Role of the A chain in thrombin function

The A chain of thrombin is covalently linked to the catalytic B chain but is separate from any known epitope for substrate recognition. In this study we present the results of the Ala replacement of 12 charged residues controlling the stability of the A chain and its interaction with the B chain. Residues Arg4 and Glu8 play a significant role in substrate recognition, even though they are located > 20 A away from residues of the catalytic triad, the primary specificity pocket and the Na+ site. The R4A mutation causes significant perturbation of Na+ binding, fibrinogen clotting and PAR1 cleavage, but modest reduction of protein C activation in the presence of thrombomodulin. These findings challenge our current paradigm of thrombin structure-function relations focused exclusively on the properties of the catalytic B chain, and explain why certain naturally occurring mutations of the A chain cause serious bleeding.     

Displacement activity of some natural cularine alkaloids at striatal3H-SCH 23 390 and3H-raclopride binding sites

Five natural cularines isolated from the aerial parts ofSarcocapnos crassifolia (Fumariaceae) and a cularioid isolated from the bark ofGuatteria ouregou (Annonaceae) were tested for their ability to displace³H-SCH 23 390 and³H-raclopride from their striatal binding sites. Celtisine, breoganine and cularidine were able to inhibit the binding at D-1 and D-2 dopaminergic sites at nanomolar concentrations. Other alkaloids were active at micromolar concentrations. These data suggest that the presence of an oxepine system in the isoquinoline skeleton could lead to compounds which would be very active and possibly selective at dopaminergic receptor sites.     

Strategies for the design of inhibitors of aldose reductase, an enzyme showing pronounced induced-fit adaptations

Aldose reductase is involved in the polyol pathway, catalyzing the reduction of glucose to sorbitol. However, due to pronounced binding site adaptations, the enzyme can operate on a broad palette of structurally diverse substrates ranging from small aliphatic and aromatic aldehydes up to steroid-type ligands. A comparative analysis of the presently accessible crystal structures of aldose reductase complexes reveals four binding-competent protein conformations. Additional relevant conformers are detected through molecular dynamics simulations. They indicate an equilibrium of several conformers which is shifted towards the binding-competent geometries upon ligand binding. Such a manifold system with several alternative binding site conformers requires some tailored concepts in virtual screening. We followed two strategies, both successfully suggesting new micromolar inhibitors. In a first attempt, we concentrated on one preferred conformer and performed a virtual screening, assuming that the binding pocket of aldose reductase adopts only this conformation. In a second approach, we followed a ligand superpositioning method. Ligands were extracted in their bound conformations from three different crystal structures, all accommodating the ligands with different active site conformations. After merging these ligands into one supermolecule, mutual alignments were computed, taking candidate ligands from a screening database. The latter strategy also retrieved several structurally new inhibitors of micromolar potency.     

What’s new in the renin-angiotensin system?

Angiotensin-converting enzyme (ACE) is a zinc- and chloride-dependent metallopeptidase that plays a vital role in the metabolism of biologically active peptides. Until recently, much of the inhibitor design and mechanism of action of this ubiquitous enzyme was based on the structures of carboxypeptidase A and thermolysin. When compared to the recently solved structures of the testis isoform of ACE (tACE) and its Drosophila homologue (AnCE), carboxypeptidase A showed little structural homology outside of the active site, while thermolysin revealed significant but less marked overall similarity. The ellipsoid-shaped structure of tACE, which has a preponderance of -helices, is characterised by a core channel that has a constriction approximately 10 Å from its opening where the zinc-binding active site is located. Comparison of the native protein with the inhibitor-bound form (lisinopril-tACE) does not reveal any striking differences in the conformation of the inhibitor binding site, disfavouring an open and closed configuration. However, the inhibitor complex does provide insights into the network of hydrogen-bonding and ionic interactions in the active site as well as the mechanism of ACE substrate hydrolysis. The three-dimensional structure of ACE now paves the way for the rational design of a new generation of domain-selective ACE inhibitors.     

High affinity of quinidine for a stereoselective microsomal binding site as determined by a radioreceptor assay

Summary The techniques of the radioreceptor binding assay were applied to detect stereoselective binding of quinidine and quinine to a site on human liver microsomes. Binding of³H-dihydroquinidine was 50% inhibited by 20–100 nM quinidine, while its enantiomer quinine did not displace the³H-ligand at concentrations up to 500 nM. This stereoselectivity agreed with the affinity values measured by functional enzyme assays of cytochrome P450 activity using sparteine or debrisoquine as substrates.Acknowledgments. We thank C. Ulpian for advice and assistance. We also thank Dr M. Robinette of Metro Organ Retrieval and Exchange and Dr T. Inaba for making human hepatic tissue available. This work was supported by grants from the Medical Research Council of Canada.     

