6,3'-dibromoflavone and 6-nitro-3'-bromoflavone: new additions to the 6,3'-disubstituted flavone family of high-affinity ligands of the brain benzodiazepine binding site with agonistic properties

6,3'-dibromoflavone and 6-nitro-3'-bromoflavone inhibited [(3)H]flunitrazepam binding to the benzodiazepine binding site of the gamma amino butyric acid receptor complex with K(i) values between 17 and 36 nM in different brain regions. Their gamma amino butyric acid ratio for [(3)H]flunitrazepam binding to cerebral cortex membranes indicated partial agonistic properties. Both compounds had similar pharmacological effects: they produced anxiolytic-like effects at low doses but did not alter locomotor activity or muscle tonicity; sedation was caused only at doses higher than 30 mg/kg in mice. These synthetic flavone derivatives join an existing family of 6,3'-disubstituted flavone compounds with high affinity for the benzodiazepine binding site and partial agonistic profiles.     

New dinuclear Co(II) and Mn(II) complexes of the phenol-based compartmental ligand containing formyl and amine functions: structural, spectroscopic and magnetic properties

The phenol-based compartmental ligand Hpy2ald contains a tridentate amino arm and a weak donor aldehyde group at the 2^′ and at the 6^′ positions of the phenol ring, respectively. This ligand reacts with cobalt(II) perchlorate, cobalt(II) tetrafluoroborate and manganese(II) perchlorate, yielding dinuclear complexes, where two metal ions are doubly bridged by two deprotonated cresolate moieties. The coordination environment around the metal ions is then completed to a very distorted octahedron by three nitrogen donor atoms from the pendant amino arm and the oxygen atom of the aldehyde group. The crystal structures of the complexes, their spectroscopic and magnetic properties are reported.     

The use of magnetic resonance imaging to predict ACL graft structural properties

Magnetic resonance imaging (MRI) could potentially be used to non-invasively predict the strength of an ACL graft after ACL reconstruction. We hypothesized that the volume and T2 relaxation parameters of the ACL graft measured with MRI will predict the graft structural properties and anteroposterior (AP) laxity of the reconstructed knee. Nine goats underwent ACL reconstruction using a patellar tendon autograft augmented with a collagen or collagen-platelet composite. After 6 weeks of healing, the animals were euthanized, and the reconstructed knees were retrieved and imaged on a 3T scanner. AP laxity was measured prior to dissecting out the femur-graft-tibia constructs which were then tested to tensile failure to determine the structural properties. Regression analysis indicated a statistically significant relationship between the graft volume and the failure load (r(2)=0.502; p=0.049). When graft volume was normalized to the T2 relaxation time, the relationship was even greater (r(2)=0.687; p=0.011). There was a significant correlation between the graft volume and the linear stiffness (r(2)=0.847; p<0.001), which remained significant with T2 normalization (r(2)=0.764; p=0.002). For AP laxity at 30° flexion, there was not a significant correlation with graft volume, but there was a significant correlation with volume normalized by the T2 relaxation time (r(2)=0.512; p=0.046). These results suggest that MRI volumetric measures combined with graft T2 properties may be useful in predicting the structural properties of ACL grafts.     

Discovering structural motifs using a structural alphabet: Application to magnesium-binding sites

Background  

For many metalloproteins, sequence motifs characteristic of metal-binding sites have not been found or are so short that they would not be expected to be metal-specific. Striking examples of such metalloproteins are those containing Mg²⁺, one of the most versatile metal cofactors in cellular biochemistry. Even when Mg²⁺-proteins share insufficient sequence homology to identify Mg²⁺-specific sequence motifs, they may still share similarity in the Mg²⁺-binding site structure. However, no structural motifs characteristic of Mg²⁺-binding sites have been reported. Thus, our aims are (i) to develop a general method for discovering structural patterns/motifs characteristic of ligand-binding sites, given the 3D protein structures, and (ii) to apply it to Mg²⁺-proteins sharing <30% sequence identity. Our motif discovery method employs structural alphabet encoding to convert 3D structures to the corresponding 1D structural letter sequences, where the Mg²⁺-structural motifs are identified as recurring structural patterns.     

