Tetramerization and Cooperativity in Plasmodium falciparum Glutathione S-Transferase Are Mediated by Atypic Loop 113�C119

Glutathione S-transferase of Plasmodium falciparum (PfGST) displays a peculiar dimer to tetramer transition that causes full enzyme inactivation and loss of its ability to sequester parasitotoxic hemin. Furthermore, binding of hemin is modulated by a cooperative mechanism. Site-directed mutagenesis, steady-state kinetic experiments, and fluorescence anisotropy have been used to verify the possible involvement of loop 113–119 in the tetramerization process and in the cooperative phenomenon. This protein segment is one of the most prominent structural differences between PfGST and other GST isoenzymes. Our results demonstrate that truncation, increased rigidity, or even a simple point mutation of this loop causes a dramatic change in the tetramerization kinetics that becomes at least 100 times slower than in the native enzyme. All of the mutants tested have lost the positive cooperativity for hemin binding, suggesting that the integrity of this peculiar loop is essential for intersubunit communication. Interestingly, the tetramerization process of the native enzyme that occurs rapidly when GSH is removed is prevented not only by GSH but even by oxidized glutathione. This result suggests that protection by PfGST against hemin is independent of the redox status of the parasite cell. Because of the importance of this unique segment in the function/structure of PfGST, it could be a new target for the development of antimalarial drugs.Approximately two million deaths in the world per year are caused by Plasmodium falciparum, the parasite responsible for tropical malaria (1, 2). In the last years, increasing interest has been developing for the peculiar glutathione S-transferase (PfGST)³ expressed by this parasite. Expressed in almost all living organisms, GSTs represent a large superfamily of multifunctional detoxifying enzymes that are able to conjugate GSH to a lot of toxic electrophilic compounds, thus facilitating their excretion. Many other protection roles of GSTs have been described, including the enzymatic reduction of organic peroxides (3–5), the inactivation of the proapoptotic JNK through a GST·JNK complex (6), and the protection of the cell from excess nitric oxide (7). The mammalian cytosolic GSTs are dimeric proteins grouped into eight species-independent classes termed Alpha, Kappa, Mu, Omega, Pi, Sigma, Theta, and Zeta on the basis of sequence similarity, immunological reactivity, and substrate specificity (3, 8–11). PfGST is one of the most abundant proteins expressed by P. falciparum (from 1 to 10%, i.e. from 0.1 to 1 mm) (12), and different from what occurs in many organisms, it is the sole GST isoenzyme expressed by this parasite. Despite its structural similarity to the Mu class GST, this specific isoenzyme cannot be assigned to any known GST class (13). The interest in this enzyme is due to its particular protective role in the parasite. In fact, in addition to the usual GST activity that promotes the conjugation of GSH to electrophilic centers of toxic compounds, this protein efficiently binds hemin, and thus it could protect the parasite (that resides in the erythrocytes) from the parasitotoxic effect of this heme by-product (14). Specific compounds that selectively inhibit its catalytic activity or hemin binding could be promising candidates as antimalarial drugs. In this context, the discovery of structural or mechanistic properties of this enzyme that are not found in other GSTs may be important for designing selective inhibitors that are toxic to the parasite but harmless for the host cells. Two properties never observed in other members of the GST superfamily are of particular interest. The first property is that this enzyme, in the absence of GSH, is inactivated in a short time and loses its ability to bind hemin (15). Recent studies indicated that the inactivation process is related to a dimer to tetramer transition (13, 16, 17). The second property is the strong positive homotropic phenomenon that modulates the affinity of the two subunits for hemin (15). The x-ray crystal structure of PfGST, solved by two different groups (13, 18), provides insights into this effect. From a structural point of view, the most intriguing differences of PfGST when compared with other GSTs are a more solvent-exposed H-site and an atypic extra loop connecting helix α-4 and helix α-5 (residues 113–119; see also Fig. 1) that could be involved in the dimer-dimer interaction. Actually, in the absence of ligands, two biological dimers form a tetramer, and these homodimers are interlocked with each other by loop 113–119 of one homodimer, which occupies an H-site of the other homodimer (13, 18). Upon binding of S-hexylglutathione, loop 113–119 rearranges; residues Asn-114, Leu-115, and Phe-116 form an additional coil in helix α-4; and the side chains of Asn-111, Phe-116, and Tyr-211 flip into the H-site of the same dimer (17, 18). The changed course of residues 113–119 in the liganded enzyme prevents the interlocking of the dimers.Open in a separate windowFIGURE 1.A, structural changes of loop 113–119 occurring in the dimer (light blue model and yellow loop; Protein Data Bank code 2AAW) to tetramer (blue model and orange loop; Protein Data Bank code 1OKT) transition. Red spheres indicate the amino acids replaced in this study to obtain mutants A, B, and C. B, model of hemin·PfGST complex obtained by docking simulation using the crystal structure for Protein Data Bank code 1Q4J (15). Hemin is shown in red, loop 113–119 is in orange, and GSH is shown as yellow sticks.In this paper, by means of site-directed mutagenesis, fluorescence anisotropy, kinetic studies, and size exclusion chromatography, we check the influence of selected mutations of this atypic loop in the tetramerization process and the possible involvement of this protein segment in the cooperative phenomenon that characterizes hemin binding. In addition we describe that the tetramerization process is inhibited not only by GSH but even by GSSG. This finding suggests that hemin binding of PfGST is independent of the redox status of the cell. Finally, we demonstrate that the presence of GSH (or GSSG) in the active site is not essential for hemin binding, but this interaction only requires an active dimeric conformation.     