The crystal structure of the AgamOBP1•Icaridin complex reveals alternative binding modes and stereo-selective repellent recognition

Anopheles gambiae Odorant Binding Protein 1 in complex with the most widely used insect repellent DEET, was the first reported crystal structure of an olfactory macromolecule with a repellent, and paved the way for OBP1-structure-based approaches for discovery of new host-seeking disruptors. In this work, we performed STD-NMR experiments to directly monitor and verify the formation of a complex between AgamOBP1 and Icaridin, an efficient DEET alternative. Furthermore, Isothermal Titration Calorimetry experiments provided evidence for two Icaridin-binding sites with different affinities (Kd = 0.034 and 0.714 mM) and thermodynamic profiles of ligand binding. To elucidate the binding mode of Icaridin, the crystal structure of AgamOBP1?Icaridin complex was determined at 1.75 Å resolution. We found that Icaridin binds to the DEET-binding site in two distinct orientations and also to a novel binding site located at the C-terminal region. Importantly, only the most active 1R,2S-isomer of Icaridin’s equimolar diastereoisomeric mixture binds to the AgamOBP1 crystal, providing structural evidence for the possible contribution of OBP1 to the stereoselectivity of Icaridin perception in mosquitoes. Structural analysis revealed two ensembles of conformations differing mainly in spatial arrangement of their sec-butyl moieties. Moreover, structural comparison with DEET indicates a common recognition mechanism for these structurally related repellents. Ligand interactions with both sites and binding modes were further confirmed by 2D ¹H-¹⁵N HSQC NMR spectroscopy. The identification of a novel repellent-binding site in AgamOBP1 and the observed structural conservation and stereoselectivity of its DEET/Icaridin-binding sites open new perspectives for the OBP1-structure-based discovery of next-generation insect repellents.     

Purification from human plasma of endogenous dodium transport inhibitor(s)

Summary Na⁺, K⁺-ATPase inhibitors extracted from plasma of healthy human subjects displaced³H-ouabain binding to human erythrocytes and inhibited the Na⁺ efflux catalyzed by the Na⁺, K⁺-pump and unexpectedly the Na⁺, K⁺-cotransport system without alteration of the Na⁺, Na⁺-exchange or the Na⁺ passive permeability. This suggests the presence in healthy human plasma of endogenous factors with ouabain-like and furosemide-like activities.Acknowledgments. We are indebted to Dr M. A. Devynck for her advice on chemical measurements and to Dr R. P. Garay for his help with flux measurements     

Structural determinants of a conserved enantiomer-selective carvone binding pocket in the human odorant receptor OR1A1

Chirality is a common phenomenon within odorants. Most pairs of enantiomers show only moderate differences in odor quality. One example for enantiomers that are easily discriminated by their odor quality is the carvones: humans significantly distinguish between the spearmint-like (R)-(?)-carvone and caraway-like (S)-(+)-carvone enantiomers. Moreover, for the (R)-(?)-carvone, an anosmia is observed in about 8% of the population, suggesting enantioselective odorant receptors (ORs). With only about 15% de-orphaned human ORs, the lack of OR crystal structures, and few comprehensive studies combining in silico and experimental approaches to elucidate structure–function relations of ORs, knowledge on cognate odorant/OR interactions is still sparse. An adjusted homology modeling approach considering OR-specific proline-caused conformations, odorant docking studies, single-nucleotide polymorphism (SNP) analysis, site-directed mutagenesis, and subsequent functional studies with recombinant ORs in a cell-based, real-time luminescence assay revealed 11 amino acid positions to constitute an enantioselective binding pocket necessary for a carvone function in human OR1A1 and murine Olfr43, respectively. Here, we identified enantioselective molecular determinants in both ORs that discriminate between minty and caraway odor. Comparison with orthologs from 36 mammalian species demonstrated a hominid-specific carvone binding pocket with about 100% conservation. Moreover, we identified loss-of-function SNPs associated with the carvone binding pocket of OR1A1. Given carvone enantiomer-specific receptor activation patterns including OR1A1, our data suggest OR1A1 as a candidate receptor for constituting a carvone enantioselective phenotype, which may help to explain mechanisms underlying a (R)-(?)-carvone-specific anosmia in humans.     

