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At least 865 records · Page 48Linked to original sources
PubMed1999

Probing the activation of protein C by the thrombin-thrombomodulin complex using structural analysis, site-directed mutagenesis, and computer modeling.

Protein C (PC) is activated to an essential anticoagulant enzyme (activated PC or APC) by thrombin (T) bound to thrombomodulin (TM), a membrane receptor present on the surface of endothelial cells. The understanding of this complex biological system is in part limited due to the lack of integration of experimental and structural data. In the work presented here, we analyze the PC-T-TM pathway in the context of both types of information. First, structural analysis of the serine protease domain of PC suggests that a positively charged cluster of amino acids could be involved in the activation process. To investigate the importance of these basic amino acids, two recombinant PC mutants were constructed using computer-guided site-directed mutagenesis. The double mutant had the K62[217]N/K63[218]D substitution and in the single mutant, K86[241] was changed to S. Both mutants were activated by free thrombin at rates equivalent to that of wild-type PC (wt-PC) and they demonstrated similar calcium-dependent inhibition of their activation. The K86[241]S mutant and wt-PC were activated by thrombin bound to soluble TM at a similar rate. In contrast, the K62[217]N/ K63[218]D mutant was activated by the T-TM complex at a 10-fold lower catalytic efficiency due to a lowering in k(cat) and increase in Km. Molecular models for PC and thrombin bound to a segment of TM were developed. The experimental results and the modeling data both indicate that electrostatic interactions are of crucial importance to orient PC onto the T-TM complex. A key electropositive region centered around loops 37[191] and 60[214] of PC is defined. PC loop 37[191] is located 7-8 A from the TM epidermal growth factor (EGF) 4 while the loop 60[214] is about 10 A away from TM EGF4. Both loops are far from thrombin. A key function of TM could be to create an additional binding site for PC. The Gla domain of PC points toward the membrane and away from thrombin or the EGF modules of TM during the activation process.

Cell Line↗
PubMed2002

Mechanism of hyaluronan degradation by Streptococcus pneumoniae hyaluronate lyase. Structures of complexes with the substrate.

Hyaluronate lyase enzymes degrade hyaluronan, the main polysaccharide component of the host connective tissues, predominantly into unsaturated disaccharide units, thereby destroying the normal connective tissue structure and exposing the tissue cells to various endo- and exogenous factors, including bacterial toxins. The crystal structures of Streptococcus pneumoniae hyaluronate lyase with tetra- and hexasaccharide hyaluronan substrates bound in the active site were determined at 1.52- and 2.0-A resolution, respectively. Hexasaccharide is the longest substrate segment that binds entirely within the active site of these enzymes. The enzyme residues responsible for substrate binding, positioning, catalysis, and product release were thereby identified and their specific roles characterized. The involvement of three residues in catalysis, Asn(349), His(399), and Tyr(408), is confirmed, and the details of proton acceptance and donation within the catalytic machinery are described. The mechanism of processivity of the enzyme is analyzed. The flexibility (allosteric) behavior of the enzyme may be understood in terms of the results of flexibility analysis of this protein, which identified two modes of motion that are also proposed to be involved in the hyaluronan degradation process. The first motion describes an opening and closing of the catalytic cleft located between the alpha- and beta-domains. The second motion demonstrates the mobility of a binding cleft, which may facilitate the binding of the negatively charged hyaluronan to the enzyme.

Amino Acid Sequence↗
PubMed1996

Synthesis, Conformational Studies, X-ray Structures, and Complexation Properties of Semirigid Macrocyclic Phosphonamides.

