Insight into the inhibition mechanism and structure–activity relationship of 2,6-dipicolinic acid and its analogue to New Delhi metallo-β-lactamase-1

In previous literature, it was found that the activity of New Delhi Metallo-β-lactamase-1 (NDM-1) was inhibited by 2,6-dipicolinic acid (DPA) derivatives. To identify the mechanism of interaction between the inhibitors and NDM-1, molecular dynamics simulations were performed for the complex systems. Via the molecular modelling, inhibitors were found to be able bind to the region of catalytic activity of NDM-1. However, the detailed binding sites of the inhibitors differed with their structures. It was determined that His189, Lys211, Met248, Ser249, His250, and Ser251 are key residues for the binding of inhibitor 36 with NDM-1, and Asp124 is the only critical residue in the NDM-1-DPA complex. Furthermore, because of the interaction of the benzene ring in inhibitor 36 with the side chain of Lys211, inhibitor 36 can form 4 strong hydrogen bonds with protein. For the NDM-1-DPA complex, owing to the absence of the aniline group, DPA can only form a weak interaction with the residues around the binding site of NDM-1, except for Asp124, leading to a weaker inhibitory activity. Therefore, we believe that the strong interaction of the inhibitor with Lys211 results in effective inhibition, and the aniline group is the element required for the inhibitory activity.     

Structure and catalytic mechanism of the β-carbonic anhydrases

The β-carbonic anhydrases (β-CAs) are a diverse but structurally related group of zinc-metalloenzymes found in eubacteria, plant chloroplasts, red and green algae, and in the Archaea. The enzyme catalyzes the rapid interconversion of CO₂ and H₂O to HCO₃⁻ and H⁺, and is believed to be associated with metabolic enzymes that consume or produce CO₂ or HCO₃⁻. For many organisms, β-CA is essential for growth at atmospheric concentrations of CO₂. Of the five evolutionarily distinct classes of carbonic anhydrase, β-CA is the only one known to exhibit allosterism. Here we review the structure and catalytic mechanism of β-CA, including the structural basis for allosteric regulation.     

Molecular Modelling reveals the inhibition mechanism and structure–activity relationship of curcumin and its analogues to Staphylococcal aureus Sortase A

Previous studies found that the activity of Sortase A, a bacterial surface protein from Staphylococcus aureus, was inhibited by curcumin and its analogues. To explore this inhibitory mechanism, Sortase A and its inhibitors in complex systems were studied by molecular docking, molecular modelling, binding energy decomposition calculation and steered molecular dynamics simulations. Energy decomposition analysis indicated that PRO-163, LEU-169, GLN-172, ILE-182 and ILE-199 are key residues in Sortase A-inhibitor complexes. Furthermore, interactions between the methoxyl group on the benzene ring in the conjugated molecule (curcumin, demethoxycurcumin, bisdemethoxycurcumin) and VAL-168, LEU-169 and GLN-172 induce the inhibitory activity based on the energy decomposition and distance analyses between the whole residues and inhibitors. However, because of its coiled structure, the non-conjugated molecule, tetrahydrocurcumin, with key residues in the binding sites of Sortase A, interacted weakly with SrtA, leading to the loss of inhibitory activity. Based on these results, the methoxyl group on the benzene ring in the conjugated molecule largely influenced the inhibitory activity of the Sortase A inhibitors.     

Structure and function of γ-secretase

The γ-secretase complex is a prime target for pharmacological intervention in Alzheimer’s disease and so far drug discovery efforts have yielded a large variety of potent and rather specific inhibitors of this enzymatic activity. However, as γ-secretase is able to cleave a wide variety of physiological important substrates, the real challenge is to develop substrate-specific compounds. Therefore, obtaining structural information about γ-secretase is indispensable. As crystal structures of the complex will be difficult to achieve, applied biochemical approaches need to be integrated with structural information obtained from other intramembrane-cleaving proteases. Here we review current knowledge about the structure and function of γ-secretase and discuss the value of these findings for the mechanistic understanding of this unusual protease.     

Structure and mechanism of the γ-secretase intramembrane protease complex

γ-Secretase is a membrane protein complex that proteolyzes within the transmembrane domain of >100 substrates, including those derived from the amyloid precursor protein and the Notch family of cell surface receptors. The nine-transmembrane presenilin is the catalytic component of this aspartyl protease complex that carries out hydrolysis in the lipid bilayer. Advances in cryoelectron microscopy have led to the elucidation of the structure of the γ-secretase complex at atomic resolution. Recently, structures of the enzyme have been determined with bound APP- or Notch-derived substrates, providing insight into the nature of substrate recognition and processing. Molecular dynamics simulations of substrate-bound enzymes suggest dynamic mechanisms of intramembrane proteolysis. Structures of the enzyme bound to small-molecule inhibitors and modulators have also been solved, setting the stage for rational structure-based drug discovery targeting γ-secretase.     

