The denatured state of HIV-1 protease under native conditions.

The denatured state of several proteins has been shown to display transient structures that are relevant for folding, stability, and aggregation. To detect them by nuclear magnetic resonance (NMR) spectroscopy, the denatured state must be stabilized by chemical agents or changes in temperature. This makes the environment different from that experienced in biologically relevant processes. Using high-resolution heteronuclear NMR spectroscopy, we have characterized several denatured states of a monomeric variant of HIV-1 protease, which is natively structured in water, induced by different concentrations of urea, guanidinium chloride, and acetic acid. We have extrapolated the chemical shifts and the relaxation parameters to the denaturant-free denatured state at native conditions, showing that they converge to the same values. Subsequently, we characterized the conformational properties of this biologically relevant denatured state under native conditions by advanced molecular dynamics simulations and validated the results by comparison to experimental data. We show that the denatured state of HIV-1 protease under native conditions displays rich patterns of transient native and non-native structures, which could be of relevance to its guidance through a complex folding process.

Covalent inhibition of P. falciparum ferredoxin-NADP+ reductase: Exploring alternative strategies for the development of new antimalarial drugs.

The protozoan Plasmodium falciparum is the main aetiological agent of tropical malaria. Characteristic of the phylum is the presence of a plastid-like organelle which hosts several homologs of plant proteins, including a ferredoxin (PfFd) and its NADPH-dependent reductase (PfFNR). The PfFNR/PfFd redox system is essential for the parasite, while mammals share no homologous proteins, making the enzyme an attractive target for novel and much needed antimalarial drugs. Based on previous findings, three chemically reactive residues important for PfFNR activity were identified: namely, the active-site Cys99, responsible for hydride transfer; Cys284, whose oxidation leads to an inactive dimeric form of the protein; and His286, which is involved in NADPH binding. These amino acid residues were probed by several residue-specific reagents and the two cysteines were shown to be promising targets for covalent inhibition. The quantitative and qualitative description of the reactivity of few compounds, including a repurposed drug, set the bases for the development of more potent and specific antimalarial leads.

Reactions of Plasmodium falciparum Ferredoxin:NADP+ Oxidoreductase with Redox Cycling Xenobiotics: A Mechanistic Study.

Ferredoxin:NADP+ oxidoreductase from Plasmodium falciparum (PfFNR) catalyzes the NADPH-dependent reduction of ferredoxin (PfFd), which provides redox equivalents for the biosynthesis of isoprenoids and fatty acids in the apicoplast. Like other flavin-dependent electrontransferases, PfFNR is a potential source of free radicals of quinones and other redox cycling compounds. We report here a kinetic study of the reduction of quinones, nitroaromatic compounds and aromatic N-oxides by PfFNR. We show that all these groups of compounds are reduced in a single-electron pathway, their reactivity increasing with the increase in their single-electron reduction midpoint potential (E17). The reactivity of nitroaromatics is lower than that of quinones and aromatic N-oxides, which is in line with the differences in their electron self-exchange rate constants. Quinone reduction proceeds via a ping-pong mechanism. During the reoxidation of reduced FAD by quinones, the oxidation of FADH. to FAD is the possible rate-limiting step. The calculated electron transfer distances in the reaction of PfFNR with various electron acceptors are similar to those of Anabaena FNR, thus demonstrating their similar "intrinsic" reactivity. Ferredoxin stimulated quinone- and nitro-reductase reactions of PfFNR, evidently providing an additional reduction pathway via reduced PfFd. Based on the available data, PfFNR and possibly PfFd may play a central role in the reductive activation of quinones, nitroaromatics and aromatic N-oxides in P. falciparum, contributing to their antiplasmodial action.

Antiplasmodial Activity of Nitroaromatic Compounds: Correlation with Their Reduction Potential and Inhibitory Action on Plasmodium falciparum Glutathione Reductase.

