Publications by authors named "Adriana E Miele"

29 Publications

  • Page 1 of 1

Biophysical characterization of the complex between the iron-responsive transcription factor Fep1 and DNA.

Eur Biophys J 2021 Jan 4. Epub 2021 Jan 4.

Department of Biochemical Sciences 'A. Rossi Fanelli', Sapienza University of Rome, P.le Aldo Moro 5, 00185, Rome, Italy.

Fep1 is an iron-responsive GATA-type transcriptional repressor present in numerous fungi. The DNA-binding domain of this protein is characterized by the presence of two zinc fingers of the Cys-Cys type and a Cys-X-Cys-X-Cys-X-Cys motif located between the two zinc fingers, that is involved in binding of a [2Fe-2S] cluster. In this work, biophysical characterization of the DNA-binding domain of Pichia pastoris Fep1 and of the complex of the protein with cognate DNA has been undertaken. The results obtained by analytical ultracentrifugation sedimentation velocity, small-angle X-ray scattering and differential scanning calorimetry indicate that Fep1 is a natively unstructured protein that is able to bind DNA forming 1:1 and 2:1 complexes more compact than the individual partners. Complex formation takes place independently of the presence of a stoichiometric [2Fe-2S] cluster, suggesting that the cluster may play a role in recruiting other protein(s) required for regulation of transcription in response to changes in intracellular iron levels.
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http://dx.doi.org/10.1007/s00249-020-01489-yDOI Listing
January 2021

Omic approaches to decipher the molecular mechanisms of fibrosis, and design new anti-fibrotic strategies.

Semin Cell Dev Biol 2020 05 27;101:161-169. Epub 2019 Dec 27.

Univ Lyon, University Claude Bernard Lyon 1, CNRS, INSA Lyon, CPE, Institute of Molecular and Supramolecular Chemistry and Biochemistry, UMR 5246, F-69622 Villeurbanne Cedex, France. Electronic address:

We review here omics approaches including transcriptomics, proteomics, glycomics, metabolomics and interactomics, databases and computational tools for omic and multi-omic investigations of fibrosis to understand the molecular mechanisms underlying fibrogenesis and fibrosis, to identify biomarkers of diagnosis, prognosis or disease progression, and new therapeutic targets and to design new anti-fibrotic drugs. We also provide perspectives for future studies including lipid and glycosaminoglycan profiling, and the design of virtual patient models as a basis for personalised medicine and virtualisation of drug development.
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http://dx.doi.org/10.1016/j.semcdb.2019.12.009DOI Listing
May 2020

A comparative analysis of secreted protein disulfide isomerases from the tropical co-endemic parasites Schistosoma mansoni and Leishmania major.

Sci Rep 2019 07 2;9(1):9568. Epub 2019 Jul 2.

ICBMS UMR 5246, CNRS - Université de Lyon, 43 Boulevard du 11 Novembre 1918, Villeurbanne, cedex, 69622, France.

The human parasites Schistosoma mansoni and Leishmania major are co-endemic and a major threat to human health. Though displaying different tissue tropisms, they excrete/secrete similar subsets of intracellular proteins that, interacting with the host extracellular matrix (ECM), help the parasites invading the host. We selected one of the most abundant proteins found in the secretomes of both parasites, protein disulfide isomerase (PDI), and performed a comparative screening with surface plasmon resonance imaging (SPRi), looking for ECM binding partners. Both PDIs bind heparan sulfate; none of them binds collagens; each of them binds further ECM components, possibly linked to the different tropisms. We investigated by small-angle X-ray scattering both PDIs structures and those of a few complexes with host partners, in order to better understand the differences within this conserved family fold. Furthermore, we highlighted a previously undisclosed moonlighting behaviour of both PDIs, namely a concentration-dependent switch of function from thiol-oxidoreductase to holdase. Finally, we have tried to exploit the differences to look for possible compounds able to interfere with the redox activity of both PDI.
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http://dx.doi.org/10.1038/s41598-019-45709-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6606611PMC
July 2019

Insights into the structure and dynamics of lysyl oxidase propeptide, a flexible protein with numerous partners.

Sci Rep 2018 08 6;8(1):11768. Epub 2018 Aug 6.

Univ Lyon, University Claude Bernard Lyon 1, CNRS, INSA Lyon, CPE, Institute of Molecular and Supramolecular Chemistry and Biochemistry, UMR 5246, F-69622, Villeurbanne cedex, France.

Lysyl oxidase (LOX) catalyzes the oxidative deamination of lysine and hydroxylysine residues in collagens and elastin, which is the first step of the cross-linking of these extracellular matrix proteins. It is secreted as a proenzyme activated by bone morphogenetic protein-1, which releases the LOX catalytic domain and its bioactive N-terminal propeptide. We characterized the recombinant human propeptide by circular dichroism, dynamic light scattering, and small-angle X-ray scattering (SAXS), and showed that it is elongated, monomeric, disordered and flexible (D: 11.7 nm, R: 3.7 nm). We generated 3D models of the propeptide by coarse-grained molecular dynamics simulations restrained by SAXS data, which were used for docking experiments. Furthermore, we have identified 17 new binding partners of the propeptide by label-free assays. They include four glycosaminoglycans (hyaluronan, chondroitin, dermatan and heparan sulfate), collagen I, cross-linking and proteolytic enzymes (lysyl oxidase-like 2, transglutaminase-2, matrix metalloproteinase-2), a proteoglycan (fibromodulin), one growth factor (Epidermal Growth Factor, EGF), and one membrane protein (tumor endothelial marker-8). This suggests new roles for the propeptide in EGF signaling pathway.
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http://dx.doi.org/10.1038/s41598-018-30190-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6078952PMC
August 2018

Fragment-Based Discovery of a Regulatory Site in Thioredoxin Glutathione Reductase Acting as "Doorstop" for NADPH Entry.