The effects and mechanism of flavonoid–rePON1 interactions. Structure–activity relationship study

Flavonoids are plant phenolic secondary metabolites that are widely distributed in the human diet. These antioxidants have received much attention because of their neuroprotective, cardioprotective, and chemopreventive actions. While a major focus has been on the flavonoids’ antioxidant properties, there is an emerging view that many of the potential health benefits of flavonoids and their in vivo metabolites are due to modulatory actions in cells through direct interactions with proteins, and not necessarily due to their antioxidant function. This view relies on the observations that flavonoids are present in the circulation at very low concentrations, which are not sufficient to exert effective antioxidant effects. The enzyme paraoxonase 1 (PON1) is associated with high-density lipoprotein (HDL), and is responsible for many of HDLs’ antiatherogenic properties. We previously showed that the flavonoid glabridin binds to rePON1 and affects the enzyme’s 3D structure. This interaction protects the enzyme from inhibition by an atherogenic component of the human carotid plaque. Here, we broadened our study to an investigation of the structure–activity relationships (SARs) of 12 flavonoids from different subclasses with rePON1 using Trp-fluorescence quenching, modeling calculations and Cu²⁺-induced low-density lipoprotein (LDL) oxidation methods. Our findings emphasize the ‘protein-binding’ mechanism by which flavonoids exert their beneficial biological role toward rePON1. Flavonoids’ capacity to interact with the enzyme’s rePON1 hydrophobic groove mostly dictates their pro/antioxidant behavior.     

Structure–function analysis of peroxidasin provides insight into the mechanism of collagen IV crosslinking

Basement membranes provide structural support and convey regulatory signals to cells in diverse tissues. Assembly of collagen IV into a sheet-like network is a fundamental mechanism during the formation of basement membranes. Peroxidasin (PXDN) was recently described to catalyze crosslinking of collagen IV through the formation of sulfilimine bonds. Despite the significance of this pathway in tissue genesis, our understanding of PXDN function is far from complete. In this work we demonstrate that collagen IV crosslinking is a physiological function of mammalian PXDN. Moreover, we carried out structure–function analysis of PXDN to gain a better insight into its role in collagen IV synthesis. We identify conserved cysteines in PXDN that mediate the oligomerization of the protein into a trimeric complex. We also demonstrate that oligomerization is not an absolute requirement for enzymatic activity, but optimal collagen IV coupling is only catalyzed by the PXDN trimers. Localization experiments of different PXDN mutants in two different cell models revealed that PXDN oligomers, but not monomers, adhere on the cell surface in “hot spots,” which represent previously unknown locations of collagen IV crosslinking.     

Structure–function relationship of three neurotoxins from the venom of Naja kaouthia: a comparison between the NMR-derived structure of NT2 with its homologues,NT1 and NT3

Three homologous short-chain neurotoxins, named NT1, NT2 and NT3, were purified from the venom of Naja kaouthia. NT1 has an identical amino acid sequence to cobrotoxin from Naja naja atra [Biochemistry 32 (1993) 2131]. NT3 shares the same sequence with cobrotoxin b [J. Biochem. (Tokyo) 122 (1997) 1252], whereas NT2 is a novel 61-residue neurotoxin. Tests of their physiological functions indicate that NT1 shows a greater inhibition of muscle contraction induced by electrical stimulation of the nerve than do NT2 and NT3. Homonuclear proton two-dimensional NMR methods were utilized to study the solution tertiary structure of NT2. A homology model-building method was employed to predict the structure of NT3. Comparison of the structures of these three toxins shows that the surface conformation of NT1 facilitates the substituted base residues, Arg28, Arg30, and Arg36, to occupy the favorable spatial location in the central region of loop II, and the cation groups of all three arginines face out of the molecular surface of NT1. This may contribute greatly to the higher binding of NT1 with AchR compared to NT2 and NT3.     