Lipid characterization and14C-acetate metabolism in catfish taste epithelium

Summary The catfish,Ictalurus punctatus is an important model system for the study of the biochemical mechanisms of taste reception. A detailed lipid analysis of epithelial tissue from the taste organ (barbel) of the catfish has been performed. Polar lipids account for 62±1% of the total, neutrals for 38±1%. Phosphatidyl-cholines, serines and ethanolamines are the major constitutents of the polar fraction. Plasmalogen concentration is high relative to that of non-neural tissues. [¹⁴C]-Acetate is incorporated into cell lipid fractions after incubation of barbel tissue at 37°C for 60 min. Percentage amounts of most lipids change with time during this in vitro incubation. The phospholipids are the most metabolically active fractions. This work yields information for continuing reconstitution experiments and indicates that the taste epithelium of this important model system is a metabolically active tissue capable of supporting lipid turnover/synthesis.This work was supported in part by NIH Research Grant No. NS-23622, NS-22620, and by the Veterans Administration.     

Ca2+/Calmodulin-dependent Protein Kinases

In this article the calcium/calmodulin-dependent protein kinases are reviewed. The primary focus is on the structure and function of this diverse family of enzymes, and the elegant regulation of their activity. Structures are compared in order to highlight the conserved architecture of their catalytic domains with respect to each other as well as protein kinase A, a prototype for kinase structure. In addition to reviewing structure and function in these enzymes, the variety of biological processes for which they play a mediating role are also examined. Finally, how the enzymes become activated in the intracellular setting is considered by exploring the reciprocal interactions that exist between calcium binding to calmodulin when interacting with the CaM-kinases.     

Tauroursodeoxycholic acid prevents E22Q Alzheimer’s Aβ toxicity in human cerebral endothelial cells

The vasculotropic E22Q mutant of the amyloid-β (Aβ) peptide is associated with hereditary cerebral hemorrhage with amyloidosis Dutch type. The cellular mechanism(s) of toxicity and nature of the AβE22Q toxic assemblies are not completely understood. Comparative assessment of structural parameters and cell death mechanisms elicited in primary human cerebral endothelial cells by AβE22Q and wild-type Aβ revealed that only AβE22Q triggered the Bax mitochondrial pathway of apoptosis. AβE22Q neither matched the fast oligomerization kinetics of Aβ42 nor reached its predominant β-sheet structure, achieving a modest degree of oligomerization with a secondary structure that remained a mixture of β and random conformations. The endogenous molecule tauroursodeoxycholic acid (TUDCA) was a strong modulator of AβE22Q-triggered apoptosis but did not significantly change the secondary structures and fibrillogenic propensities of Aβ peptides. These data dissociate the pro-apoptotic properties of Aβ peptides from their distinct mechanisms of aggregation/fibrillization in vitro, providing new perspectives for modulation of amyloid toxicity. Received 20 November 2008; received after revision 12 December 2008; accepted 12 January 2009     

On the binding of steroid sulfates to albumin

3H-Labeled steroid sulfates, sulfate of estrone (E1S) or dehydroepiandrosterone (DHAS), were dialyzed against delipidated human serum albumin or human plasma in the presence of increasing amounts of competing non-labeled sulfates (DHAS or E1S). The apparent equilibrium constants (K) of the tracers did not measurably change at concentrations of the non-radioactive sulfates below 10(-5) mol/l. At higher concentrations, K decreased gradually. The apparent equilibrium constant of 3H-E1S was diminished by plasma in a similar fashion. It may be concluded that albumin possesses one strong, non-specific binding site. This site, however, does not seem to be utilized for the binding of E1S in vivo, because of its preferential occupation by other ligands. This may be true for other steroid sulfates as well, depending on their relative abundance in plasma.     

Structural and functional features of dimeric dihydrodiol dehydrogenase

Dimeric dihydrodiol dehydrogenase (DD) catalyzes the NADP(+)-dependent oxidation of trans-dihydrodiols of aromatic hydrocarbons to their corresponding catechols. The tertiary structure of dimeric DD consists of a classical dinucleotide binding domain comprising two betaalphabetaalphabeta motifs at the N-terminus, and an eight-stranded, predominantly anti-parallel beta-sheet, forming the C-terminal domain The aim of this review is to summarize the biochemical and structural properties of dimeric DD, compare it to enzymes that are structurally similar, and provide an insight into its catalytic mechanism and membership amongst a unique family of monomeric/oligomeric proteins that most likely share a common ancestry.     

