factor for the activity of the mono-Zn species is the distance between Asp90 and the Zn ion, which is controlled by Arg91: a slight movement of Asp90 impairs catalysis. The evolution of a large superfamily including Zn-beta-lactamases suggests that they may not all share the same mechanism.

Structural studies of streptococcus agalactiae hyaluronate lyase.

The bacteria Streptococcus agalactiae, part of normal human flora, produce an enzyme, hyaluronate lyase, which appears to contribute to the invasive capacity of this pathogen by degrading hyaluronan and chondroitin sulfates of the extracellular matrix of host tissues. The native enzyme, the product of the hylB(3502) allele, has a molecular mass of 111 kDa but undergoes an autocatalytic conversion to a smaller enzymatically active 92 kDa form. To initiate the determination of the catalytic mechanism of action of these enzymes, the 111 and 92 kDa enzymes were crystallized by a vapor-diffusion method using polyethylene glycol monomethyl ether 5000 and potassium thiocyanate as precipitating agents. The 111 kDa enzyme crystals are of poor quality and diffract X-rays to a very low resolution. However, the crystals of the truncated 92 kDa enzyme diffract X-rays to 2.1 A resolution. The crystal symmetry is C222(1) and the unit-cell parameters are a = 51.69, b = 157.03, c = 239.20 A (alpha = beta = gamma = 90 degrees ). The V(m) of 2.64 A(3) Da(-1) is consistent with the presence of one molecule of hyaluronate lyase in the asymmetric unit and a crystal solvent content of 53%. An isomorphous ethylmercuricthiosalicylic acid heavy-atom derivative diffraction data set has been obtained in order to solve the structure.

Production and characterization of the functional fragment of pneumococcal surface protein A.

Pneumococcal surface protein A (PspA) is present on the cell wall of Streptococcus pneumoniae pathogen and has an antigenetically variable N-terminal domain. This aminoterminal domain is essential for full pneumococcal virulence, and monoclonal antibodies raised against it protect mice against pneumococcal infections. We have cloned and expressed a 34-kDa N-terminal fragment of PspA in Escherichia coli in a soluble form using the T7 RNA polymerase pET-20b vector system. Nickel chelate affinity purification followed by size exclusion and anion exchange chromatography yielded large amounts of pure and homogeneous protein. Analytical ultracentrifugation sedimentation velocity band and boundary studies showed that the molecule was present in aqueous solutions in a monomeric form with an axial shape ratio of approximately 1:12, typical of fibrous proteins. Sequence analyses indicated an alpha-helical coiled-coil structure for this monomeric molecule with only few loop-type breaks in helicity. The mostly alpha-helical structure of this PspA construct was consistent with circular dichroism spectroscopy data. Based on the ultracentrifugation studies, the circular dichroism spectra, and the PspA's sequence analyses, two structural models for the amino-terminal part of the PspA molecule are proposed. The evident highly charged and polar character of the surface of the modeled structures suggests functional properties of PspA that are related to the prevention of S. pneumoniae interactions with the host complement system.

Crystal structure of the human protein kinase CK2 regulatory subunit reveals its zinc finger-mediated dimerization.

Protein kinase CK2 is a tetramer composed of two alpha catalytic subunits and two beta regulatory subunits. The structure of a C-terminal truncated form of the human beta subunit has been determined by X-ray crystallography to 1.7 A resolution. One dimer is observed in the asymmetric unit of the crystal. The most striking feature of the structure is the presence of a zinc finger mediating the dimerization. The monomer structure consists of two domains, one entirely alpha-helical and one including the zinc finger. The dimer has a crescent shape holding a highly acidic region at both ends. We propose that this acidic region is involved in the interactions with the polyamines and/or catalytic subunits. Interestingly, conserved amino acid residues among beta subunit sequences are clustered along one linear ridge that wraps around the entire dimer. This feature suggests that protein partners may interact with the dimer through a stretch of residues in an extended conformation.

Purification of histone core octamers and 2.15 A X-ray analysis of crystals in KCl/phosphate.

Intact histone octamers, produced by a new method quickly and in bulk, were crystallized in KCl/phosphate, and the X-ray data were analysed to 2.15 A, confirming a P65 space group. This environment preserves the high-resolution structure of the octamers and will be useful for studying them with other functionally important molecules. The octamers form into left-handed superhelices hexagonally spaced by 158.65 A, having a pitch of 102.57 A with six octamers per turn. A dipotassium tetraiodo mercurate derivative had good phasing power and should prove valuable in refining the structure after molecular-replacement analysis with lower resolution coordinates; the heavy atom was isomorphously placed at a unique site between the two H3-cysteine residues in the octamer.

