Identification of a novel plasmin(ogen)-binding motif in surface displayed alpha-enolase of Streptococcus pneumoniae

The interaction of Streptococcus pneumoniae with human plasmin(ogen) represents a mechanism to enhance bacterial virulence by capturing surface-associated proteolytic activity in the infected host. Plasminogen binds to surface displayed pneumococcal alpha-enolase (Eno) and is subsequently activated to the serine protease plasmin by host-derived tissue plasminogen activator (tPA) or urokinase (uPA). The C-terminal lysyl residues of Eno at position 433 and 434 were identified as a binding site for the kringle motifs of plasmin(ogen) which contain lysine binding sites. In this report we have identified a novel internal plamin(ogen)-binding site of Eno by investigating the protein-protein interaction. Plasmin(ogen)-binding activity of C-terminal mutated Eno proteins used in binding assays as well as surface plasmon resonance studies suggested that an additional binding motif of Eno is involved in the Eno-plasmin(ogen) complex formation. The analysis of spot synthesized synthetic peptides representing Eno sequences identified a peptide of nine amino acids located between amino acids 248-256 as the minimal second binding epitope mediating binding of plasminogen to Eno. Binding of radiolabelled plasminogen to viable pneumococci was competitively inhibited by a synthetic peptide FYDKERKVYD representing the novel internal plasmin(ogen)-binding motif of Eno. In contrast, a synthetic peptide with amino acid substitutions at critical positions in the internal binding motif identified by systematic mutational analysis did not inhibit binding of plasminogen to pneumococci. Pneumococcal mutants expressing alpha-enolase with amino acid substitutions in the internal binding motif showed a substantially reduced plasminogen-binding activity. The virulence of these mutants was also attenuated in a mouse model of intranasal infection indicating the significance of the novel plasminogen-binding motif in the pathogenesis of pneumococcal diseases.     

High-resolution crystal structure of apolipoprotein(a) kringle IV type 7: insights into ligand binding

Apolipoprotein(a) [apo(a)] consists of a series of tandemly repeated modules known as kringles that are commonly found in many proteins involved in the fibrinolytic and coagulation cascades, such as plasminogen and thrombin, respectively. Specifically, apo(a) contains multiple tandem repeats of domains similar to plasminogen kringle IV (designated as KIV(1) to KIV(10)) followed by sequences similar to the kringle V and protease domains of plasminogen. The KIV domains of apo(a) differ with respect to their ability to bind lysine or lysine analogs. KIV(10) represents the high-affinity lysine-binding site (LBS) of apo(a); a weak LBS is predicted in each of KIV(5)-KIV(8) and has been directly demonstrated in KIV(7). The present study describes the first crystal structure of apo(a) KIV(7), refined to a resolution of 1.45 A, representing the highest resolution for a kringle structure determined to date. A critical substitution of Tyr-62 in KIV(7) for the corresponding Phe-62 residue in KIV(10), in conjunction with the presence of Arg-35 in KIV(7), results in the formation of a unique network of hydrogen bonds and electrostatic interactions between key LBS residues (Arg-35, Tyr-62, Asp-54) and a peripheral tyrosine residue (Tyr-40). These interactions restrain the flexibility of key LBS residues (Arg-35, Asp-54) and, in turn, reduce their adaptability in accommodating lysine and its analogs. Steric hindrance involving Tyr-62, as well as the elimination of critical ligand-stabilizing interactions within the LBS are also consequences of this interaction network. Thus, these subtle yet critical structural features are responsible for the weak lysine-binding affinity exhibited by KIV(7) relative to that of KIV(10).     