The semirigid phosphonamide ligands 1-5 have been synthesized from the macrocyclic precursors 6-9 by reaction with 1,3-propanediol ditosylate or 1,2-dichloroethane. For the thiophosphoryl compounds 1 and 2, and the phosphoryl derivative 5, the reactions were carried out in biphasic aqueous NaOH solutions. The phosphoryl derivatives 3 and 4 were better obtained from NaH in anhydrous tetrahydrofuran. The conformations of the hosts in solution were deduced from low-temperature NMR and NOE difference experiments. Conformational equilibria between exo and endo forms are observed for the 18-membered macrocycles 1 and 3. The exo conformer predominates in solution for the 21-membered macrocycle 2, whereas 4 exists as rapidly exchanging conformers. The X-ray crystal structures of macrocycles 1, 2, and 5 have been determined as well as the complexes 1.Hg(SCN)(2) and 5.LiNO(3). In the Hg(2+) complex the metal ion is located out of the macrocyclic cavity and is coordinated to the thiophosphoryl unit. In 5.LiNO(3)()()the Li(+) cation is located inside the macrocyclic cavity and is coordinated to a tetrahedral array of oxygen donors. Free energies of complexation (DeltaG degrees ) of the phosphorylated ligands 3-5 with alkali metal and ammonium cations were determined in CHCl(3) saturated with H(2)O by picrate extraction experiments. The -DeltaG degrees values are greatest for 4 complexing K(+) and NH(4)(+) (7.3 and 8.0 kcal/mol, respectively). The relationships between structure and binding are discussed.

Journal Article↗
PubMed2006

Bonding trends of thiosemicarbazones in mononuclear and dinuclear copper(I) complexes: syntheses, structures, and theoretical aspects.

Reactions of copper(I) halides with a series of thiosemicarbazone ligands (Htsc) in the presence of triphenylphosphine (Ph(3)P) in acetonitrile have yielded three types of complexes: (i) monomers, [CuX(eta1-S-Htsc)(Ph3P)2] [X, Htsc = I (1), Br (2), benzaldehyde thiosemicarbazone (Hbtsc); I (5), Br (6), Cl (7), pyridine-2-carbaldehyde thiosemicarbazone (Hpytsc)], (ii) halogen-bridged dimers, [Cu2(mu2-X)2(eta1-S-Htsc)2(Ph3P)2] [X, Htsc = Br (3), Hbtsc; I (8), furan-2-carbaldehyde thiosemicarbazone (Hftsc); I (11), thiophene-2-carbaldehyde thiosemicarbazone (Httsc)], and (iii) sulfur-bridged dimers, [Cu2X2(mu2-S-Htsc)2(Ph3P)2] [X, Htsc = Cl (4), Hbtsc; Br (9), Cl (10), pyrrole-2-carbaldehyde thiosemicarbazone (Hptsc); Br (12), Httsc]. All of these complexes have been characterized with the help of elemental analysis, IR, 1H, 13C, or 31P NMR spectroscopy, and X-ray crystallography (1-12). In all of the complexes, thiosemicarbazones are acting as neutral S-donor ligands in eta()S or mu2-S bonding modes. The Cu...Cu separations in the Cu(mu2-X)2Cu and Cu(mu2-S)2Cu cores lie in the ranges 2.981(1)-3.2247(6) and 2.813(1)-3.2329(8) Angstroms, respectively. The geometry around each Cu center in monomers and dimers may be treated as distorted tetrahedral. Ab initio density functional theory calculations on model monomeric and dimeric complexes of the simplest thiosemicarbazone [H2C=N-NH-C(S)-NH2, Htsc] have revealed that monomers and halogen-bridged dimers have similar stability and that sulfur-bridged dimers are stable only when halogen atoms are engaged in hydrogen bonding with the solvent of crystallization or H2O molecules.

Copper↗
PubMed2025

SVbyEye: a visual tool to characterize structural variation among whole-genome assemblies.

MOTIVATION: We are now in the era of being able to routinely generate highly contiguous (near telomere-to-telomere) genome assemblies of human and nonhuman species. Complex structural variation and regions of rapid evolutionary turnover are being discovered for the first time. Thus, efficient and informative visualization tools are needed to evaluate and directly observe structural differences between two or more genomes. RESULTS: We developed SVbyEye, an open-source R package to visualize and annotate sequence-to-sequence alignments along with various functionalities to process these alignments. The tool facilitates the characterization of complex structural variants in the context of sequence homology helping resolve the mechanisms underlying their formation. AVAILABILITY AND IMPLEMENTATION: SVbyEye is available on GitHub (https://github.com/daewoooo/SVbyEye) and via Zenodo (https://doi.org/10.5281/zenodo.15303553).