BreaKmer: detection of structural variation in targeted massively parallel sequencing data using kmers

Genomic structural variation (SV), a common hallmark of cancer, has important predictive and therapeutic implications. However, accurately detecting SV using high-throughput sequencing data remains challenging, especially for ‘targeted’ resequencing efforts. This is critically important in the clinical setting where targeted resequencing is frequently being applied to rapidly assess clinically actionable mutations in tumor biopsies in a cost-effective manner. We present BreaKmer, a novel approach that uses a ‘kmer’ strategy to assemble misaligned sequence reads for predicting insertions, deletions, inversions, tandem duplications and translocations at base-pair resolution in targeted resequencing data. Variants are predicted by realigning an assembled consensus sequence created from sequence reads that were abnormally aligned to the reference genome. Using targeted resequencing data from tumor specimens with orthogonally validated SV, non-tumor samples and whole-genome sequencing data, BreaKmer had a 97.4% overall sensitivity for known events and predicted 17 positively validated, novel variants. Relative to four publically available algorithms, BreaKmer detected SV with increased sensitivity and limited calls in non-tumor samples, key features for variant analysis of tumor specimens in both the clinical and research settings.     

Non-invasive detection of apoptosis using magnetic resonance imaging and a targeted contrast agent.

The C2 domain of synaptotagmin I, which binds to anionic phospholipids in cell membranes, was shown to bind to the plasma membrane of apoptotic cells by both flow cytometry and confocal microscopy. Conjugation of the protein to superparamagnetic iron oxide nanoparticles allowed detection of this binding using magnetic resonance imaging. Detection of apoptotic cells, using this novel contrast agent, was demonstrated both in vitro, with isolated apoptotic tumor cells, and in vivo, in a tumor treated with chemotherapeutic drugs.     

Analysis of the eukaryotic topoisomerase II DNA gate: a single-molecule FRET and structural perspective

Type II DNA topoisomerases (topos) are essential and ubiquitous enzymes that perform important intracellular roles in chromosome condensation and segregation, and in regulating DNA supercoiling. Eukaryotic topo II, a type II topoisomerase, is a homodimeric enzyme that solves topological entanglement problems by using the energy from ATP hydrolysis to pass one segment of DNA through another by way of a reversible, enzyme-bridged double-stranded break. This DNA break is linked to the protein by a phosphodiester bond between the active site tyrosine of each subunit and backbone phosphate of DNA. The opening and closing of the DNA gate, a critical step for strand passage during the catalytic cycle, is coupled to this enzymatic cleavage/religation of the backbone. This reversible DNA cleavage reaction is the target of a number of anticancer drugs, which can elicit DNA damage by affecting the cleavage/religation equilibrium. Because of its clinical importance, many studies have sought to determine the manner in which topo II interacts with DNA. Here we highlight recent single-molecule fluorescence resonance energy transfer and crystallographic studies that have provided new insight into the dynamics and structure of the topo II DNA gate.     

Diverse structural solutions to catalysis in a family of antibodies

BACKGROUND: Small organic molecules coupled to a carrier protein elicit an antibody response on immunisation. The diversity of this response has been found to be very narrow in several cases. Some antibodies also catalyse chemical reactions. Such catalytic antibodies are usually identified among those that bind tightly to an analogue of the transition state (TSA) of the relevant reaction; therefore, catalytic antibodies are also thought to have restricted diversity. To further characterise this diversity, we investigated the structure and biochemistry of the catalytic antibody 7C8, one of the most efficient of those which enhance the hydrolysis of chloramphenicol esters, and compared it to the other catalytic antibodies elicited in the same immunisation. RESULTS: The structure of a complex of the 7C8 antibody Fab fragment with the hapten TSA used to elicit it was determined at 2.2 A resolution. Structural comparison with another catalytic antibody (6D9) raised against the same hapten revealed that the two antibodies use different binding modes. Furthermore, whereas 6D9 catalyses hydrolysis solely by transition-state stabilisation, data on 7C8 show that the two antibodies use mechanisms where the catalytic residue, substrate specificity and rate-limiting step differ. CONCLUSIONS: Our results demonstrate that substantial diversity may be present among antibodies catalysing the same reaction. Therefore, some of these antibodies represent different starting points for mutagenesis aimed at boosting their activity. This increases the chance of obtaining more proficient catalysts and provides opportunities for tailoring catalysts with different specificities.     