With the aim to clarify the mechanism(s) of action of nitroaromatic compounds against the malaria parasite Plasmodium falciparum, we examined the single-electron reduction by P. falciparum ferredoxin:NADP+ oxidoreductase (PfFNR) of a series of nitrofurans and nitrobenzenes (n = 23), and their ability to inhibit P. falciparum glutathione reductase (PfGR). The reactivity of nitroaromatics in PfFNR-catalyzed reactions increased with their single-electron reduction midpoint potential (E17). Nitroaromatic compounds acted as non- or uncompetitive inhibitors towards PfGR with respect to NADPH and glutathione substrates. Using multiparameter regression analysis, we found that the in vitro activity of these compounds against P. falciparum strain FcB1 increased with their E17 values, octanol/water distribution coefficients at pH 7.0 (log D), and their activity as PfGR inhibitors. Our data demonstrate that both factors, the ease of reductive activation and the inhibition of PfGR, are important in the antiplasmodial in vitro activity of nitroaromatics. To the best of our knowledge, this is the first quantitative demonstration of this kind of relationship. No correlation between antiplasmodial activity and ability to inhibit human erythrocyte GR was detected in tested nitroaromatics. Our data suggest that the efficacy of prooxidant antiparasitic agents may be achieved through their combined action, namely inhibition of antioxidant NADPH:disulfide reductases, and the rapid reduction by single-electron transferring dehydrogenases-electrontransferases.

Cold Denaturation of the HIV-1 Protease Monomer.

The human immunodeficiency virus-1 (HIV-1) protease is a complex protein that in its active form adopts a homodimer dominated by ß-sheet structures. We have discovered a cold-denatured state of the monomeric subunit of HIV-1 protease that is populated above 0 °C and therefore directly accessible to various spectroscopic approaches. Using nuclear magnetic resonance secondary chemical shifts, temperature coefficients, and protein dynamics, we suggest that the cold-denatured state populates a compact wet globule containing transient non-native-like α-helical elements. From the linearity of the temperature coefficients and the hydrodynamic radii, we propose that the overall architecture of the cold-denatured state is maintained over the temperature range studied.

Cellulose production is coupled to sensing of the pyrimidine biosynthetic pathway via c-di-GMP production by the DgcQ protein of Escherichia coli.

Production of cellulose, a stress response-mediated process in enterobacteria, is modulated in Escherichia coli by the activity of the two pyrimidine nucleotide biosynthetic pathways, namely, the de novo biosynthetic pathway and the salvage pathway, which relies on the environmental availability of pyrimidine nitrogenous bases. We had previously reported that prevalence of the salvage over the de novo pathway triggers cellulose production via synthesis of the second messenger c-di-GMP by the DgcQ (YedQ) diguanylate cyclase. In this work, we show that DgcQ enzymatic activity is enhanced by UTP, whilst being inhibited by N-carbamoyl-aspartate, an intermediate of the de novo pathway. Thus, direct allosteric control by these ligands allows full DgcQ activity exclusively in cells actively synthesizing pyrimidine nucleotides via the salvage pathway. Inhibition of DgcQ activity by N-carbamoyl-aspartate appears to be favoured by protein-protein interaction between DgcQ and PyrB, a subunit of aspartate transcarbamylase, which synthesizes N-carbamoyl-aspartate. Our results suggest that availability of pyrimidine bases might be sensed, somehow paradoxically, as an environmental stress by E. coli. We hypothesize that this link might have evolved since stress events, leading to extensive DNA/RNA degradation or lysis of neighbouring cells, can result in increased pyrimidine concentrations and activation of the salvage pathway.

Structural bases of the altered catalytic properties of a pathogenic variant of apoptosis inducing factor.

The apoptosis-inducing factor (AIF) is a FAD-containing protein playing critical roles in caspase-independent apoptosis and mitochondrial respiratory chain biogenesis and maintenance. While its lethal role is well known, the details of its mitochondrial function remain elusive. So far, nineteen allelic variants of AIF have been associated to human diseases, mainly affecting the nervous system. A strict correlation is emerging between the degree of impairment of its ability to stabilize the charge-transfer (CT) complex between FAD and NAD+ and the severity of the resulting pathology. Recently, we demonstrated that the G307E replacement in murine AIF (equivalent to the pathogenic G308E in the human protein) dramatically decreases the rate of CT complex formation through the destabilization of the flavoprotein interaction with NAD(H). To provide further insights into the structural bases of its altered functional properties, here we report the first crystal structure of an AIF pathogenic mutant variant in complex with NAD+ (murine AIF-G307ECT) in comparison with its oxidized form. With respect to wild type AIF, the mutation leads to an altered positioning of NAD+ adenylate moiety, which slows down CT complex formation. Moreover, the altered balance between the binding of the adenine/nicotinamide portions of the coenzyme determines a large drop in AIF-G307E ability to discriminate between NADH and NADPH.