Software↗
PubMed2004

A new tridentate pincer phosphine/n-heterocyclic carbene ligand: palladium complexes, their structures, and catalytic activities.

A new imidazolium salt, 1,3-bis(2-diphenylphosphanylethyl)-3H-imidazol-1-ium chloride (2), for the phosphine/N-heterocyclic carbene-based pincer ligand, PC(NHC)P, and its palladium complexes were reported. The complex, [Pd(PC(NHC)P)Cl]Cl (4), was prepared by the common route of silver carbene transfer reaction and a novel direct reaction between the ligand precursor, PC(NHC)P.HCl and PdCl(2) without the need of a base. Metathesis reactions of 4 with AgBF(4) in acetonitrile produced [Pd(PC(NHC)P)(CH(3)CN)](BF(4))(2) (5). The same reaction in the presence of excess pyridine gave [Pd(PC(NHC)P)(py)](BF(4))(2) (6). The X-ray structure determination on 4-6 revealed the chiral twisting of the central imidazole rings from the metal coordination plane. In solution, fast interconversion between left- and right-twisted forms occurs. The twisting reflects the weak pi-accepting property of the central NHC in PC(NHC)P. The uneven extent of twisting among the three complexes further implies the low rotational barrier about the Pd-NHC bond. Related theoretical computations confirm the small rotational energy barrier about the Pd-NHC bond (ca. 4 kcal/mol). Catalytic applications of 4 and 5 have shown that the complexes are modest catalysts in Suzuki coupling. The complexes were active catalysts in Heck coupling reactions with the dicationic complex 5 being more effective than the monocationic complex 4.

Journal Article↗
PubMed2002

Synthesis of tris- and tetrakis(pyrazol-1-yl)borate gold(III) complexes. Crystal structures of [Au[kappa(2)-N,N'-BH(Pz)(3)]Cl(2)] (pz = pyrazol-1-yl) and [Au[kappa(2)-N,N'-B(Pz)(4)](kappa(2)-C,N-C(6)H(4)CH(2)NMe(2)-2)]ClO(4).CHCl(3).

Na[BH(pz)(3)] and Na[AuCl(4)].2H(2)O react in water (1:1) to give [Au[kappa(2)-N,N'-BH(pz)(3)]Cl(2)] (1) or, in the presence of NaClO(4) (2:1:1), the cationic complex [Au[kappa(2)-N,N'-BH(pz)(3)](2)]ClO(4) (2). The reactions of Na[B(pz)(4)] with the cyclometalated gold complexes [AuRCl(2)] and NaClO(4) (1:1:1) produce [Au[kappa(2)-N,N'-B(pz)(4)](R)]ClO(4) [R = kappa(2)-C,N-C(6)H(4)CH(2)NMe(2)-2 (3)] or [Au[kappa(2)-N,N'-B(pz)(4)](R)Cl] [R = C(6)H(3)(N=NC(6)H(4)Me-4')-2-Me-5 (4)], respectively, although 4 is better obtained in the absence of NaClO(4). The crystal structures of 1 and 3.CHCl(3) are reported. Both complexes display the gold center in square planar environments, two coordination sites being occupied by the chelating poly(pyrazolyl)borate ligands.

Journal Article↗
PubMed1984

Qualitative and quantitative differences between resident peritoneal mononuclear phagocytes of beige and control mice.