Crystallization and preliminary x-ray diffraction analysis of the regulatory subunit of human protein kinase CK2.

Protein kinase CK2 is a tetramer composed of two alpha catalytic subunits and two beta regulatory subunits. A C-terminal truncated form of the beta subunit has been overproduced in Escherichia coli and purified to homogeneity. Two crystal forms of the truncated protein which diffract to at least 2 A resolution have been obtained. Form I belongs to the monoclinic space group P21, with unit-cell parameters a = 49.9, b = 92.9, c = 53.7 A, beta = 96.3 degrees, and yields plate-like crystals. Form II belongs to the tetragonal space group P42212, with unit-cell parameters a = 132.19, b = 132.19, c = 63.79 A, and produces rod-shaped crystals. Both crystal forms have a functional dimer in the crystal asymmetric unit.

Expression and purification of Streptococcus pneumoniae hyaluronate lyase from Escherichia coli.

Pneumococcal hyaluronate lyase enzyme breaks down hyaluronan of the extracellular matrix of tissues and possibly contributes to the invasion of host tissue and to the penetration of host defenses by this bacterial pathogen. In light of the emergence of increasing numbers of antibiotic-resistant strains, the understanding of the mechanism of action of hyaluronate lyase enzyme may lead to a better understanding of interactions between a host and bacterial pathogens and may contribute to more efficient treatment of bacterial infections. The native Streptococcus pneumoniae hyaluronate lyase enzyme has a molecular mass of 107 kDa but undergoes conversion to smaller enzymatically active forms. The truncated 83-kDa functional form of the enzyme has been cloned into the pET-21d vector, expressed in Escherichia coli, and purified to homogeneity using a nickel affinity column with chelating Sepharose fast flow media. The recombinant enzyme is active and stable and the availability of large quantities of the enzyme will help in its biochemical and biophysical characterization. As a number of other Gram-positive surface proteins, it appears that the enzyme is anchored via its carboxy-terminal part to the pneumococcal cell wall by a covalent linkage with peptidoglycan structures.

Crystallization and preliminary X-ray analysis of Streptococcus pneumoniae hyaluronate lyase.

A fully active 83-kDa truncated form of recombinant hyaluronate lyase from Streptococcus pneumoniae was crystallized by the hanging drop vapor diffusion method using ammonium sulfate as a precipitating agent. Crystals grew at room temperature using a variety of buffers with pH around 6. The crystals diffract X-rays beyond 2.0 A resolution using Cu K alpha radiation and a rotating-anode X-ray source. They belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions, a = 84.2, b = 104.2, c = 104.6 A, and alpha = beta = gamma = 90.0 degrees. The VM value of 2.9 A3/Da is consistent with only one molecule of the enzyme in the asymmetric unit and the solvent content of 57%. Diffraction data 94.7% complete to 2.0 A resolution with Rsym of 5.4% were collected from one native crystal at room temperature. The search for heavy-atom derivatives to solve the structure is in progress.

X-ray structure solution of amaryllis lectin by molecular replacement with only 4% of the total diffracting matter.

It is often the case that analogous proteins from different species crystallize in a different form. These structures can usually be easily solved by the molecular-replacement (MR) technique, as the protein folding is very often conserved. However, the results from MR become more uncertain as the proportion of diffracting matter decreases as a result of multimericity and/or absence of some of the atoms in the model. In this paper results are presented on the structure solution of amaryllis lectin (109 residues per monomer) containing two protein molecules in the asymmetric unit. The structure was solved by MR using the Calpha coordinates of one monomer from snowdrop lectin which has 85% amino-acid sequence identity to amaryllis lectin. This represents only 6% of the non-H atoms of the protein molecule to be used for structure determination and it is a major improvement on previous reports. Further calculations were carried out in order to establish the minimum number of atoms which could be included in the model before a clear solution to the MR problem was revealed. This study showed that the structure of amaryllis lectin could still have been solved easily with 3.85% of the model, which even in the most favourable cases, will probably constitute a minimum for molecular-replacement structure solution.

New crystals of the histone core octamer diffract to higher resolution, 2.65 A.

A new crystal form of the histone octamer, crystallized in 1.6 M KCl, 1.6 M phosphate, diffracts to appreciably better than 2.6 A resolution. The crystals have space group P6(1) or P6(5) and lattice parameters a = b = 158.29, c = 103.27 A, alpha = beta = 90, gamma = 120 degrees, with one molecule per asymmetric unit. The new crystals promise to yield more detail of the histone basic domains and a higher resolution structure for the histone octamer.