Recombinant human dihydroxyacetonephosphate acyl-transferase characterization as an integral monotopic membrane protein.

Although the precise functions of ether phospholipids are still poorly understood, significant alterations in their physiological levels are associated either to inherited disorders or to aggressive metastatic cancer. The essential precursor, alkyl-dihydroxyacetone phosphate (DHAP), for all ether phospholipids species is synthetized in two consecutive reactions performed by two enzymes sitting on the inner side of the peroxisomal membrane. Here, we report the characterization of the recombinant human DHAP acyl-transferase, which performs the first step in alkyl-DHAP synthesis. By exploring several expression systems and designing a number of constructs, we were able to purify the enzyme in its active form and we found that it is tightly bound to the membrane through the N-terminal residues.

Tools for the rational design of bivalent microtubule-targeting drugs.

Microtubule (MT) dynamic behaviour is an attractive drug target for chemotherapy, whose regulation by MT-stabilizing and destabilizing agents has been fruitfully applied in treating several types of cancers. MT-stabilizing agents are also emerging as potential remedies for neurodegenerative conditions, such as Alzheimer's and Parkinson's disease, although single-target drugs are not expected to fully cure these complex pathologies. Drug combination often displays enhanced efficacy with respect to mono-therapies. In particular, MT-targeting bivalent compounds (MTBCs) represent a promising class of molecules; however, surprisingly, the majority of MTBCs reported so far exhibit equal if not less efficacy than their building monomers. In order to shed light on MTBCs poor performance, we characterised through a set of complementary approaches thiocolchine (TH) and two bivalent TH-homodimers as prototype molecules. First, the binding affinities of these three molecules were assessed, then we obtained the crystallographic structure of a tubulin-TH complex. The binding affinities were interpreted in light of structural data and of molecular dynamics simulations. Finally, their effects on MT cytoskeleton and cell survival were validated on HeLa cells. The ensemble of these data provides chemical and structural considerations on how a successful rational design of MTBCs should be conceived.

Key Role of the Adenylate Moiety and Integrity of the Adenylate-Binding Site for the NAD(+)/H Binding to Mitochondrial Apoptosis-Inducing Factor.

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein with pro-life and pro-death activities, which plays critical roles in mitochondrial energy metabolism and caspase-independent apoptosis. Defects in AIF structure or expression can cause mitochondrial abnormalities leading to mitochondrial defects and neurodegeneration. The mechanism of AIF-induced apoptosis was extensively investigated, whereas the mitochondrial function of AIF is poorly understood. A unique feature of AIF is the ability to form a tight, air-stable charge-transfer (CT) complex upon reaction with NADH and to undergo a conformational switch leading to dimerization, proposed to be important for its vital and lethal functions. Although some aspects of interaction of AIF with NAD(+)/H have been analyzed, its precise mechanism is not fully understood. We investigated how the oxidized and photoreduced wild-type and G307A and -E variants of murine AIF associate with NAD(+)/H and nicotinamide mononucleotide (NMN(+)/H) to determine the role of the adenylate moiety in the binding process. Our results indicate that (i) the adenylate moiety of NAD(+)/H is crucial for the association with AIF and for the subsequent structural reorganization of the complex, but not for protein dimerization, (ii) FAD reduction rather than binding of NAD(+)/H to AIF initiates conformational rearrangement, and (iii) alteration of the adenylate-binding site by the G307E (equivalent to a pathological G308E mutation in human AIF) or G307A replacements decrease the affinity and association rate of NAD(+)/H, which, in turn, perturbs CT complex formation and protein dimerization but has no influence on the conformational switch in the regulatory peptide.

An improved expression system for the VC1 ligand binding domain of the receptor for advanced glycation end products in Pichia pastoris.