A qualitative and quantitative comparison of resident peritoneal macrophages (RPM) from beige (C57BL6Jbg/bg), (bg), and control black (C57BL/6J), (BL), mice has been made. Giant anomalous lysosomes as described for other leukocytes are inconsistently demonstrable. The principal morphological expression of the bg phenotype in RPM is the presence of elongated dumbbell-shaped, horseshoe-shaped, and ring-formed cytoplasmic lysosomes demonstrable by electron microscopy. Detailed analysis indicates that these are probably variations of the same basic structure, a deformed biconcave disc with differences in form resulting from their being cut in various planes of section. Additional structural complexity results from fusion between adjacent lysosomes. Thus the distinctive lysosomes of bg mouse RPM are not totally analogous to those structures described in other cell types. A morphometric analysis of control RPM compared to those from bg animals shows a significant decrease in the lysosomal volume proportion from 5.8% to 5.0% (P less than 0.02) with a corresponding increase in the mitochondrial volume proportion in cells from bg animals (P less than 0.05). There are no differences between bg and controls in regard to cellular profile areas, nuclear profile areas, nuclear volume proportions, and the volume to surface ratios of either the average cell or nucleus. This study represents the first compilation by morphometric methods of total granule content in the bg animal and demonstrates the ability of these techniques to detect significant quantitative changes in cells as structurally complex as mononuclear phagocytes.

Animals↗
PubMed2002

Analysis of the complex genomic structure of Bcl-x and its relationship to Bcl-x(gamma) expression after CD28-dependent costimulation.

The Bcl-x(gamma) cytosolic protein is essential for costimulatory activity after CD3/CD28 coligation. Here we delineate the Bcl-x(gamma)/Bcl-x genomic organization and the molecular mechanism that allows expression. We show that exon 4 of the Bcl-x gene encodes the unique C-terminal end of the Bcl-x(gamma) molecule while exons 5, 6, 7 and 8 are differentially transcribed to yield three alternative Bcl-x(gamma) 3' untranslated regions (UTR). CD28-dependent signals may increase levels of Bcl-x(gamma) protein through induction of an alternatively-spliced Bcl-x(gamma) 3' UTR that contains stem loop structures that stabilize Bcl-x(gamma) RNA. The ability receptor-induced signals to regulate the splicing pattern of the complex Bcl-x gene may allow T-cells to respond appropriately to antigenic stimuli.

3' Untranslated Regions↗
PubMed2005

Quinic acid derivatives as sialyl Lewis(x)-mimicking selectin inhibitors: design, synthesis, and crystal structure in complex with E-selectin.

A search for noncarbohydrate sLe(x) mimics led to the development of quinic acid derivatives as selectin inhibitors. At Wyeth we solved the first cocrystal structure of a small molecule, quinic acid, with E-selectin. In the cocomplex two hydroxyls of quinic acid mimic the calcium-bound fucose of the tetrasaccharide sLe(x). The X-ray structure, together with structure based computational methods, was used to design quinic acid based libraries that were synthesized and evaluated for their ability to block the interaction of sLex with P-selectin. A large number of analogues were prepared using solution-phase parallel synthesis. Selected compounds showed decrease in leukocyte rolling in the IVM mouse model. Compound 2 inhibited neutrophil influx in the murine TIP model and demonstrated good plasma exposure.

Animals↗
PubMed2003

Self-assembly in aqueous solution of wheel-shaped Mo154 oxide clusters into vesicles.

Surfactants and membrane lipids readily assemble into complex structures such as micelles, liposomes or hollow vesicles owing to their amphiphilic character-the fact that part of their structure is attracted to polar environments while another part is attracted to non-polar environments. The self-assembly of complex structures also occurs in polyoxometallate chemistry, as exemplified by the molybdenum blue solutions known for centuries. But while the presence of nanometre-sized metal oxide aggregates in these solutions has long been recognized, unravelling the composition and formation process of these aggregates proved difficult. Recent work has indicated that discrete, wheel-shaped mixed-valence polyoxomolybdate clusters of the type [Mo154] (refs 2-4) assemble into well-defined nanometre-sized aggregates, including spherical structures. Here we report light-scattering data and transmission electron microscopy images of hollow spherical structures with an average, almost monodisperse radius of about 45 nm and composed of approximately 1,165 [Mo154] wheel-shaped clusters. The clusters appear to lie flat and homogeneously distributed on the vesicle surface. Unlike conventional lipid vesicles, the structures we observe are not stabilized by hydrophobic interactions. Instead, we believe the polyoxomolybdate-based vesicles form owing to a subtle interplay between short-range van der Waals attraction and long-range electrostatic repulsion, with important further stabilization arising from hydrogen bonding involving water molecules encapsulated between the wheel-shaped clusters and in the vesicles' interior.