ACS Chem Biol 2018 08 11;13(8):2190-2202. Epub 2018 Jun 11.

Department of Life, Health and Environmental Sciences , University of L'Aquila , 67100 L'Aquila , Italy.

Members of the FAD/NAD-linked reductase family are recognized as crucial targets in drug development for cancers, inflammatory disorders, and infectious diseases. However, individual FAD/NAD reductases are difficult to inhibit in a selective manner with off-target inhibition reducing usefulness of identified compounds. Thioredoxin glutathione reductase (TGR), a high molecular weight thioredoxin reductase-like enzyme, has emerged as a promising drug target for the treatment of schistosomiasis, a parasitosis afflicting more than 200 million people. Taking advantage of small molecules selected from a high-throughput screen and using X-ray crystallography, functional assays, and docking studies, we identify a critical secondary site of the enzyme. Compounds binding at this site interfere with well-known and conserved conformational changes associated with NADPH reduction, acting as a doorstop for cofactor entry. They selectively inhibit TGR from Schistosoma mansoni and are active against parasites in culture. Since many members of the FAD/NAD-linked reductase family have similar catalytic mechanisms, the unique mechanism of inhibition identified in this study for TGR broadly opens new routes to selectively inhibit homologous enzymes of central importance in numerous diseases.
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http://dx.doi.org/10.1021/acschembio.8b00349DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6905387PMC
August 2018

Pichia pastoris Fep1 is a [2Fe-2S] protein with a Zn finger that displays an unusual oxygen-dependent role in cluster binding.

Sci Rep 2016 08 22;6:31872. Epub 2016 Aug 22.

Dip. Scienze Biochimiche 'A. Rossi Fanelli', Sapienza Università di Roma, Roma, Italy.

Fep1, the iron-responsive GATA factor from the methylotrophic yeast Pichia pastoris, has been characterised both in vivo and in vitro. This protein has two Cys2-Cys2 type zinc fingers and a set of four conserved cysteines arranged in a Cys-X5-Cys-X8-Cys-X2-Cys motif located between the two zinc fingers. Electronic absorption and resonance Raman spectroscopic analyses in anaerobic and aerobic conditions indicate that Fep1 binds iron in the form of a [2Fe-2S] cluster. Site-directed mutagenesis shows that replacement of the four cysteines with serine inactivates this transcriptional repressor. Unexpectedly, the inactive mutant is still able to bind a [2Fe-2S] cluster, employing two cysteine residues belonging to the first zinc finger. These two cysteine residues can act as alternative cluster ligands selectively in aerobically purified Fep1 wild type, suggesting that oxygen could play a role in Fep1 function by causing differential localization of the [Fe-S] cluster.
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http://dx.doi.org/10.1038/srep31872DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4992955PMC
August 2016

Uncovering new structural insights for antimalarial activity from cost-effective aculeatin-like derivatives.

Org Biomol Chem 2015 Feb;13(7):2064-77

Département de Pharmacochimie Moléculaire, Univ. Grenoble-Alpes, CNRS UMR 5063, CNRS ICMG FR 2607, bâtiment André Rassat, 470 rue de la Chimie, F-38041 Grenoble Cedex 9, France.

A series of new aculeatin-like analogues were synthesized in two steps by combining two sets of building blocks. Many compounds showed inhibitory activities in vitro against Plasmodium falciparum and have helped to gain more insight into structure-activity relationships around the spirocyclohexadienone pharmacophoric scaffold. Plasmodium falciparum thioredoxin reductase (PfTrxR) has been investigated as a putative cellular target. Moreover, a new aculeatin-like scaffold without Michael acceptor properties, efficient at 0.86 μM against P. falciparum 3D7, was identified and raises the prospect of developing a new antimalarial agent.
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http://dx.doi.org/10.1039/c4ob02459aDOI Listing
February 2015

Selenocysteine robustness versus cysteine versatility: a hypothesis on the evolution of the moonlighting behaviour of peroxiredoxins.

Biochem Soc Trans 2014 Dec;42(6):1768-72

*Istituto Pasteur-Fondazione Cenci-Bolognetti and Dipartimento di Scienze Biochimiche "A. Rossi Fanelli", Sapienza Università di Roma, Piazzale A. Moro 5, 00185 Rome, Italy.

Peroxiredoxins (Prxs) and glutathione peroxidases (Gpxs) provide the majority of peroxides reducing activity in the cytoplasm. Both are peroxidases but differences in the chemical mechanism of reduction of oxidative agents, as well as in the reactivity of the catalytically active residues, confer peculiar features on them. Ultimately, Gpx should be regarded as an efficient peroxides scavenger having a high-reactive selenocysteine (Sec) residue. Prx, by having a low pKa cysteine, is less efficient than Gpx in reduction of peroxides under physiological conditions, but the chemistry of the sulfur together with the peculiar structural arrangement of the active site, in typical Prxs, make it suitable to sense a redox environment and to switch-in-function so as to exert holdase activity under redox-stress conditions. The complex macromolecular assembly would have evolved the chaperone holdase function and the moonlighting behaviour typical of many Prxs.
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http://dx.doi.org/10.1042/BST20140212DOI Listing
December 2014

Thioredoxin reductase and its inhibitors.