The receptor for the advanced glycation end products (RAGE) is a type I transmembrane glycoprotein belonging to the immunoglobulin superfamily and binds a variety of unrelated ligands sharing a negative charge. Most ligands bind to the extracellular V or VC1 domains of the receptor. In this work, V and VC1 of human RAGE were produced in the methylotrophic yeast Pichia pastoris and directed to the secretory pathway. Fusions to a removable C-terminal His-tag evidenced proteolytic processing of the tag by extracellular proteases and also intracellular degradation of the N-terminal portion of V-His. Expression of untagged forms was attempted. While the V domain was retained intracellularly, VC1 was secreted into the medium and was functionally active in binding AGEs. The glycosylation state of VC1 was analyzed by mass spectrometry and peptide-N-glycosidase F digestion. Like RAGE isolated from mammalian sources, the degree of occupancy of the N-glycosylation sites was full at Asn25 and partial at Asn81 which was also subjected to non-enzymatic deamidation. A simple procedure for the purification to homogeneity of VC1 from the medium was developed. The folded state of the purified protein was assessed by thermal shift assays. Recombinant VC1 from P. pastoris showed a remarkably high thermal stability as compared to the protein expressed in bacteria. Our in vivo approach indicates that the V and C1 domains constitute a single folding unit. The stability and solubility of the yeast-secreted VC1 may be beneficial for future in vitro studies aimed to identify new ligands or inhibitors of RAGE.

Precursor of ether phospholipids is synthesized by a flavoenzyme through covalent catalysis.

The precursor of the essential ether phospholipids is synthesized by a peroxisomal enzyme that uses a flavin cofactor to catalyze a reaction that does not alter the redox state of the substrates. The enzyme crystal structure reveals a V-shaped active site with a narrow constriction in front of the prosthetic group. Mutations causing inborn ether phospholipid deficiency, a very severe genetic disease, target residues that are part of the catalytic center. Biochemical analysis using substrate and flavin analogs, absorbance spectroscopy, mutagenesis, and mass spectrometry provide compelling evidence supporting an unusual mechanism of covalent catalysis. The flavin functions as a chemical trap that promotes exchange of an acyl with an alkyl group, generating the characteristic ether bond. Structural comparisons show that the covalent versus noncovalent mechanistic distinction in flavoenzyme catalysis and evolution relies on subtle factors rather than on gross modifications of the cofactor environment.

CHCHD4 binding affects the active site of apoptosis inducing factor (AIF): Structural determinants for allosteric regulation.

Apoptosis-inducing factor (AIF), which is confined to mitochondria of normal healthy cells, is the first identified caspase-independent cell death effector. Moreover, AIF is required for the optimal functioning of the respiratory chain machinery. Recent findings have revealed that AIF fulfills its pro-survival function by interacting with CHCHD4, a soluble mitochondrial protein which promotes the entrance and the oxidative folding of different proteins in the inner membrane space. Here, we report the crystal structure of the ternary complex involving the N-terminal 27-mer peptide of CHCHD4, NAD+, and AIF harboring its FAD (flavin adenine dinucleotide) prosthetic group in oxidized form. Combining this information with biophysical and biochemical data on the CHCHD4/AIF complex, we provide a detailed structural description of the interaction between the two proteins, validated by both chemical cross-linking mass spectrometry analysis and site-directed mutagenesis.

A single tyrosine hydroxyl group almost entirely controls the NADPH specificity of Plasmodium falciparum ferredoxin-NADP+ reductase.

Plasmodium falciparum ferredoxin-NADP(+) reductase (FNR) is a FAD-containing enzyme that, in addition to be a promising target of novel antimalarial drugs, represents an excellent model of plant-type FNRs. The cofactor specificity of FNRs depends on differences in both k(cat) and K(m) values for NADPH and NADH. Here, we report that deletion of the hydroxyl group of the conserved Y258 of P. falciparum FNR, which interacts with the 2'-phosphate group of NADPH, selectively decreased the k(cat) of the NADPH-dependent reaction by a factor of 2 to match that of the NADH-dependent one. Rapid-reaction kinetics, active-site titrations with NADP(+), and anaerobic photoreduction experiments indicated that this effect may be the consequence of destabilization of the catalytically competent conformation of bound NADPH. Moreover, because the Y258F replacement increased the K(m) for NADPH 4-fold and decreased that for NADH 3-fold, it led to a drop in the ability of the enzyme to discriminate between the coenzymes from 70- to just 1.5-fold. The impact of the Y258F change was not affected by the presence of the H286Q mutation, which is known to enhance the catalytic activity of the enzyme. Our data highlight the major role played by the Y258 hydroxyl group in determining the coenzyme specificity of P. falciparum FNR. From the general standpoint of engineering the kinetic properties of plant-type FNRs, although P. falciparum FNR is less strictly NADPH-dependent than its homologues, the almost complete abolishment of coenzyme selectivity reported here has never been accomplished before through a single mutation.