Journal Article↗
PubMed2001

Single particle macromolecular structure determination via electron microscopy.

Three-dimensional structure determination of macromolecules and macromolecular complexes is an integral part of understanding biological functions. For large protein and macromolecular complexes structure determination is often performed using electron cryomicroscopy where projection images of individual macromolecular complexes are combined to produce a three-dimensional reconstruction. Single particle methods have been devised to perform this structure determination for macromolecular complexes with little or no underlying symmetry. These computational methods generally involve an iterative process of aligning unique views of the macromolecular images followed by determination of the angular components that define those views. In this review, this structure determination process is described with the aim of clarifying a seemingly complex structural method.

Bacteriophages↗
PubMed2005

Uncovering the design principles of circadian clocks: mathematical analysis of flexibility and evolutionary goals.

In this paper, we present the mathematical details underlying both an approach to the flexibility of regulatory networks and an analytical characterization of evolutionary goals of circadian clock networks. A fundamental problem in cellular regulation is to understand the relation between the form of regulatory networks and their function. Circadian clocks present a particularly interesting instance of this. Recent work has shown that they have complex structures involving multiple interconnected feedback loops with both positive and negative feedback. We address the question of why they have such a complex structure and argue that it is to provide the flexibility necessary to simultaneously attain multiple key properties of circadian clocks such as robust entrainment and temperature compensation. To do this we address two fundamental problems: (A) to understand the relationships between the key evolutionary aims of the clock and (B) to ascertain how flexible the clock's structure is. To address the first problem we use infinitesimal response curves (IRCs), a tool that we believe will be of general utility in the analysis of regulatory networks. To understand the second problem we introduce the flexibility dimension d, show how to calculate it and then use it to analyse a range of models. We believe our results will generalize to a broad range of regulatory networks.

Animals↗
PubMed1989

Three-dimensional structure of complexes of single-stranded DNA-binding proteins with DNA. IKe and fd gene 5 proteins form left-handed helices with single-stranded DNA.

Specimen-tilting in an electron microscope was used to determine the three-dimensional architecture of the helical complexes formed with DNA by the closely related single-stranded DNA binding proteins of fd and IKe filamentous viruses. The fd gene 5 protein is the only member of the DNA-helix-destabilizing class of proteins whose structure has been determined crystallographically, and yet a parameter essential to molecular modeling of the co-operative interaction of this protein with DNA, the helix handedness, has not been available prior to this work. We find that complexes formed by titrating fd viral DNA with either the fd or IKe gene 5 protein have a left-handed helical sense. Complexes isolated from Escherichia coli infected by fd virus are also found to be left-handed helical; hence, the left-handed fd helices are not an artefact of reconstitution in vitro. Because the proteins and nucleic acid of the complexes are composed of asymmetric units which cannot be fitted equivalently to right-handed and left-handed helices, these results rule out a previous computer graphics atomic model for the helical fd complexes: a right-handed helix had been assumed for the model. Our work provides a defined three-dimensional structural framework within which to model the protein-DNA and protein-protein interactions of two structurally related proteins that bind contiguously and co-operatively on single-stranded DNAs.

DNA, Bacterial↗
PubMed1998

Implications for function and therapy of a 2.9 A structure of binary-complexed antithrombin.

The crystal structure of a binary complex of human antithrombin with a peptide of the same sequence as its reactive loop (P14-P3) has been determined at 2.9 A. The peptide binds as the middle strand s4A in the A beta-sheet, homologously to that of the reactive loop in the latent and cleaved forms of antithrombin. Peptide binding results in the complete expulsion of the hinge region of the loop from the A beta-sheet although the conformation differs from that of heparin-activated antithrombin. The 36-fold increase in the rate of reaction of the binary complex with factor Xa indicates that full loop expulsion alone is not sufficient for complete heparin activation of antithrombin but that this is also dependent on the overall conformation of the molecule. Previous studies have demonstrated that reactive loop peptides can block or reverse the polymerisation of serpins associated with cirrhosis and thrombosis. The antithrombin binary complex structure defines the precise localisation of the blocking peptide in a serpin and provides the basis for rational drug design for mimetics that will prevent polymerisation in vivo and so ameliorate the associated disease.