Curr Protein Pept Sci 2014 ;15(6):621-46

Istituto Pasteur - Fondazione Cenci-Bolognetti, Istituto di Biologia e Medicina Molecolare del CNR, Dipartimento di Scienze Biochimiche "A. Rossi Fanelli", Sapienza Università di Roma, Piazzale A. Moro 5, 00185 Rome, Italy.

Thioredoxin plays a crucial role in a wide number of physiological processes, which span from reduction of nucleotides to deoxyriboucleotides to the detoxification from xenobiotics, oxidants and radicals. The redox function of Thioredoxin is critically dependent on the enzyme Thioredoxin NADPH Reductase (TrxR). In view of its indirect involvement in the above mentioned physio/pathological processes, inhibition of TrxR is an important clinical goal. As a general rule, the affinities and mechanisms of binding of TrxR inhibitors to the target enzyme are known with scarce precision and conflicting results abound in the literature. A relevant analysis of published results as well as the experimental procedures is therefore needed, also in view of the critical interest of TrxR inhibitors. We review the inhibitors of TrxR and related flavoreductases and the classical treatment of reversible, competitive, non competitive and uncompetitive inhibition with respect to TrxR, and in some cases we are able to reconcile contradictory results generated by oversimplified data analysis.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4275836PMC
http://dx.doi.org/10.2174/1389203715666140530091910DOI Listing
April 2015

Crystal structure of Plasmodium falciparum thioredoxin reductase, a validated drug target.

Biochem Biophys Res Commun 2012 Sep 6;425(4):806-11. Epub 2012 Aug 6.

Department of Biochemical Sciences and Istituto Pasteur - Fondazione Cenci Bolognetti, Sapienza University of Rome, 00185 Rome, Italy.

Plasmodium falciparum is the vector of the most prevalent and deadly form of malaria, and, among the Plasmodium species, it is the one with the highest rate of drug resistance. At the basis of a rational drug design project there is the selection and characterization of suitable target(s). Thioredoxin reductase, the first protection against reactive oxygen species in the erythrocytic phase of the parasite, is essential for its survival. Hence it represents a good target for the design of new anti-malarial active compounds. In this paper we present the first crystal structure of recombinant P. falciparum thioredoxin reductase (PfTrxR) at 2.9Å and discuss its differences with respect to the human orthologue. The most important one resides in the dimer interface, which offers a good binding site for selective non competitive inhibitors. The striking conservation of this feature among the Plasmodium parasites, but not among other Apicomplexa parasites neither in mammals, boosts its exploitability.
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http://dx.doi.org/10.1016/j.bbrc.2012.07.156DOI Listing
September 2012

Moonlighting by different stressors: crystal structure of the chaperone species of a 2-Cys peroxiredoxin.

Structure 2012 Mar;20(3):429-39

Department of Biochemical Sciences, Sapienza University of Rome and Istituto Pasteur-Fondazione Cenci Bolognetti, P.le Aldo Moro 5, 00185 Rome, Italy.

2-Cys peroxiredoxins (Prxs) play two different roles depending on the physiological status of the cell. They are thioredoxin-dependent peroxidases under low oxidative stress and ATP-independent chaperones upon exposure to high peroxide concentrations. These alternative functions have been associated with changes in the oligomerization state from low-(LMW) to high-molecular-weight (HMW) species. Here we present the structures of Schistosoma mansoni PrxI in both states: the LMW decamer and the HMW 20-mer formed by two stacked decamers. The latter is the structure of a 2-Cys Prx chaperonic form. Comparison of the structures sheds light on the mechanism by which chemical stressors, such as high H(2)O(2) concentration and acidic pH, are sensed and translated into a functional switch in this protein family. We also propose a model to account for the in vivo formation of long filaments of stacked Prx rings.
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http://dx.doi.org/10.1016/j.str.2012.01.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3299984PMC
March 2012

On the mechanism and rate of gold incorporation into thiol-dependent flavoreductases.

J Inorg Biochem 2012 Mar 27;108:105-11. Epub 2011 Nov 27.

Department of Biochemical Sciences A. Rossi Fanelli, Sapienza University of Rome, Rome, Italy.

NADPH-dependent flavoreductases are important drug targets. During their enzymatic cycle thiolates and selenolates that have high affinity for transition metals are generated. Auranofin (AF), a gold-containing compound, is classified by the World Health Organization as an antirheumatic agent and it is indicated as the scaffold for the development of new anticancer and antiparasitic drugs. AF inhibits selenocysteine-containing flavoreductases (thioredoxin reductase and thioredoxin glutathione reductase) more effectively than non Se-containing ones (glutathione reductase); this preference has been ascribed to the high affinity of selenium for gold. We solved the 3D structure of the Se-containing Thioredoxin Glutathione Reductase from the human parasite Schistosoma mansoni complexed with Au and our results challenge this view: we believe that the relative velocity of the reaction rather than the relative affinity, depends on the presence of Sec residues, which appear to dictate AF selectivity.
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http://dx.doi.org/10.1016/j.jinorgbio.2011.11.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3396563PMC
March 2012

Structural and functional characterization of Schistosoma mansoni Thioredoxin.