Ligand Binding in Allosteric Flavoproteins: Part 1. Quantitative Analysis of the Interaction with NAD+ of the Apoptosis Inducing Factor (AIF) Harboring FAD in the Reduced State.

To perform their action, flavoproteins usually interact with a variety of low molecular weight partners, including electron transporters, yielding transient complexes whose tightness is often controlled by the redox state of the bound flavin cofactor. As a case study, here we describe the quantitative analysis of the redox-dependent interaction of the mammalian apoptosis inducing factor (AIF) with its NAD+ ligand. In particular, we report a protocol for the spectrophotometric titration of AIF in its reduced state under anaerobic conditions with NAD+, in order to determine the dissociation constant of the resulting complex.

Ligand Binding in Allosteric Flavoproteins: Part 2. Quantitative Analysis of the Redox-Dependent Interaction of the Apoptosis-Inducing Factor (AIF) with Its Protein Partner.

To perform their action usually flavoproteins interact transiently with a variety of molecular partners, whose binding is reciprocally affected and often controlled by the redox state of the bound flavin cofactor. As a case study, here we describe an approach for the quantitative characterization of the redox-controlled interaction of the mammalian apoptosis inducing factor (AIF) with one of its known protein partners, namely, the mitochondrial coiled-coil-helix-coiled-coil-helix domain-containing protein 4 (CHCHD4). In particular, we report a protocol for the titration of the flavoprotein in both in its oxidized and reduced states with CHCHD4, using an implementation of the MicroScale Thermophoresis (MST) technique.

Plasmodium falciparum Ferredoxin-NADP+ Reductase-Catalyzed Redox Cycling of Plasmodione Generates Both Predicted Key Drug Metabolites: Implication for Antimalarial Drug Development.

Plasmodione (PD) is a potent antimalarial redox-active 3-benzyl-menadione acting at low nanomolar range concentrations on different malaria parasite stages. The specific bioactivation of PD was proposed to occur via a cascade of redox reactions starting from one-electron reduction and then benzylic oxidation, leading to the generation of several key metabolites including corresponding benzylic alcohol (PD-bzol, for PD benzhydrol) and 3-benzoylmenadione (PDO, for PD oxide). In this study, we showed that the benzylic oxidation of PD is closely related to the formation of a benzylic semiquinone radical, which can be produced under two conditions: UV photoirradiation or catalysis by Plasmodium falciparum apicoplast ferredoxin-NADP+ reductase (PfFNR) redox cycling in the presence of oxygen and the parent PD. Electrochemical properties of both PD metabolites were investigated in DMSO and in water. The single-electron reduction potential values of PD, PD-bzol, PDO, and a series of 3-benzoylmenadiones were determined according to ascorbate oxidation kinetics. These compounds possess enhanced reactivity toward PfFNR as compared with model quinones. Optimal conditions were set up to obtain the best conversion of the starting PD to the corresponding metabolites. UV irradiation of PD in isopropanol under positive oxygen pressure led to an isolated yield of 31% PDO through the transient semiquinone species formed in a cascade of reactions. In the presence of PfFNR, PDO and PD-bzol could be observed during long lasting redox cycling of PD continuously fueled by NADPH regenerated by an enzymatic system. Finally, we observed and quantified the effect of PD on the production of oxidative stress in the apicoplast of transgenic 3D7[Api-roGFP2-hGrx1]P. falciparum parasites by using the described genetically encoded glutathione redox sensor hGrx1-roGFP2 methodology. The observed fast reactive oxygen species (ROS) pulse released in the apicoplast is proposed to be mediated by PD redox cycling catalyzed by PfFNR.

Antiplasmodial activity of quinones: roles of aziridinyl substituents and the inhibition of Plasmodium falciparum glutathione reductase.