A novel fibrin(ogen)olytic trypsin-like protease from Chinese oak silkworm (Antheraea pernyi): Purification and characterization

A novel fibrin(ogen)olytic protease from Antheraea pernyi (important economically insect), named cocoonase, was isolated by a combination of ion-exchange chromatography and gel filtration. Furthermore, the characterization of cocoonase was investigated using fibrin(ogen)olytic, thrombolysis, and hemorrhagic assays. The NH₂-terminal sequence (IVGGY SVTID KAPYQ) was established by Edman degradation. Based on the N-terminal sequencing, cocoonase cDNA has been cloned by means of RT-PCR and 5′RACE. It is composed of 261 amino acid residues and possesses the structural features of trypsin-like serine protease. The purified cocoonase showed specific esterase activity on N-β-benzoyl-l-arginine ethyl (BAEE), and the kinetic constants, Km and Vmax were 2.577 × 10⁻³ mol/L and 4.09 × 10⁻³ μmol/L/s, respectively. Cocoonase showed strong activities on both fibrin and fibrinogen, preferentially hydrolyzed Aα and Bβ chains followed by γ-chains of fibrinogen. Cocoonase exhibited a thrombolysis activity both in vitro (blood-clot lysis activity assay) and in vivo (carrageenan-induced thrombosis model). These findings indicate that A. pernyi cocoonase ia a novel fibrin(ogen)olytic enzyme and may have a potential clinical application as an antithrombotic agent.     

Solution structure of the C-terminal domain from poly(A)-binding protein in Trypanosoma cruzi: a vegetal PABC domain

PABC is a phylogenetically conserved peptide-binding domain primarily found within the C terminus of poly(A)-binding proteins (PABPs). This domain recruits a series of translation factors including poly(A)-interacting proteins (Paip1 and Paip2) and release factor 3 (RF3/GSPT) to the initiation complex on mRNA. Here, we determine the solution structure of the Trypanosoma cruzi PABC domain (TcPABC), a representative of the vegetal class of PABP proteins. TcPABC is similar to human PABC (hPABC) and consists of five alpha-helices, in contrast to the four helices observed in PABC domains from yeast (yPABC) and hyper plastic disk proteins (hHYD). A mobile N-terminal helix is observed in TcPABC that does not pack against the core of the protein, as found in hPABC. Characteristic to all PABC domains, the last four helices of TcPABC fold into a right-handed super coil. TcPABC demonstrates high-affinity binding to PABP interacting motif-2 (PAM-2) and reveals a peptide-binding surface homologous to that of hPABC. Our results demonstrate the last four helices in TcPABC are sufficient for peptide recognition and we predict a similar binding mode in PABC domains. Furthermore, these results point to the presence of putative PAM-2 site-containing proteins in trypanosomes.     

Gas phase assembly and X-ray crystal structure of copper(I) 3,5-bis(trifluoromethyl)benzoate network with corannulene

Gas-phase co-deposition of [Cu(O₂C(3,5-CF₃)₂C₆H₃)] (1) with C₂₀H₁₀ at 170 °C affords crystals of the first copper(I)-corannulene adduct [Cu₆(O₂C(3,5-CF₃)₂C₆H₃)₆](C₂₀H₁₀)₂ (2). The X-ray crystallographic characterization of 2 reveals its main structural building blocks: a planar hexanuclear metal core supported by bridging 3,5-bis(trifluoromethyl)benzoate groups and two corannulene molecules. The Cu?Cu distances within the core of 2.6826(8)-2.7607(8) Å fall within the range of cuprophilic interactions. Several intermolecular Cu?C contacts between the cyclic Cu₆-unit and corannulene ranging from 2.799(5) to 3.266(5) Å can be identified. The shortest ones lying within the sum of the van der Waals radii for Cu and C (Σr_vdW (Cu, C) = 3.10 Å) are to the rim sites of corannulene. A noticeable flattening of the C₂₀H₁₀-bowl in 2 is also observed.     

Triboluminescence and crystal structure of the centrosymmetric complex [Tb(NO3)2(Acac)(Phen)2]·H2O

The atomic structure of crystals of the complex [Tb(NO₃)₂(Acac)(Phen)₂]·H₂O, (AA – acetylacetonate anion, Phen – 1,10‐phenanthroline) characterized by an intensive luminescence and triboluminescence has been determined by means of an X‐ray structural analysis method. Centrosymmetric crystals have a monoclinic syngony: a = 11.2298(1), b = 9.6492(1), c = 13.2745(1) Å, β = 101.290(1), space group P2/n, Z = 2, ρ_calc = 1.790 g/cm³. The crystal structure is represented by individual С₂₉Н₂₅N₆O₉Tb complexes linked through van der Waals interactions with clearly expressed cleavage planes. The Tb(III) atom coordination polyhedron reflects the state of a distorted square antiprism. The structural aspects of the suggested model of formation of the triboluminescent properties were considered and the role of the cleavage planes discussed. Copyright © 2016 John Wiley & Sons, Ltd.     