Protein Sci 2011 Jun 5;20(6):1069-76. Epub 2011 May 5.

Dipartimento di Scienze Biochimiche and Istituto Pasteur-Fondazione Cenci Bolognetti, "Sapienza" University of Rome, P.le Aldo Moro 5, 00185 Rome, Italy.

Schistosomiasis, the human parasitosis caused by various species of the blood-fluke Schistosoma, is a debilitating disease affecting 200 million people in tropical areas. The massive administration of the only effective drug, praziquantel, leads to the appearance of less sensitive parasite strains, thus, making urgent the search for new therapeutic approaches and new suitable targets. The thiol-mediated detoxification pathway has been identified as a promising target, being essential during all the parasite developmental stages and sufficiently different from the host counterpart. As a part of a project aimed at the structural characterization of all the proteins involved in this pathway, we describe hereby the high-resolution crystal structure of Schistosoma mansoni Thioredoxin (SmTrx) in three states, namely: the wild-type oxidized adult enzyme and the oxidized and reduced forms of a juvenile isoform, carrying an N-terminal extension. SmTrx shows a typical thioredoxin fold, highly similar to the other components of the superfamily. Although probably unlikely to be a reasonable drug target given its high similarity with the human counterpart, SmTrx completes the characterization of the whole set of thiol-mediated detoxification pathway components. Moreover, it can reduce oxidized glutathione and is one of the few defence proteins expressed in mature eggs and in the hatch fluid, thus confirming an important role in the parasite. We believe its crystal structure may provide clues for the formation of granulomas and the pathogenesis of the chronic disease.
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http://dx.doi.org/10.1002/pro.634DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3104236PMC
June 2011

Mapping the catalytic cycle of Schistosoma mansoni thioredoxin glutathione reductase by X-ray crystallography.

J Biol Chem 2010 Oct 21;285(42):32557-67. Epub 2010 Jul 21.

Department of Biochemical Sciences A. Rossi Fanelli, CNR Institute of Molecular Biology and Pathology and Istituto Pasteur-Fondazione Cenci Bolognetti, Sapienza University of Rome, P.le Aldo Moro 5, 00185 Rome, Italy.

Schistosomiasis is the second most widespread human parasitic disease. It is principally treated with one drug, praziquantel, that is administered to 100 million people each year; less sensitive strains of schistosomes are emerging. One of the most appealing drug targets against schistosomiasis is thioredoxin glutathione reductase (TGR). This natural chimeric enzyme is a peculiar fusion of a glutaredoxin domain with a thioredoxin selenocysteine (U)-containing reductase domain. Selenocysteine is located on a flexible C-terminal arm that is usually disordered in the available structures of the protein and is essential for the full catalytic activity of TGR. In this study, we dissect the catalytic cycle of Schistosoma mansoni TGR by structural and functional analysis of the U597C mutant. The crystallographic data presented herein include the following: the oxidized form (at 1.9 Å resolution); the NADPH- and GSH-bound forms (2.3 and 1.9 Å, respectively); and a different crystal form of the (partially) reduced enzyme (3.1 Å), showing the physiological dimer and the entire C terminus of one subunit. Whenever possible, we determined the rate constants for the interconversion between the different oxidation states of TGR by kinetic methods. By combining the crystallographic analysis with computer modeling, we were able to throw further light on the mechanism of action of S. mansoni TGR. In particular, we hereby propose the putative functionally relevant conformational change of the C terminus after the transfer of reducing equivalents from NADPH to the redox sites of the enzyme.
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http://dx.doi.org/10.1074/jbc.M110.141960DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2952258PMC
October 2010

Inhibition of Schistosoma mansoni thioredoxin-glutathione reductase by auranofin: structural and kinetic aspects.

J Biol Chem 2009 Oct 26;284(42):28977-85. Epub 2009 Aug 26.

Department of Biochemical Sciences A. Rossi Fanelli, Sapienza University of Rome and Istituto Pasteur-Fondazione Cenci Bolognetti, P. Le Aldo Moro 5, 00185 Rome, Italy.

Schistosomiasis is a parasitic disease affecting over 200 million people currently treated with one drug, praziquantel. A possible drug target is the seleno-protein thioredoxin-glutathione reductase (TGR), a key enzyme in the pathway of the parasite for detoxification of reactive oxygen species. The enzyme is a unique fusion of a glutaredoxin domain with a thioredoxin reductase domain, which contains a selenocysteine (Sec) as the penultimate amino acid. Auranofin (AF), a gold-containing compound already in clinical use as an anti-arthritic drug, has been shown to inhibit TGR and to substantially reduce worm burden in mice. Using x-ray crystallography we solved (at 2.5 A resolution) the structure of wild type TGR incubated with AF. The electron density maps show that the actual inhibitor is gold, released from AF. Gold is bound at three different sites not directly involving the C-terminal Sec residue; however, because the C terminus in the electron density maps is disordered, we cannot exclude the possibility that gold may also bind to Sec. To investigate the possible role of Sec in the inactivation kinetics, we tested the effect of AF on a model enzyme of the same superfamily, i.e. the naturally Sec-lacking glutathione reductase, and on truncated TGR. We demonstrate that the role of selenium in the onset of inhibition by AF is catalytic and can be mimicked by an external source of selenium (benzeneselenol). Therefore, we propose that Sec mediates the transfer of gold from its ligands in AF to the redox-active Cys couples of TGR.
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http://dx.doi.org/10.1074/jbc.M109.020701DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2781444PMC
October 2009

Investigating the structural plasticity of a cytochrome P450: three-dimensional structures of P450 EryK and binding to its physiological substrate.