Characterization of a second carotenoid β-hydroxylase gene from Arabidopsis and its relationship to the LUT1 locus

Xanthophylls are oxygenated carotenoids that perform critical roles in plants. -carotene hydroxylases (-hydroxylases) add hydroxyl groups to the -rings of carotenes and have been cloned from several bacteria and plants, including Arabidopsis. The lut1 mutation of Arabidopsis disrupts -ring hydroxylation and has been suggested to identify a related carotene hydroxylase that functions specifically on -ring structures. We have used library screening and genomics-based approaches to isolate a second -hydroxylase genomic clone and its corresponding cDNA from Arabidopsis. The encoded protein is 70% identical to the previously reported Arabidopsis -hydroxylase 1. Phylogenetic analysis indicates a common origin for the two proteins, however, their different chromosomal locations, intron positions and intron sizes suggest their duplication is not recent. Although both hydroxylases are expressed in all Arabidopsis tissues analyzed, -hydroxylase 1 mRNA is always present at higher levels. Both cDNAs encode proteins that efficiently hydroxylate the C-3 position of -ring containing carotenes and are only weakly active towards -ring containing carotenes. Neither -hydroxylase cDNA maps to the LUT1 locus, and the genomic region encompassing the LUT1 locus does not contain a third related hydroxylase. These data indicate that the LUT1 locus encodes a protein necessary for -ring hydroxylation but unrelated to -hydroxylases at the level of amino acid sequence.     

Structure of the zooplankton community in a subtropical shallow lake (Itapeva Lake – South of Brazil) and its relationship to hydrodynamic aspects

The structure of the zooplankton community in Itapeva Lake was formed by four groups and more than 127 zooplankton species, in which microplankton was the predominant size structure. The largest richness recorded was of the protists group and in autumn seasonal campaign. Protists were characteristic of the lake, regarding density, except during spring at the Center point (copepods) and autumn at the South point (rotifers). The seasonal distribution revealed that during summer, mean density increased in the zooplankton community, exactly the opposite of phytoplankton (that blooms during the cold season). However, the maximum density was recorded during autumn. High density was recorded for the ciliate Codonella sp. at all points and during all seasons. The abundance of the tecamoeba Difflugia tuberculata was strongly associated with the maximum effects of fetch in the lake. Rotifers were generally the second most representative groups in terms of density. Rotifers and Cladocera were more abundant in summer, whereas copepods were in spring and winter/98. The Shannon–Wiener index showed that the smallest zooplankton diversity average occurred during the winter/98 (H=1.44), while in autumn the largest zooplankton diversity average (H=2.36) was observed. Correlations (r-Pearson, p<0.05) with wind velocity were significant for zooplankton density (groups and/or abundant species), diversity, and richness. The analysis of variance (ANOVA) showed a seasonally significant spatial-temporal variation for the factors sampling point, day and shift (p<0.01). Temporal alterations in density, diversity and richness were closely dependent on the hydrodynamic action induced by the wind on the spatial distribution of the zooplankton community in the Itapeva Lake.     

Are the health attributes of lycopene related to its antioxidant function?

A variety of epidemiological trials have suggested that higher intake of lycopene-containing foods (primarily tomato products) or blood lycopene concentrations are associated with decreased cardiovascular disease and prostate cancer risk. Of the carotenoids tested, lycopene has been demonstrated to be the most potent in vitro antioxidant leading many researchers to conclude that the antioxidant properties of lycopene are responsible for disease prevention. In our review of human and animal trials with lycopene, or lycopene-containing extracts, there is limited support for the in vivo antioxidant function for lycopene. Moreover, tissue levels of lycopene appear to be too low to play a meaningful antioxidant role. We conclude that there is an overall shortage of supportive evidence for the “antioxidant hypothesis” as lycopene’s major in vivo mechanism of action. Our laboratory has postulated that metabolic products of lycopene, the lycopenoids, may be responsible for some of lycopene’s reported bioactivity.     

Structure–function relationship in P-type ATPases—a biophysical approach

P-type ATPases are a large family of membrane proteins that perform active ion transport across biological membranes. In these proteins the energy-providing ATP hydrolysis is coupled to ion-transport that builds up or maintains the electrochemical potential gradients of one or two ion species across the membrane. P-type ATPases are found in virtually all eukaryotic cells and also in bacteria, and they are transporters of a broad variety of ions. So far, a crystal structure with atomic resolution is available only for one species, the SR Ca-ATPase. However, biochemical and biophysical studies provide an abundance of details on the function of this class of ion pumps. The aim of this review is to summarize the results of preferentially biophysical investigations of the three best-studied ion pumps, the Na,K-ATPase, the gastric H,K-ATPase, and the SR Ca-ATPase, and to compare functional properties to recent structural insights with the aim of contributing to the understanding of their structure–function relationship.     

Structure,function, and formation of extrusive organelles — microtoxicysts in the rhizopodPenardia cometa

Summary A study of the structure, function, and development of extrusive organelles (microtoxicysts) in the unusual bacteriovorous rhizopodPenardia cometa is performed. Microtoxicysts are located in the cortical cytoplasm of the cell body and in special thickenings on reticulopodia. Similar types of extrusomes have been observed in some cercomonads. The microtoxicysts are organized as membrane-bound vesicles of a complex form. An oviform-conical axial element lies inside each vesicle and is directed with its narrower end towards the plasma membrane. An internal cylindrical tube occupies the central part of the axial element; it is turned out as the organelle is shot out. The extrusomes ofP. cometa originate from and develop in derivates of the endoplasmic reticulum, the initial diameter of proextrusome vesicles is twice the diameter of the mature organelles. At late stages of maturation the microtoxicysts adopt their characteristic form and orientation. The mode of construction, ejection and development is compared with that in some other carnivorous and bacterivorous protists.     