Synthesis and crystal structure of tetrameric silver(I) 3,5-di-tert-butyl-pyrazolate

A tetrameric [Ag(μ-3,5-^tBu₂pz)]₄ · CH₂Cl₂ (1 · CH₂Cl₂) has been prepared and structurally characterized. The four Ag-atoms are in an approximate rhombic arrangement with pyrazolato bridges alternating on either side of the Ag₄-plane. A ¹H NMR study shows partial decomposition of 1 to the mononuclear [Ag(3,5-^tBu₂pzH)₂]⁺ in solution.     

Triboluminescence and crystal structure of the complex [Eu(NО3)3(HMPA)3]: role of cleavage planes

The atomic structure of crystals of the [Eu(NО₃)₃(HMPA)₃] [hexamethylphosphotriamide (HMPA)] complex characterized by an intensive luminescence and triboluminescence was determined using X‐ray structural analysis. Noncentrosymmetric crystals have a monoclinic syngony: a = 16.0686 (3), b = 11.0853 (2), c = 20.9655 Å (4), β = 93.232° (1), space group P2₁, Z = 4, ρ_calc = 1.560 g/cm³_. The crystal structure is represented by individual С₁₈Н₅₄EuN₁₂O₁₂P₃ complexes linked through van der Waals interactions with clearly expressed cleavage planes. The Eu(III) atom coordination polyhedron reflected the state of a distorted square antiprism. Structural aspects of the suggested model, including formation of triboluminescence properties, were considered and the role of the cleavage planes was discussed.     

Radioimmunoassay and chemical properties of glucose-6-phosphate dehydrogenase and of a specific NADP(H)-binding protein (FX) from human erythrocytes

Two sensitive radioimmunoassays, based on a double-antibody technique, were developed which allow detection of nanogram amounts of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and of a so far unknown NADP(H)-binding protein present in human erythrocytes (designated FX).The two proteins isolated in homogeneous form from human erythrocytes were iodinated with ¹²⁵I by means of lactoperoxidase. Antisera to both purified proteins were raised in rabbits and sequentially adsorbed on human erythrocytes and on human serum before use. No cross-reaction between the two proteins was apparent.Hemolysates from normal as well as from glucose-6-phosphate dehydrogenase-deficient subjects were investigated for their content in both immunoreactive proteins using the two radioimmunoassay methods. This preliminary study showed significantly lowered levels of immunoreactive glucose-6-phosphate dehydrogenase in erythrocytes from subjects carrying the Mediterranean variant of this enzyme (characterized by severe deficiency of catalytic activity), compared with normal subjects. This figure was reversed as concerns the content of immunoreactive FX which was found to be twice as high in glucose-6-phosphate dehydrogenase Mediterranean erythrocytes as in normal ones.The two purified proteins were submitted to a comparative analysis of their chemical properties including NH₂-terminal residues, CNBr peptides and tryptic fingerprints. These studies revealed significant differences in the primary structures of the two proteins and therefore tend to exclude FX'x being a discrete product arising from degradation of native glucose-6-phosphate dehydrogenase. Moreover, amino axid analysis and tryptic fingerprints indicated that FX, as well as glucose-6-phosphatase dehydrogenase, is composed of very similar and possibly identical polypeptide chains.     

The preparation and crystal structure of acetatobis(l-arginine)zinc(II) acetate trihydrate, the first reported X-ray structure of a zinc(II)-arginine complex

The synthesis and X-ray crystal structure of acetatobis(l-arginine)zinc(II) acetate trihydrate, [Zn(OAc)(l-Arg)₂]OAc·3H₂O is reported. In this structure, the first of a zinc(II)-arginine complex to be reported, the geometry around zinc(II) is distorted square-pyramidal containing two trans-N,O chelated l-Arg ligands in the basal plane and the acetato ligand in an axial position. The structure contains a second acetate which is salt-bridged to the δ and ω NH groups of the guanidinium side chain of an arginine ligand and also contains three hydrogen bonded water molecules.     