Interaction of Mycobacterium tuberculosis CYP130 with Heterocyclic Arylamines

The Mycobacterium tuberculosis P450 enzymes are of interest for their pharmacological development potential, as evidenced by their susceptibility to inhibition by antifungal azole drugs that normally target sterol 14α-demethylase (CYP51). Although antifungal azoles show promise, direct screening of compounds against M. tuberculosis P450 enzymes may identify novel, more potent, and selective inhibitory scaffolds. Here we report that CYP130 from M. tuberculosis has a natural propensity to bind primary arylamines with particular chemical architectures. These compounds were identified via a high throughput screen of CYP130 with a library of synthetic organic molecules. As revealed by subsequent x-ray structure analysis, selected compounds bind in the active site by Fe-coordination and hydrogen bonding of the arylamine group to the carbonyl oxygen of Gly²⁴³. As evidenced by the binding of structural analogs, the primary arylamine group is indispensable, but synergism due to hydrophobic contacts between the rest of the molecule and protein amino acid residues is responsible for a binding affinity comparable with that of the antifungal azole drugs. The topology of the CYP130 active site favors angular coordination of the arylamine group over the orthogonal coordination of azoles. Upon substitution of Gly²⁴³ by an alanine, the binding mode of azoles and some arylamines reverted from type II to type I because of hydrophobic and steric interactions with the alanine side chain. We suggest a role for the conserved Ala(Gly)²⁴³-Gly²⁴⁴ motif in the I-helix in modulating both the binding affinity of the axial water ligand and the ligand selectivity of cytochrome P450 enzymes.CYP130 is one of the 20 Mycobacterium tuberculosis cytochrome P450 (P450, CYP)² enzymes and is one of three (CYP51, CYP121, and CYP130) that have been studied as individually expressed proteins at the structural level. Evidence has accumulated for the importance of M. tuberculosis P450 enzymes in virulence (CYP132) (1), host infection (CYP125) (2), and pathogen viability (CYP128, CYP121) (3, 4), although neither their exact biological functions nor any of the endogenous substrates upon which these enzymes operate have yet been established. However, it has recently been shown in vitro that CYP121 catalyzes a C–C coupling reaction between two tyrosine groups (5). CYP130 is absent from the genome of Mycobacterium bovis, suggesting that it might play specific role(s) in the infection of the human host and thus constitute a potential therapeutic target.The potential of M. tuberculosis P450 enzymes for pharmacologic development was initially suggested by their susceptibility to inhibition by antifungal azole drugs such as fluconazole, econazole, and clotrimazole. These drugs block sterol 14α-demethylase CYP51 in fungi (6), tightly bind to M. tuberculosis P450 proteins (7, 8), and display inhibitory potential against latent and multidrug-resistant forms of tuberculosis both in vitro and in tuberculosis-infected mice (9–14).The substantial differences between fungal CYP51 and the potential P450 targets in microbial pathogens, including M. tuberculosis, suggest that the direct screening of compounds against M. tuberculosis CYP enzymes could identify novel inhibitory scaffolds that are more potent and selective than antifungal drugs. Structurally characterized screening targets are advantageous, as the already defined purification and crystallization protocols can be applied to obtain co-crystal structures and to elucidate the binding modes of screening hits. This approach has been successfully applied to CYP51, resulting in identification of novel inhibitory scaffolds for CYP51 therapeutic targets (15, 16).Toward this goal, the property of P450 enzymes to shift the ferric heme iron Soret band on ligand binding (17) provides an experimental platform for high throughput screening of compound libraries to select chemotypes with high binding affinities for the target. Expulsion of the heme iron axial water ligand from the Fe-coordination sphere by the incoming substrate followed by transition of the ferric heme from the low-spin hexacoordinated to the high-spin pentacoordinated state characterize type I spectral shifts and are a prerequisite for P450 catalytic activity. Replacement of a weak axial ligand, the water molecule, with a stronger one possessing a nitrogen-containing aliphatic or aromatic group coordinating to the heme iron characterizes type II spectral shifts.To find new high affinity ligands of CYP130, a commercial library of 20,000 small organic molecules comprising a large selection of molecular scaffolds was screened against the enzyme. In contrast to the results with CYP51, no type I binding hits were identified. Screening produced about a dozen structurally diverse type II hits that were unexpectedly devoid of the usual aromatic nitrogen atoms readily accessible for axial coordination of the heme iron, suggesting an alternative coordination mode. High resolution x-ray structure analysis determined that two compounds coordinated to the heme iron via a primary arylamine group, providing the first structural evidence on P450-heterocyclic arylamine interactions.     