Structure and function of BamE within the outer membrane and the β-barrel assembly machine

Insertion of folded proteins into the outer membrane of Gram-negative bacteria is mediated by the essential β-barrel assembly machine (Bam). Here, we report the native structure and mechanism of a core component of this complex, BamE, and show that it is exclusively monomeric in its native environment of the periplasm, but is able to adopt a distinct dimeric conformation in the cytoplasm. BamE is shown to bind specifically to phosphatidylglycerol, and comprehensive mutagenesis and interaction studies have mapped key determinants for complex binding, outer membrane integrity and cell viability, as well as revealing the role of BamE within the Bam complex.     

Structure and function of the N‐terminal domain of the human mitochondrial calcium uniporter

The mitochondrial calcium uniporter (MCU) is responsible for mitochondrial calcium uptake and homeostasis. It is also a target for the regulation of cellular anti‐/pro‐apoptosis and necrosis by several oncogenes and tumour suppressors. Herein, we report the crystal structure of the MCU N‐terminal domain (NTD) at a resolution of 1.50 Å in a novel fold and the S92A MCU mutant at 2.75 Å resolution; the residue S92 is a predicted CaMKII phosphorylation site. The assembly of the mitochondrial calcium uniporter complex (uniplex) and the interaction with the MCU regulators such as the mitochondrial calcium uptake‐1 and mitochondrial calcium uptake‐2 proteins (MICU1 and MICU2) are not affected by the deletion of MCU NTD. However, the expression of the S92A mutant or a NTD deletion mutant failed to restore mitochondrial Ca²⁺ uptake in a stable MCU knockdown HeLa cell line and exerted dominant‐negative effects in the wild‐type MCU‐expressing cell line. These results suggest that the NTD of MCU is essential for the modulation of MCU function, although it does not affect the uniplex formation.     

Structure and mechanism of the 2′,3′ phosphatase component of the bacterial Pnkp-Hen1 RNA repair system

Pnkp is the end-healing and end-sealing component of an RNA repair system present in diverse bacteria from many phyla. Pnkp is composed of three catalytic modules: an N-terminal polynucleotide 5′ kinase, a central 2′,3′ phosphatase and a C-terminal ligase. The phosphatase module is a Mn²⁺-dependent phosphodiesterase–monoesterase that dephosphorylates 2′,3′-cyclic phosphate RNA ends. Here we report the crystal structure of the phosphatase domain of Clostridium thermocellum Pnkp with Mn²⁺ and citrate in the active site. The protein consists of a core binuclear metallo-phosphoesterase fold (exemplified by bacteriophage λ phosphatase) embellished by distinctive secondary structure elements. The active site contains a single Mn²⁺ in an octahedral coordination complex with Asp187, His189, Asp233, two citrate oxygens and a water. The citrate fills the binding site for the scissile phosphate, wherein it is coordinated by Arg237, Asn263 and His264. The citrate invades the site normally occupied by a second metal (engaged by Asp233, Asn263, His323 and His376), and thereby dislocates His376. A continuous tract of positive surface potential flanking the active site suggests an RNA binding site. The structure illuminates a large body of mutational data regarding the metal and substrate specificity of Clostridium thermocellum Pnkp phosphatase.     

Positive and negative affect during the school day and its relationship to morningness–eveningness

We carried out a field study in adolescents in a German school to assess their actual affective state during the first and last lesson of a school day. Also, we wanted to assess if chronotype or sleep duration has an influence on affect. One hundred and nine girls and 110 boys participated in the study (mean age: 14.47). The composite scale of morningness (CSM) was used to assess morningness–eveningness and the positive and negative affect schedule (PANAS) was used for affect measurement. Sleep habits were assessed as both, habitual and actual sleep–wake rhythm. Positive affect (PA) was higher in the sixth lesson compared to the first one, but negative affect (NA) was not. CSM scores were positively correlated with PA during the first and last lesson. The correlation coefficient was higher in the first lesson of the day. NA was unrelated to CSM scores. Pupils with a shorter actual sleep duration had a higher NA. Habitual sleep duration was unrelated to affect measures. The results showed no synchrony effect; thus, evening types have still a lower PA in the last lesson than morning types. We emphasise the importance of mood and its relationship with morningness in school children, and we hypothesise that this may also have an influence on functioning during the school day.