Synthesis, characterization and crystal structure of manganese (IV) complex derived from salicylic acid

The binuclear manganese (IV) [Mn₂(Hsal)₄(OH)₄] (H₂sal = salicylic acid) complex has been obtained from a complex reaction mixture in methanol consisting of Mn(II)(OAc)₂ · 4H₂O, GS ( a reagent obtained by refluxing glycine and salicylaldehyde in 1:1 molar ratio in methanol), monosodium salicylate and pyridine. The compound contains a distorted octahedral MnO₆ coordination unit of potential importance to high oxidation state manganese bimolecules.     

The crystal structure of cobalt-substituted pseudoazurin from Alcaligenes faecalis

The Cu(II) center at the active site of the blue copper protein pseudoazurin from Alcaligenes faecalis has been substituted by Co(II) via denaturing of the protein, chelation and removal of copper by EDTA and refolding of the apo‐protein, followed by addition of an aqueous solution of CoCl₂. Sitting drop vapour diffusion experiments produced green hexagonal crystals, which belong to space group P6₅, with unit cell dimensions a = b = 50.03, c = 98.80 Å. Diffraction data, collected at 291 K on a copper rotating anode X‐ray source, were phased by the anomalous signal of the cobalt atom. The structure was built automatically, fitted manually and subsequently refined to 1.86 Å resolution. The Co‐substituted protein exhibits similar overall geometry to the native structure with copper. Cobalt binds more strongly to the axial Met86‐Sδ and retains the tetrahedral arrangement with the four ligand atoms, His40‐Nδ₁, Cys78‐Sγ, His81‐Nδ₁, and 86Met‐Sδ, although the structure is less distorted than the native copper protein. The structure reported herein, is the first crystallographic structure of a Co(II)‐substituted pseudoazurin. © 2010 Wiley Periodicals, Inc. Biopolymers 95: 202–207, 2011.     

X-ray structure and photoluminescence of copper(I) 2,6-bis(trifluoromethyl)benzoate

This report describes synthesis, X-ray crystallographic characterization, and photoluminescence of copper(I) 2,6-bis(trifluoromethyl)benzoate, [Cu(O₂C(2,6-CF₃)₂C₆H₃)] (1). Complex 1 has a polymeric structure comprised of axially bound dinuclear [Cu₂(O₂C(2,6-CF₃)₂C₆H₃)₂] units that is reminiscent of the well-known structure of copper(I) acetate, [Cu₂(O₂CMe)₂] (2). However, a close comparison of two analogues revealed a non-planarity of 1D chains and their orthogonal disposition in the crystal structure of 1 versus parallel alignment of planar chains in 2. Complex 1 exhibits green photoluminescence at ca. 560 nm upon UV-radiation (λ_ex = 350 nm) in the solid state.     

Synthesis, X-ray crystal structure and magnetic study of a dicyanamido bridged 1D chain nickel(II) complex

Reaction of Ni(NO₃)₂ · 6H₂O and sodium dicyanamide (Nadca) yields a 1D infinite chain complex {[Ni(dien)(μ_1,5-dca)(H₂O)](NO₃)}_n (1) (where dien = diethylenetriamine). The coordination environment in complex 1 around the nickel(II) ions is distorted octahedron. Three nitrogen atoms of the ligand diethylenetriamine and an oxygen atom of H₂O molecule constitute the four coordination sites of the basal plane of the octahedron. Of two axial positions of the octahedron, one position is occupied by the nitrogen atom of a μ_1,5-dca anion the remaining coordination site is occupied by a nitrogen atom of another end-to-end bridging dca from an adjacent [Ni(dien)(μ_1,5-dca)(H₂O)] moiety, yielding 1D infinite chains which propagate parallel to crystallographic a-axis. No measurable magnetic interaction was evidenced through variable temperature magnetic susceptibility measurements (4-300 K). However, the magnetic susceptibility of the compound can be explained in terms of single-ion anisotropic model with zero-field splitting for nickel(II) ions.     