Ubiquitin family proteins and their relationship to the proteasome: a structural perspective

Many biological processes rely on targeted protein degradation, the dysregulation of which contributes to the pathogenesis of various diseases. Ubiquitin plays a well-established role in this process, in which the covalent attachment of polyubiquitin chains to protein substrates culminates in their degradation via the proteasome. The three-dimensional structural topology of ubiquitin is highly conserved as a domain found in a variety of proteins of diverse biological function. Some of these so-called "ubiquitin family proteins" have recently been shown to bind components of the 26S proteasome via their ubiquitin-like domains, thus implicating proteasome activity in pathways other than protein degradation. In this chapter, we provide a structural perspective of how the ubiquitin family of proteins interacts with the proteasome.     

Toward a structural understanding of arf family:effector specificity

Arf family proteins are critical regulators of intracellular trafficking and actin cytoskeleton dynamics. To carry out their cellular functions, Arf family proteins interact with various effectors that differ in nature and structure. Understanding how these proteins interact with structurally different partners and are distinguished by specific effectors while being closely related requires a structural characterization and comparison of the various Arf family:effector complexes. Recent structural reports of Arf and Arl proteins in complex with different downstream effectors shed new light on general and specific structural recognition determinants characteristic of Arf family proteins.     

Synthesis, structural determination and magnetic properties of a heterobimetallic CuRe complex containing a macrocyclic ligand

Reaction of the rhenium(IV) compound, [Bu₄N]₂ReCl₄ox, with the highly unsaturated tetraazabismacrocyclic copper(II) complex cation [CuCuL]⁴⁺ (L = 6,13-Bis(dodecylaminomethylidene)-1,4,8,11-tetraazacyclotetradeca-4,7,11,14-tetraene) produced a new kind of heterobimetallic compound: [CuCuL][ReCl₄ox]₂ · 2DMF in which [ReCl₄ox]²⁻ anions and [CuCuL]⁴⁺ cations are linked by electrostatic forces. The crystal structure of this compound was determined at 173(2) K. It crystallizes triclinic, space group , with a = 9.441(4), b = 11.032(5), c = 15.261(7) Å, α = 89.05(1)°, β = 88.93(1)°, γ = 77.09(1)°, Z = 1, R₁ = 0.0557, wR₂ = 0.1332. The magnetic behavior of this compound has been investigated over the temperature range 1.72-300 K. The compound behaves as a ferrimagnetic Cu^IIRe^IV bimetallic, chain with intrachain antiferromagnetic coupling.     

The 4-oxalocrotonate tautomerase family of enzymes: how nature makes new enzymes using a beta-alpha-beta structural motif

4-Oxalocrotonate tautomerase (4-OT) catalyzes the isomerization of beta,gamma-unsaturated enones to their alpha,beta-isomers. The enzyme is part of a plasmid-encoded pathway, which enables bacteria harboring the plasmid to use various aromatic hydrocarbons as their sole sources of carbon and energy. Among isomerases and enzymes in general, 4-OT is unusual for two reasons: it has one of the smallest known monomer sizes (62 amino acids) and the amino-terminal proline functions as the catalytic base. In addition to Pro-1, three other residues (Arg-11, Arg-39, and Phe-50) have been identified as critical catalytic residues by kinetic analysis, site-directed mutagenesis, chemical synthesis, NMR, and crystallographic studies. Arginine-39 functions as the general acid catalyst (assisted by an ordered water molecule) in the reaction while Arg-11 plays a role in substrate binding and facilitates catalysis by acting as an electron sink. Finally, the hydrophobic nature of the active site, which lowers the pK(a) of Pro-1 to approximately 6.4 and provides a favorable environment for catalysis, is largely maintained by Phe-50. 4-OT is also the title enzyme of the 4-OT family of enzymes. The chromosomal homologues in this family are composed of monomers ranging in size from 61 to 79 amino acids, which code a beta-alpha-beta structural motif. The homologues all retain Pro-1 and generally have an aromatic or hydrophobic amino acid at the Phe-50 position. Characterization of representative members has uncovered mechanistic and structural diversity. A new activity, a trans-3-chloroacrylic acid dehalogenase, has been identified in addition to the previously known tautomerase and isomerase activities. Two new structures have also been found, along with the 4-OT hexamer. The dehalogenase functions as a heterohexamer while the Escherichia coli homologue, designated YdcE, functions as a dimer. Moreover, both 4-OT and the Bacillus subtilis homologue, designated YwhB, exhibit low-level dehalogenase activity. Amplification of this activity could have produced the full-fledged dehalogenase. The sum of these observations indicates that Nature uses the beta-alpha-beta structural motif as a building block in a variety of manners to create new enzymes.     