J Biol Chem 2009 Oct 22;284(42):29170-9. Epub 2009 Jul 22.

CNR Institute of Molecular Biology and Pathology, P.le A. Moro 5, 00185 Rome, Italy.

Cytochrome P450s are heme-containing proteins that catalyze the oxidative metabolism of many physiological endogenous compounds. Because of their unique oxygen chemistry and their key role in drug and xenobiotic metabolism, particular attention has been devoted in elucidating their mechanism of substrate recognition. In this work, we analyzed the three-dimensional structures of a monomeric cytochrome P450 from Saccharopolyspora erythraea, commonly called EryK, and the binding kinetics to its physiological ligand, erythromycin D. Three different structures of EryK were obtained: two ligand-free forms and one in complex with its substrate. Analysis of the substrate-bound structure revealed the key structural determinants involved in substrate recognition and selectivity. Interestingly, the ligand-free structures of EryK suggested that the protein may explore an open and a closed conformation in the absence of substrate. In an effort to validate this hypothesis and to investigate the energetics between such alternative conformations, we performed stopped-flow absorbance experiments. Data demonstrated that EryK binds erythromycin D via a mechanism involving at least two steps. Contrary to previously characterized cytochrome P450s, analysis of double jump mixing experiments confirmed that this complex scenario arises from a pre-existing equilibrium between the open and closed subpopulations of EryK, rather than from an induced-fit type mechanism.
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http://dx.doi.org/10.1074/jbc.M109.003590DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2781461PMC
October 2009

Pattern of cavities in globins: the case of human hemoglobin.

Biopolymers 2009 Dec;91(12):1097-107

University of Rome, P.le Aldo Moro 5, 00185 Rome, Italy.

Our aim is to shed light on the conservation of potential ligand docking sites that play an important role in ligand dynamics of globins by using the technique of filling internal cavities naturally present in hemoglobin and myoglobin with xenon atoms. In particular, we present the high resolution structures of the Xe-adduct of deoxygenated wild type human hemoglobin and a quadruple mutant (L(B10)Y and H(E7)Q in alpha and beta chains). For the sake of comparison we also determined under the same experimental conditions the xenon complex of wild type sperm whale myoglobin. The analysis revealed that the number and position of Xe binding cavities are different in the alpha and beta subunits, the latter being more similar to myoglobin. Notably, no proximal Xe docking site was detected in hemoglobin, at variance with myoglobin. The pattern of internal cavities accessibility and affinity for xenon suggests a different role for the dynamics of ligand migration in the two types of hemoglobin chains as compared to myoglobin. The number and position of hydrophobic cavities in hemoglobin are briefly discussed also in comparison with the data available for other members of the globin superfamily.
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http://dx.doi.org/10.1002/bip.21201DOI Listing
December 2009

Cloning, expression, purification, crystallization and preliminary X-ray crystallographic analysis of C-12 hydroxylase EryK from Saccharopolyspora erythraea.

Protein Pept Lett 2008 ;15(10):1138-41

Istituto di Biologia e Patologia Molecolari, CNR, and Dipartimento di Scienze Biochimiche, "Sapienza" Università di Roma, P.le A. Moro 5, I-00185 Rome, Italy.

Erythromycin A is produced by Saccharopolyspora erythraea via a secondary metabolic pathway using several steps including glycosylations and hydroxylations of the first macrolide intermediate 6-deoxyerythronolide B. Erythromycin C-12 hydroxylase (CYP113A1), the P450 cytochrome active in the final stages of erythromycin biosynthesis, was cloned and expressed in E. coli. Different crystal forms were harvested from distinct crystallization conditions: two ligand-free forms, one substrate bound and two inhibitors-bound. All crystals belong either to the monoclinc P2(1)or to the orthorhombic P2(1)2(1)2(1) space groups, and exhibit diffraction limits ranging from 2.3 to 1.6 A. The structures will be determined by molecular replacement.
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http://dx.doi.org/10.2174/092986608786071201DOI Listing
January 2009

Will new antischistosomal drugs finally emerge?

Trends Parasitol 2008 Sep 31;24(9):379-82. Epub 2008 Jul 31.

Institute of Cell Biology, CNR, 32 Via Ramarini, 00015 Monterotondo, Rome, Italy.

It has been often observed that the chemotherapeutic armamentarium against an important disease such as schistosomiasis consists of just one drug, praziquantel. Thus, development of drug resistance is an impending danger, with serious implications for the health protection of many millions of people. This rational and legitimate concern might now begin to be relieved by the recent proposal of a new class of compounds that could represent a novel source of drugs against schistosomiasis.
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http://dx.doi.org/10.1016/j.pt.2008.05.006DOI Listing
September 2008

Glutathione reductase and thioredoxin reductase at the crossroad: the structure of Schistosoma mansoni thioredoxin glutathione reductase.

Proteins 2008 Aug;72(3):936-45

Department of Biochemical Sciences A Rossi Fanelli, Istituto Pasteur Fondazione Cenci-Bolognetti and CNR Institute of Molecular Biology and Pathology, Sapienza University of Rome, Piazzale Aldo Moro 5, 00185 Rome, Italy.