Decreased monosaccharide transport in renal brush-border membrane vesicles of spontaneously hypertensive rats

Na(+)-dependent D-glucose and D-galactose transport were studied in brush-border membrane vesicles (BBMVs) from kidney cortex isolated from both spontaneously hypertensive rats (SHR) and their normotensive genetic control Wistar-Kyoto (WKY) rats. Initial rates and accumulation ratios of Na(+)-dependent D-glucose and D-galactose transport were significantly lower in SHR compared with WKY, the observed decreases being similar for both substrates. To explain the reduction in sugar transport by renal BBMVs, the density of Na(+)-dependent sugar cotransporters was studied in BBMVs from kidney cortex isolated from SHR and WKY rats. Phlorizin-specific binding and Western blot analysis indicated a reduction in the density of the cotransporters in SHR relative to WKY rats. This reduction was similar to those found for the initial rates and accumulation ratios for D-glucose and D-galactose in SHR. Na+ uptake, studied using 22Na+, was significantly increased in SHR, so the observed reduction in sugar transport could be due to disruption of the Na+ gradient between renal BBMVs in SHR. Furthermore, a significant decrease in the activity of Na(+)-K(+)-ATPase was observed in SHR. In conclusion, changes in the density of the Na(+)-dependent sugar cotransporter and in the Na+ gradient across the brush-border membranes might be involved in the observed reduction in sugar transport by renal BBMVs from SHR.     

Molecular mechanisms of thrombin function

The discovery of thrombin as a Na⁺-dependent allosteric enzyme has revealed a novel strategy for regulating protease activity and specificity. The allosteric nature of this enzyme influences all its physiologically important interactions and rationalizes a large body of structural and functional information. For the first time, a coherent mechanistic framework is available for understanding how thrombin interacts with fibrinogen, thrombomodulin and protein C, and how Na⁺ binding influences the specificity sites of the enzyme. This information can be used for engineering thrombin mutants with selective specificity towards protein C and for the rational design of potent active site inhibitors. Thrombin also serves as a paradigm for allosteric proteases. Elucidation of the molecular basis of the Na⁺-dependent allosteric regulation of catalytic activity, based on the residue present at position 225, provides unprecedented insights into the function and evolution of serine proteases. This mechanism represents one of the simplest and most important structure-function correlations ever reported for enzymes in general. All vitamin K-dependent proteases and some complement factors are subject to the Na⁺-dependent regulation discovered for thrombin. Na⁺ is therefore a key factor in the activation of zymogens in the coagulation and complement systems.     

The E-site story: the importance of maintaining two tRNAs on the ribosome during protein synthesis

In the sixties James Watson suggested a twosite model for the ribosome comprising the P site for the peptidyl transfer RNA (tRNA) before peptide-bond formation and the A site, where decoding takes place according to the codon exposed there. In the eighties a third tRNA binding site was detected, the E site, which was specific for deacylated tRNA and turned out to be a universal feature of ribosomes. However, despite having three tRNA binding sites, only two tRNAs occupy the ribosome at a time during protein synthesis: at the A and P sites before translocation (PRE state) and at the P and E sites after translocation (POST state). The importance of having two tRNAs in the POST state has been revealed during the last 25 years, showing that the E site contributes two fundamental features: (i) the fact that incorporation of a wrong amino acid is not harmful for the cell (only 1 in about 400 misincorporations destroys the function of a protein) stems from the presence of an E-tRNA; (ii) maintenance of the reading frame is one of the most remarkable achievements of the ribosome, essential for faithful translation of the genetic information. The presence of the POST state E-tRNA prevents loss of the reading frame. Received 14 March 2006; received after revision 8 June 2006; accepted 4 August 2006     

Structure/function analysis of a critical disulfide bond in the active site of l-xylulose reductase

l-Xylulose reductase (XR) is involved in water re-absorption and cellular osmoregulation. The crystal structure of human XR complemented with site-directed mutagenesis (Cys138Ala) indicated that the disulfide bond in the active site between Cys138 and Cys150 is unstable and may affect the reactivity of the enzyme. The effects of reducing agents on the activities of the wild-type and mutant enzymes indicated the reversibility of disulfide-bond formation, which resulted in three-fold decrease in catalytic efficiency. Furthermore, the addition of cysteine (>2 mM) inactivated human XR and was accompanied by a 10-fold decrease in catalytic efficiency. TOF-MS analysis of the inactivated enzyme showed the S-cysteinylation of Cys138 in the wild-type and Cys150 in the mutant enzymes. Thus, the action of human XR may be regulated by cellular redox conditions through reversible disulfide-bond formation and by S-cysteinylation. Received 25 January 2009; received after revision 12 February 2009; accepted 16 February 2009 H.-T. Zhao, S. Endo: These two authors contribute equally to this work.