Two new copper(II) polyamine compounds: Synthesis, characterization and crystal structure

Two new compounds of Cu(II) of stoichiometry CuCl₄(polyamineH₂) containing the polyamines (PA): spermidine or spermine were prepared. Their synthesis, spectroscopic and structural characterization are herein described. The obtained complex with spermidine was characterized by elemental and thermogravimetric analysis, electronic and infrared spectroscopy. In the case of the compound with spermine, crystals were obtained. So, beside all other techniques the compound was also characterized by single crystal X-ray diffractometry. In both cases the species [CuCl₄]²⁻ is present and displays a similar polymeric structure. The X-ray, infrared and electronic spectra are herein discussed based on structural peculiarities of the compounds.     

Crystal structure of penicillin-binding protein 1a (PBP1a) reveals a mutational hotspot implicated in beta-lactam resistance in Streptococcus pneumoniae

Streptococcus pneumoniae is a major human pathogen whose infections have been treated with beta-lactam antibiotics for over 60 years, but the proliferation of strains that are highly resistant to such drugs is a problem of worldwide concern. Beta-lactams target penicillin-binding proteins (PBPs), membrane-associated enzymes that play essential roles in the peptidoglycan biosynthetic process. Bifunctional PBPs catalyze both the polymerization of glycan chains (glycosyltransfer) and the cross-linking of adjacent pentapeptides (transpeptidation), while monofunctional enzymes catalyze only the latter reaction. Although S. pneumoniae has six PBPs, only three (PBP1a, PBP2x, PBP2b) are major resistance determinants, with PBP1a being the only bifunctional enzyme. PBP1a plays a key role in septum formation during the cell division cycle and its modification is essential for the development of high-level resistance to penicillins and cephalosporins. The crystal structure of a soluble form of pneumococcal PBP1a (PBP1a*) has been solved to 2.6A and reveals that it folds into three domains. The N terminus contains a peptide from the glycosyltransfer domain bound to an interdomain linker region, followed by a central, transpeptidase domain, and a small C-terminal unit. An analysis of PBP1a sequences from drug-resistant clinical strains in light of the structure reveals the existence of a mutational hotspot at the entrance of the catalytic cleft that leads to the modification of the polarity and accessibility of the mutated PBP1a active site. The presence of this hotspot in all variants sequenced to date is of key relevance for the development of novel antibiotherapies for the treatment of beta-lactam-resistant pneumococcal strains.     

Identification and characterization of proteins that interact with the carboxy terminus of poly(A)-binding protein and inhibit translation in vitro

Poly(A)-binding proteins (PABPs) are multifunctional proteins that play important roles in mRNA stability and protein translation. Two cucumber ( Cucumis sativus L.) proteins, PCI6 (PABP-CT-interacting) and PCI243 were identified based on ability to interact with the carboxy terminus (CT) of PABP in yeast two-hybrid and in vitro binding assays. PCI6 and PCI243 share a conserved amino acid domain (SxLnpnApxFxP) in common with human PABP-CT interactors, and with Arabidopsis ERD15 (early-responsive to dehydration). Deletion analysis and point mutations indicate that presence of this domain is necessary for the interaction, and tests with ERD15 demonstrate that it is predictive of interaction. Other plant proteins possessing this domain fall into two categories: small, acidic proteins like PCI6, PCI243 and ERD15, and larger neutral proteins that also include an RNA recognition motif. PCI6 is expressed in a range of tissues, e.g., leaves, roots, stems and flowers, and follows a diurnal pattern of expression, increasing during light hours and declining overnight. In wheat germ and mouse ascites Krebs-2 in vitro translation systems, PCI6 inhibited translation whereas the non-interacting mutant, PCI6-23A, did not or had a greatly reduced effect. The activity of PCI6, therefore, is reminiscent of that of human PABP-interacting protein 2 (Paip2). These results demonstrate a novel interaction between PABP and several plant proteins sharing a SxLnpxApxFxP motif, with possible implications for translational regulation.     

Identification of the reactive (inhibitory) sites of chymotryptic inhibitor I from potatoes

Chymotryptic Inhibitor I from potato tubers was subjected to limited hydrolysis with catalytic amounts of chymotrypsin and trypsin at pH 3. The fragmen