Growth of human cultured cells exposed to a non-homogeneous static magnetic field generated by Sm-Co magnets.

A static magnetic field, with a strong spatial gradient, was established on the surface of cell culture dishes by use of a gilded iron needle set vertically above an Sm-Co magnet. The calculated magnetic flux density was more than 1.5 T at the center of the needle tip, and the products of the flux density and its gradient were about 200 and 60 T2/m at distances of 0.1 and 0.3 mm, respectively, from the center. The DNA content, DNA synthesis and labeling index of cultured cells located within 0.1 mm from the center of the needle, and the growth rate of cells located within 0.3 mm from the center, were measured. HeLa cells grew at a normal rate for 96 h in the magnetic field and showed no significant change in shape, detectable by scanning electron microscopy. The growth of HeLa cells was not influenced by exposure to the magnetic field. Similarly, exposure for 48 h to the magnetic field had no effect on growth of normal human gingival fibroblasts (Gin-1). The DNA content, assayed by microfluorometry of the nuclei of both types of cells stained by the Feulgen reaction, was not significantly different from that of controls. Moreover, exposure to the magnetic field had no effect on DNA synthesis or the labeling index of HeLa cells assayed by autoradiography of incorporated [3H]thymidine. It is concluded that a non-homogeneous magnetic field of the intensity and the gradient used in this study does not significantly influence the growth of HeLa cells or Gin-1 cells.     

The transthyretin-related protein: structural investigation of a novel protein family

The transthyretin-related protein (TRP) family comprises proteins predicted to be structurally related to the homotetrameric transport protein transthyretin (TTR). The function of TRPs is not yet fully established, but recent data suggest that they are involved in purine catabolism. We have determined the three-dimensional structure of the Escherichia coli TRP in two crystal forms; one at 1.65 A resolution in the presence of zinc, and the other at 2.1 A resolution in the presence of zinc and bromide. The structures revealed five zinc-ion-binding sites per monomer. Of these, the zinc ions bound at sites I and II are coordinated in tetrahedral geometries to the side chains of residues His9, His96, His98, Ser114, and three water molecules at the putative ligand-binding site. Of these four residues, His9, His98, and Ser114 are conserved. His9 and His98 bind the central zinc (site I) together with two water molecules. The side chain of His98 also binds to the zinc ion at site II. Bromide ions bind at site I only, replacing one of the water molecules coordinated to the zinc ion. The C-terminal four amino acid sequence motif Y-[RK]-G-[ST] constitutes the signature sequence of the TRP family. Two Tyr111 residues form direct hydrogen bonds to each other over the tetramer interface at the area, which in TTR constitutes the rear part of its thyroxine-binding channel. The putative substrate/ligand-binding channel of TRP is consequently shallower and broader than its counterpart in TTR.     

The tRNase Z family of proteins: physiological functions, substrate specificity and structural properties

tRNase Z is the endoribonuclease that generates the mature 3'-end of tRNA molecules by removal of the 3'-trailer elements of precursor tRNAs. This enzyme has been characterized from representatives of all three domains of life (Bacteria, Archaea and Eukarya), as well as from mitochondria and chloroplasts. tRNase Z enzymes come in two forms: short versions (280-360 amino acids in length), present in all three kingdoms, and long versions (750-930 amino acids), present only in eukaryotes. The recently solved crystal structure of the bacterial tRNase Z provides the structural basis for the understanding of central functional elements. The substrate is recognized by an exosite that protrudes from the main protein body and consists of a metallo-beta-lactamase domain. Cleavage of the precursor tRNA occurs at the binuclear zinc site located in the other subunit of the functional homodimer. The first gene of the tRNase Z family was cloned in 2002. Since then a comprehensive set of data has been acquired concerning this new enzyme, including detailed functional studies on purified recombinant enzymes, mutagenesis studies and finally the determination of the crystal structure of three bacterial enzymes. This review summarizes the current knowledge about these exciting enzymes.     