Although quinones have been the subject of great interest as possible antimalarial agents, the mechanism of their antimalarial activity is poorly understood. Flavoenzyme electrontransferase-catalyzed redox cycling of quinones, and their inhibition of the antioxidant flavoenzyme glutathione reductase (GR, EC 1.8.1.7) have been proposed as possible mechanisms. Here, we have examined the activity of a number of quinones, including the novel antitumor agent RH1, against the malaria parasite Plasmodium falciparum strain FcB1 in vitro, their single-electron reduction rates by P. falciparum ferredoxin:NADP(+) reductase (PfFNR, EC 1.18.1.2), and their ability to inhibit P. falciparum GR. The multiparameter statistical analysis of our data implies, that the antiplasmodial activity of fully-substituted quinones (n=15) is relatively independent from their one-electron reduction potential (E(7)(1)). The presence of aziridinyl groups in quinone ring increased their antiplasmodial activity. Since aziridinyl-substituted quinones do not possess enhanced redox cycling activity towards PfFNR, we propose that they could act as as DNA-alkylating agents after their net two-electron reduction into aziridinyl-hydroquinones. We found that under the partial anaerobiosis, i.e., at the oxygen concentration below 40-50 microM, this reaction may be carried out by single-electron transferring flavoenzymes present in P. falciparum, like PfFNR. Another parameter increasing the antiplasmodial activity of fully-substituted quinones is an increase in their potency as P. falciparum GR inhibitors, which was revealed using multiparameter regression analysis. To our knowledge, this is the first quantitative demonstration of a link between the antiplasmodial activity of compounds and GR inhibition.

Synthesis of human renalase1 in Escherichia coli and its purification as a FAD-containing holoprotein.

Renalase is a protein ubiquitous in vertebrates, which has been proposed to modulate blood pressure and heart rate, and whose downregulation might result in hypertension. Despite its potential relevance for human health, the biochemical characterization of renalase is still lacking, possibly due to difficulties in obtaining it in recombinant form. By expressing two different gene constructs, we found that the major isoform of human renalase, renalase1, is mainly produced in Escherichia coli in inclusion bodies. However, by optimizing the expression conditions, significant amounts of soluble products were obtained. Both soluble renalase forms have been purified to homogeneity exploiting their N-terminal His-tag. Linking of the protein of interest to the SUMO protein did not improve solubility, but yielded untagged renalase1 after proteolytic processing of the fusion product. The two recombinant renalase forms displayed the same molecular properties. They bind equimolar amounts of FAD and appear to be correctly folded by various criteria. The procedures for the production and isolation of recombinant renalase1 here reported are expected to boost the much awaited biochemical studies on this remarkable protein.

Plasmodium falciparum ferredoxin-NADP+ reductase His286 plays a dual role in NADP(H) binding and catalysis.

The NADP-binding site of Plasmodium falciparum ferredoxin-NADP(+) reductase contains two basic residues, His286 and Lys249, conserved within the Plasmodium genus, but not in other plant-type homologues. Previous crystal studies indicated that His286 interacts with the adenine ring and with the 5'-phosphate of 2'-P-AMP, a ligand that mimics the adenylate moiety of NADP(H). Here we show that replacement of His286 with aliphatic residues results both in a decrease in the affinity of the enzyme for NADPH and in a decrease in k(cat), due to a lowered hydride-transfer rate. Unexpectedly, the mutation to Gln produces an enzyme more active than the wild-type one, whereas the change to Lys destabilizes the nicotinamide-isoalloxazine interaction, decreasing k(cat). On the basis of the crystal structure of selected mutants complexed with 2'-P-AMP, we conclude that the His286 side chain plays a dual role in catalysis both by providing binding energy for NADPH and by favoring the catalytically competent orientation of its nicotinamide ring. For the latter function, the H-bonding potential rather than the positively charged state of the His286 imidazole seems sufficient. Furthermore, we show that the Lys249Ala mutation decreases K(m)(NADPH) and K(d) for NADP(+) or 2'-P-AMP by a factor of 10. We propose that the Lys249 side chain participates in substrate recognition by interacting with the 2'-phosphate of NADP(H) and that this interaction was not observed in the crystal form of the enzyme-2'-P-AMP complex due to a conformational perturbation of the substrate-binding loop induced by dimerization.