Amino Acid Sequence↗
PubMed2004

Structural basis for the selective inhibition of JNK1 by the scaffolding protein JIP1 and SP600125.

The c-jun N-terminal kinase (JNK) signaling pathway is regulated by JNK-interacting protein-1 (JIP1), which is a scaffolding protein assembling the components of the JNK cascade. Overexpression of JIP1 deactivates the JNK pathway selectively by cytoplasmic retention of JNK and thereby inhibits gene expression mediated by JNK, which occurs in the nucleus. Here, we report the crystal structure of human JNK1 complexed with pepJIP1, the peptide fragment of JIP1, revealing its selectivity for JNK1 over other MAPKs and the allosteric inhibition mechanism. The van der Waals contacts by the three residues (Pro157, Leu160, and Leu162) of pepJIP1 and the hydrogen bonding between Glu329 of JNK1 and Arg156 of pepJIP1 are critical for the selective binding. Binding of the peptide also induces a hinge motion between the N- and C-terminal domains of JNK1 and distorts the ATP-binding cleft, reducing the affinity of the kinase for ATP. In addition, we also determined the ternary complex structure of pepJIP1-bound JNK1 complexed with SP600125, an ATP-competitive inhibitor of JNK, providing the basis for the JNK specificity of the compound.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Role of HGF/SF and c-Met in morphogenesis and metastasis of epithelial cells.

We have analysed the role of hepatocyte growth factor/scatter factor (HGF/SF) in the process of morphogenesis and metastasis of epithelial (carcinoma) cells. HGF/SF induces various morphogenic responses in epithelial cells that derive from different tissues when these are grown in three-dimensional gels, e.g. branching tubules in kidney, breast, and prostate epithelial cells, crypt-like structures with brush border in colon epithelial cells, and alveolar-like aggregates in lung and pancreas cells. Epithelial cells are thus able to form complex structures in vitro which resemble the structures formed in the organ they originate from. We also examined the response of human breast carcinoma cells to HGF/SF in vivo. MDA MB 435 cells transfected with HGF/SF were injected into the mammary fat pad of nude mice, where they form tumours which spontaneously metastasize to the lungs. We found that expression of HGF/SF promoted metastasis whereas expression of the cell adhesion molecule E-cadherin was inhibitory. Moreover, expression of E-cadherin reconstituted the ability of the cells to form complex structures in response to HGF/SF in vitro. These data demonstrate that the different responses to HGF/SF depend on the state of the epithelial cells: morphogenesis requires epithelial differentiation and cell polarity, whereas metastasis is observed when the cells have lost their epithelial characteristics. Moreover, we have recently identified Gab-1 as a direct-binding substrate of the c-Met receptor. Gab-1 binds to c-Met phosphorylated on tyrosine residues, but not to a number of other tyrosine kinases from different subfamilies. A newly identified proline-rich domain of Gab-1 is responsible for the binding to the bidentate docking site in c-Met. Expression of Gab-1 in epithelial cells is sufficient to induce c-Met-specific cellular responses which include the formation of branching tubules. Thus, Gab-1 seem to correspond to the substrate of the c-Met receptor tyrosine kinase that mediates the epithelial morphogenesis.

Animals↗
PubMed2006

A stable neutral stannaaromatic compound: synthesis, structure and complexation of a kinetically stabilized 2-stannanaphthalene.

A kinetically stabilized 2-stannanaphthalene, the first example of a stable, neutral aromatic compound containing a tin atom, has been synthesized and fully characterized. The results of spectroscopic and crystallographic structural analyses of the compound and theoretical calculations using model compounds strongly suggest that it has a delocalized 10pi-electron ring system as does naphthalene. In addition, it was found to behave as an eta6-arene ligand in the ligand exchange reaction with [Cr(CH3CN)3(CO)3] giving the first stable eta6-2-stannanaphthalene chromium complex.

Journal Article↗