MotifAdjuster: a tool for computational reassessment of transcription factor binding site annotations

Valuable binding-site annotation data are stored in databases. However, several types of errors can, and do, occur in the process of manually incorporating annotation data from the scientific literature into these databases. Here, we introduce MotifAdjuster , a tool that helps to detect these errors, and we demonstrate its efficacy on public data sets.     

Detection of Chlamydia trachomatis-DNA in synovial fluid: evaluation of the sensitivity of different DNA extraction methods and amplification systems

Introduction  

Polymerase chain reaction (PCR) and ligase chain reaction (LCR) are used in research for detection of Chlamydia trachomatis (C. tr.) in synovial fluid (SF). However there is no standardized system for diagnostic use in clinical practice, therefore this study aimed at determining the molecular biology method best suited to detect C. tr. from SF.     

In Vivo Imaging of Far-red Fluorescent Proteins after DNA Electrotransfer to Muscle Tissue

DNA electrotransfer to muscle tissue yields long-term, high levels of gene expression; showing great promise for future gene therapy. We want to characterize the novel far-red fluorescent protein Katushka as a marker for gene expression using time domain fluorescence in vivo imaging. Highly efficient transgenic expression was observed after DNA electrotransfer with 100-fold increase in fluorescent intensity. The fluorescent signal peaked 1 week after transfection and returned to background level within 4 weeks. Katushka expression was not as stable as GFP expression, which was detectable for 8 weeks. Depth and 3D analysis proved that the expression was located in the target muscle. In vivo bio-imaging using the novel Katushka fluorescent protein enables excellent evaluation of the transfection efficacy, and spatial distribution, but lacks long-term stability.     

Modeling system states in liver cells: Survival,apoptosis and their modifications in response to viral infection

Background  

The decision pro- or contra apoptosis is complex, involves a number of different inputs, and is central for the homeostasis of an individual cell as well as for the maintenance and regeneration of the complete organism.