Thioredoxin glutathione reductase (TGR) is a key flavoenzyme expressed by schistosomes that bridges two detoxification pathways crucial for the parasite survival in the host's organism. In this article we report the crystal structure (at 2.2 A resolution) of TGR from Schistosoma mansoni (SmTGR), deleted in the last two residues. The structure reveals the peculiar architecture of this chimeric enzyme: the small Glutaredoxin (Grx) domain at the N-terminus is joined to the large thioredoxin reductase (TR) one via an extended complementary surface, involving residues not conserved in the Grx superfamily; the TR domain interacts with an identical partner via its C-terminal domain, forming a dimer with a twisted "W" shape. Although lacking the penultimate Selenocysteine residue (Sec), the enzyme is still able to reduce oxidized glutathione. These data update the interpretation of the interdomain communication in TGR enzymes. The possible function of this enzyme in pathogenic parasites is discussed.
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http://dx.doi.org/10.1002/prot.21986DOI Listing
August 2008

The three-dimensional structure of two redox states of cyclophilin A from Schistosoma mansoni. Evidence for redox regulation of peptidyl-prolyl cis-trans isomerase activity.

J Biol Chem 2007 Aug 25;282(34):24851-7. Epub 2007 Jun 25.

Istituto Pasteur-Fondazione Cenci Bolognetti, Department of Biochemical Sciences "A. Rossi Fanelli," "Sapienza" University of Rome, Piazzale Aldo Moro 5, 00185 Rome, Italy.

Treatment of schistosomiasis, a widespread human parasitic disease caused by the helminth parasites of the genus Schistosoma, relies mainly on one chemotherapeutic agent, praziquantel, although several other compounds exert anti-parasitic effects. One such compound is the immunosuppressant cyclosporin A, which has been shown to significantly diminish worm burden in mice infected with Schistosoma mansoni. Given the well established interaction between cyclosporin A and the cyclophilin superfamily of peptidylprolyl cis-trans isomerases, we solved the structure of cyclophilin A from S. mansoni (SmCypA) by x-ray crystallography in the reduced and oxidized states at 1.5 and 1.8 A of resolution, respectively. Oxidized SmCypA contains a disulfide bridge between two C-terminal cysteines (Cys-122 and Cys-126). This is the first example of a cyclophilin containing this disulfide bridge. Parallel functional studies suggest a mechanism for regulation of SmCypA activity via oxidation of its thiol groups; in fact, whereas oxidized SmCypA is inactive, reduced SmCypA is an efficient isomerase active at nanomolar levels with a k(cat)/K(m) of 1.1 x 10(7) M(-1) s(-1), and it is inhibited by cyclosporin A (IC(50) of 14 +/- 4 nM). The lack of conservation of this cysteine couple within the CypA superfamily, their close proximity to the active site, and the importance of thiol groups for peptidyl-prolyl cis-trans isomerase activity render this structural feature a challenge for the development of alternative and more effective anti-schistosomiasis inhibitors and may in addition imply an alternative function of SmCypA in the schistosome.
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http://dx.doi.org/10.1074/jbc.M702714200DOI Listing
August 2007

Probing the mechanism of GSH activation in Schistosoma haematobium glutathione-S-transferase by site-directed mutagenesis and X-ray crystallography.

J Mol Biol 2006 Jul 2;360(3):678-89. Epub 2006 Jun 2.

Department of Biochemical Sciences A. Rossi Fanelli and Istituto Pasteur- Fondazione Cenci Bolognetti, University of Rome La Sapienza, Rome, Italy.

During turnover, the catalytic tyrosine residue (Tyr10) of the sigma class Schistosoma haematobium wild-type glutathione-S-transferase is expected to switch alternately in and out of the reduced glutathione-binding site (G-site). The Tyrout10 conformer forms a pi-cation interaction with the guanidinium group of Arg21. As in other similar glutathione-S-transferases, the catalytic Tyr has a low pKa of 7.2. In order to investigate the catalytic role of Tyr10, and the structural and functional roles of Arg21, we carried out structural studies on two Arg21 mutants (R21L and R21Q) and a Tyr10 mutant, Y10F. Our crystallographic data for the two Arg21 mutants indicate that only the Tyrout10 conformation is populated, thereby excluding a role of Arg21 in the stabilisation of the out conformation. However, Arg21 was confirmed to be catalytically important and essential for the low pKa of Tyr10. Upon comparison with structural data generated for reduced glutathione-bound and inhibitor-bound wild-type enzymes, it was observed that the orientations of Tyr10 and Arg35 are concerted and that, upon ligand binding, minor rearrangements occur within conserved residues in the active site loop. These rearrangements are coupled to quaternary rigid-body movements at the dimer interface and alterations in the localisation and structural order of the C-terminal domain.
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http://dx.doi.org/10.1016/j.jmb.2006.05.040DOI Listing
July 2006

Schistosoma mansoni fatty acid binding protein: specificity and functional control as revealed by crystallographic structure.

Biochemistry 2004 Oct;43(41):13000-11

Department of Biochemical Sciences A. Rossi Fanelli, CNR Institute of Molecular Biology and Pathology and Istituto Pasteur-Fondazione Cenci Bolognetti, University of Rome La Sapienza, Piazzale Aldo Moro 5, 00185 Rome, Italy.