In vivo detection of c-MET expression in a rat hepatocarcinogenesis model using molecularly targeted magnetic resonance imaging

The multifunctional growth factor scatter factor/hepatocyte growth factor and its tyrosine kinase receptor, c-MET, have been implicated in the genesis and malignant progression of numerous human malignancies, including hepatocellular carcinomas. The incidence of hepatocellular carcinomas in the United States has increased noticeably over the past two decades and is listed as the fifth major cancer in men worldwide. In this study, we used a choline-deficient l-amino acid (CDAA)-defined rat hepatocarcinogenesis model to visualize increased in vivo expression of the c-MET antigen in neoplastic lesion formation with the use of a super paramagnetic iron oxide (SPIO)-anti-c-MET molecularly targeted magnetic resonance imaging (MRI) contrast agent. SPIO-anti-c-MET was used for the first time to detect overexpression of c-MET in neoplastic nodules and tumors within the livers of CDAA-treated rats, as determined by a decrease in MRI signal intensity and a decrease in regional T(2) values. Specificity for the binding of the molecularly targeted anti-c-MET contrast agent was determined using rat hepatoma (H4-II-E-C3) cell cultures and immunofluorescence microscopic imaging of the targeting agents within neoplastic liver tissue 1 to 2 hours following intravenous administration of SPIO-anti-c-MET and MRI investigation. This method has the ability to visualize in vivo the overexpression of c-MET at early developmental stages of tumor formation.     

Molecular and structural characterization of a surfactant-stable high-alkaline protease AprB with a novel structural feature unique to subtilisin family

High-alkaline proteases are of great importance because of their proteolytic activity and stability under high-alkaline condition. We have previously isolated a new protease (AprB) which has potential industrial applications based on its high-alkaline adaptation. However, the molecular and structural basis for alkaline adaptation of this enzyme has not been fully elucidated. In the present study, AprB gene was cloned and expressed in the Bacillus subtilis WB600. This gene codes for a protein of 375 amino acids comprised with a 28-residual signal peptide, a 78-residual pro-peptide, and a 269-residual mature protein. The deduced amino acid sequence has the highest homology of 63.2% with that of the high-alkaline proteases. Recombinant AprB was purified and determined to be monomeric with molecular mass of 26.755 kDa. The NH₂-terminal sequence of the purified AprB was A-Q-S-I-P-W-G-I-E-R. This enzyme exhibited high catalytic efficiencies (K_cat/K_m) towards natural, modified, and synthesis substrates with optimal activity at 60 °C and pH 10. AprB was stable over a wide range of pH 5 to 11 and various surfactants, and could be activated by Mg²⁺, Ca²⁺ and Ba²⁺. The structural properties of AprB, like a higher ratio of R/(R + K), a larger area of hydrophobic surface, increased number of ion pairs formed by Arg residue, and the exposure of Asp active residue on the surface, might be responsible for its alkaline adaptation. In contrast with members of subtilisin family, such as M-protease and subtilisin BPN′, AprB harbored a high content of Glu and Asp residues, and a low content of Arg and Lys residues on the surface. Interestingly, these structural characters were similar with that of psychrophilic proteases, which suggested that these molecular factors were not restricted in the psychrophilic proteases, and therefore were not solely responsible for their cold-adaptation. Our results reveal a novel structural feature of AprB unique to subtilisin family and provide clues for its alkaline adaptation.     

A wide family of pyridoxal thiosemicarbazone ferric complexes: Syntheses, structures and magnetic properties

This study reports the syntheses and the characterization of 12 ferric complexes of pyridoxal thiosemicarbazone. The richness of the coordination chemistry of this ligand is highlighted by the modulation of its charge from neutral H₂L to anionic L²⁻, thus leading to a wide family of ferric compounds with charge varying from +3 to −1. The structures of complexes [Fe(HL)₂]ClO₄ · 2H₂O and [Fe(HL)L] · 4.5H₂O were solved and discussed with a particular attention brought to the intermolecular interactions occurring between the complexes. The investigation of magnetic properties of these compounds revealed that two of them are in the HS state at any temperature, whereas the others are in the LS state. These results are discussed in relation to the modulation of the electronic properties of the ligand.     

The structural biology of the dynamin‐related proteins: New insights into a diverse,multitalented family

Dynamin‐related proteins are multidomain, mechanochemical GTPases that self‐assemble and orchestrate a wide array of cellular processes. Over the past decade, structural insights from X‐ray crystallography and cryo‐electron microscopy have reshaped our mechanistic understanding of these proteins. Here, we provide a historical perspective on these advances that highlights the structural attributes of different dynamin family members and explores how these characteristics affect GTP hydrolysis, conformational coupling and oligomerization. We also discuss a number of lingering challenges remaining in the field that suggest future directions of study.