Schistosoma mansoni fatty acid binding protein (Sm14) was crystallized with bound oleic acid (OLA) and arachidonic acid (ACD), and their structures were solved at 1.85 and 2.4 A resolution, respectively. Sm14 is a vaccine target for schistosomiasis, the second most prevalent parasitic disease in humans. The parasite is unable to synthesize fatty acids depending on the host for these nutrients. Moreover, arachidonic acid (ACD) is required to synthesize prostaglandins employed by schistosomes to evade the host's immune defenses. In the complex, the hydrocarbon tail of bound OLA assumes two conformations, whereas ACD adopts a unique hairpin-looped structure. ACD establishes more specific interactions with the protein, among which the most important is a pi-cation bond between Arg78 and the double bond at C8. Comparison with homologous fatty acid binding proteins suggests that the binding site of Sm14 is optimized to fit ACD. To test the functional implications of our structural data, the affinity of Sm14 for 1,8-anilinonaphthalenesulfonic acid (ANS) has been measured; moreover the binding constants of six different fatty acids were determined from their ability to displace ANS. OLA and ACD exhibited the highest affinities. To determine the rates of fatty acid binding and dissociation we carried out stopped flow kinetic experiments monitoring displacement by (and of) ANS. The binding rate constant of ligands is controlled by a slow pH dependent conformational change, which we propose to have physiological relevance.
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http://dx.doi.org/10.1021/bi048505fDOI Listing
October 2004

Two distinct interaction motifs in amphiphysin bind two independent sites on the clathrin terminal domain beta-propeller.

Nat Struct Mol Biol 2004 Mar 15;11(3):242-8. Epub 2004 Feb 15.

MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK.

During the assembly of clathrin-coated vesicles, many peripheral membrane proteins, including the amphiphysins, use LLDLD-type clathrin-box motifs to interact with the N-terminal beta-propeller domain (TD) of clathrin. The 2.3 A-resolution structure of the clathrin TD in complex with a TLPWDLWTT peptide from amphiphysin 1 delineates a second clathrin-binding motif, PWXXW (the W box), that binds at a site on the TD remote from the clathrin box-binding site. The presence of both sequence motifs within the unstructured region of the amphiphysins allows them to bind more tightly to free TDs than do other endocytic proteins that contain only clathrin-box motifs. This property, along with the propensity of the N-terminal BAR domain to bind curved membranes, will preferentially localize amphiphysin and its partner, dynamin, to the periphery of invaginated clathrin lattices.
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http://dx.doi.org/10.1038/nsmb736DOI Listing
March 2004

Complex landscape of protein structural dynamics unveiled by nanosecond Laue crystallography.

Proc Natl Acad Sci U S A 2003 Jul 7;100(15):8704-9. Epub 2003 Jul 7.

Laboratoire de Cristallographie et de Cristallogénèse des Protéines, UMR 9015, Institut de Biologie Structurale/Commissariat à l'Energie Atomique/Université Joseph Fourier, and European Synchrotron Radiation Facility, Grenoble, France.

Although conformational changes are essential for the function of proteins, little is known about their structural dynamics at atomic level resolution. Myoglobin (Mb) is the paradigm to investigate conformational dynamics because it is a simple globular heme protein displaying a photosensitivity of the iron-ligand bond. Upon laser photodissociation of carboxymyoglobin Mb a nonequilibrium population of protein structures is generated that relaxes over a broad time range extending from picoseconds to milliseconds. This process is associated with migration of the ligand to cavities in the matrix and with a reduction in the geminate rebinding rate by several orders of magnitude. Here we report nanosecond time-resolved Laue diffraction data to 1.55-A resolution on a Mb mutant, which depicts the sequence of structural events associated with this extended relaxation. Motions of the distal E-helix, including the mutated residue Gln-64(E7), and of the CD-turn are found to lag significantly (100-300 ns) behind local rearrangements around the heme such as heme tilting, iron motion out of the heme plane, and swinging of the mutated residue Tyr-29(B10), all of which occur promptly (< or =3 ns). Over the same delayed time range, CO is observed to migrate from a cavity distal to the heme known to bind xenon (called Xe4) to another such cavity proximal to the heme (Xe1). We propose that the extended relaxation of the globin moiety reflects reequilibration among conformational substates known to play an essential role in controlling protein function.
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http://dx.doi.org/10.1073/pnas.1430900100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC166376PMC
July 2003

Analysis of the effect of microgravity on protein crystal quality: the case of a myoglobin triple mutant.

Acta Crystallogr D Biol Crystallogr 2003 Jun 23;59(Pt 6):982-8. Epub 2003 May 23.

Dipartimento di Scienze Biochimiche and CNR Istituto di Biologia e Patologia Molecolari, Universita di Roma La Sapienza, P.le A. Moro 5, 00185 Rome, Italy.

Crystals of the Met derivative of the sperm whale myoglobin triple mutant Mb-YQR [L(B10)Y, H(E7)Q and T(E10)R] were grown under microgravity conditions and on earth by vapour diffusion. A comparison of crystal quality after complete data collection and processing shows how microgravity-grown crystals diffract to better resolution and lead to considerably improved statistics for X-ray diffraction data compared with crystals grown on earth under the same conditions. The same set of experiments was reproduced on two different Spacelab missions (ISS 6A and ISS 8A) in 2001 and 2002. The structure of this mutant myoglobin, refined using data collected at ELETTRA (Trieste, Italy) from both kinds of crystals, shows that X-ray diffraction from microgravity-grown crystals leads to better defined electron-density maps as well as improved geometrical quality of the refined model. Improvement of the stereochemical parameters of a protein structure is fundamental to quantitative analysis of its function and dynamics and hence to thorough understanding of the molecular mechanisms of action.
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http://dx.doi.org/10.1107/s0907444903005924DOI Listing
June 2003

The structure of ActVA-Orf6, a novel type of monooxygenase involved in actinorhodin biosynthesis.

EMBO J 2003 Jan;22(2):205-15

Dipartimento di Scienze Biochimiche and CNR, Centro di Studi sulla Biologia Molecolare, Università di Roma 'La Sapienza', Piazzale A.Moro, 5, 00185 Roma, Italy.

ActVA-Orf6 monooxygenase from Streptomyces coelicolor that catalyses the oxidation of an aromatic intermediate of the actinorhodin biosynthetic pathway is a member of a class of small monooxygenases that carry out oxygenation without the assistance of any of the prosthetic groups, metal ions or cofactors normally associated with activation of molecular oxygen. The overall structure is a ferredoxin-like fold with a novel dimeric assembly, indicating that the widely represented ferredoxin fold may sustain yet another functionality. The resolution (1.3 A) of the enzyme structure and its complex with substrate and product analogues allows us to visualize the mechanism of binding and activation of the substrate for attack by molecular oxygen, and utilization of two gates for the reaction components including a proton gate and an O(2)/H(2)O gate with a putative protein channel. This is the first crystal structure of an enzyme involved in the tailoring of a type II aromatic polyketide and illustrates some of the enzyme-substrate recognition features that may apply to a range of other enzymes involved in modifying a polyketide core structure.
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http://dx.doi.org/10.1093/emboj/cdg031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC140106PMC
January 2003

The carbon monoxide derivative of human hemoglobin carrying the double mutation LeuB10-->Tyr and HisE7-->Gln on alpha and beta chains probed by infrared spectroscopy.

Arch Biochem Biophys 2002 Jun;402(1):59-64

Department of Biochemical Sciences and CNR Center of Molecular Biology, University La Sapienza, 00185 Rome, Italy.

The fine structural properties of the distal heme pocket have been probed by infrared spectroscopy of ferrous carbon monoxy human hemoglobin mutants carrying the mutations LeuB10-->Tyr and HisE7-->Gln on the alpha, beta, and both chains, respectively. The stretching frequency of iron-bound carbon monoxide occurs as a single broad band around 1943 cm(-1) in both the alpha and the beta mutated chains. Such a frequency value indicates that no direct hydrogen bonding exists between the bound CO molecule and the TyrB10 phenolic oxygen, at variance with other naturally occurring TyrB10, GlnE7 nonvertebrate hemoglobins. The rates of carbon monoxide release have been determined for the first time by a Fourier transform infrared spectroscopy stopped-flow technique that allowed us to single out the heterogeneity in the kinetics of CO release in the alpha and beta chains for the mutated proteins and for native HbA. The rates of CO release are 15- to 20-fold faster for the mutated alpha or beta chains with respect to the native ones consistent with the lack of distal stabilization on the iron-bound CO molecule. The present results demonstrate that residues in key topological positions (namely E7 and B10) for the distal steric control of the iron-bound ligand are not interchangeable among hemoglobins from different species.
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http://dx.doi.org/10.1016/S0003-9861(02)00061-9DOI Listing
June 2002

Structural dynamics of myoglobin: ligand migration among protein cavities studied by Fourier transform infrared/temperature derivative spectroscopy.

J Biol Chem 2002 Apr 15;277(14):11636-44. Epub 2002 Jan 15.

Department of Biophysics, University of Ulm, D-89069 Ulm, Germany.

Fourier transform infrared (FTIR) spectroscopy in the CO stretch bands combined with temperature derivative spectroscopy (TDS) was used to characterize intermediate states obtained by photolysis of two sperm whale mutant myoglobins, YQR (L29(B10)Y, H64(E7)Q, T67(E10)R) and YQRF (with an additional I107(G8)F replacement). Both mutants assume two different bound-state conformations, A(0) and A(3), which can be distinguished by their different CO bands near 1965 and 1933 cm(-1). They most likely originate from different conformations of the Gln-64 side chain. Within each A substate, a number of photoproduct states have been characterized on the basis of the temperature dependence of recombination in TDS experiments. Different locations and orientations of the ligand within the protein can be distinguished by the infrared spectra of the photolyzed CO. Recombination from the primary docking site, B, near the heme dominates below 50 K. Above 60 K, ligand rebinding occurs predominantly from a secondary docking site, C', in which the CO is trapped in the Xe4 cavity on the distal side, as shown by crystallography of photolyzed YQR and L29W myoglobin CO. Another kinetic state (C") has been identified from which rebinding occurs around 130 K. Moreover, a population appearing above the solvent glass transition at approximately 180 K (D state) is assigned to rebinding from the Xe1 cavity, as suggested by the photoproduct structure of the L29W sperm whale myoglobin mutant. For both the YQR and YQRF mutants, rebinding from the B sites near the heme differs for the two A substates, supporting the view that the return of the ligand from the C', C", and D states is not governed by the recombination barrier at the heme iron but rather by migration to the active site. Comparison of YQR and YQRF shows that access to the Xe4 site (C') is severely restricted by introduction of the bulky Phe side chain at position 107.
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http://dx.doi.org/10.1074/jbc.M109892200DOI Listing
April 2002