Characterization of Schistosoma mansoni Peroxiredoxin 1: the assembling and switching between the alternative structures and functions of a moonlighting enzyme Fulvio Saccoccia 1,2 , Patrizio Di Micco 1,3 , Matteo Ardini 4 , Giovanna Boumis 1,2 , Luana Di Leandro 4 , Veronica Morea 3 , Adriana E. Miele 1,2 , David L. Williams 5 , Rodolfo Ippoliti 4 , Maurizio Brunori 1,2 , Andrea Bellelli 1,2 , Francesco Angelucci 4 1. Biochemical Sciences, Sapienza University of Rome, Rome, Italy; 2. Institute Pasteur-Fondazione Cenci-Bolognettii, Rome, Italy; 3. National Research Council of Italy, Institute of Molecular Biology and Pathology, Rome, Italy; 4. Life, health and environment, University of L'Aquila, L'Aquila, Italy; 5. Immunology-Microbiology, Rush University Medical Center, Chicago, U.S.A . Neglected Tropical Diseases 1 Dengue, rabies, trachoma, Buruli ulcer, treponematoses, leprosy, Chagas disease, human African trypanosomiasis, leishmaniasis, cysticercosis, drucunculiasis, echinococcosis, food-borne trematode infections, lymphatic filariasis, onchocerciasis, schistosomiasis , soil-transmitted helminthiases The life cycle of Schistosoma mansoni 2 : - two hosts - many stages The thioredoxin system - redox omeostasis - Glutathione peroxidase (Gpx) and Peroxiredoxin (Prx) provide peroxidase activity against ROS 3 Prx is a validated drug target against Schistosomiasis 4 Schistosomiasis affects 300 million people with 280000 deaths /year Background: the biological problem MOONLIGHTING BY DIFFERENT STRESSORS: THE SWITCHING IN STRUCTURE AND FUNCTION OF SmPRXI In reducing conditions Cys P takes the fully-folded (FF) conformation. After substrate reduction, oxidation of Cys P by Cys R occurs and unwinding of the first turn of the α2 helix leads the locally-unfolded (LU) conformation 3 . Fully Folded Locally Unfolded The reduced Prx exists as a pentamer of dimers arranged in a ring-like structure that is responsible for the peroxidase activity. Once the disulfide bond is formed, the decamer dissociates into dimers. Both decameric and dimeric forms are LMW forms but evidences suggested that a HMW was formed exerting holdase function upon overoxidation 5 . The first crystallographic structure of the holdase form: SmPrxI in acidic conditions From peroxidase to chaperone 2-Cys peroxiredoxins (Prx) are thought to play alternative roles in cell's physiology: under low oxidative stress conditions they assemble in a low molecular weight (LMW) toroid-shaped decameric form and function as thioredoxin-dependent peroxidases, whereas under high oxidative stress conditions they undergo a structural change and function as high molecular weight (HMW) ATP-independent chaperones. Neither the precise structure of the HMW complexes nor the binding sites for unfolded proteins were known. By means of X-ray crystallography, transmission electron microscopy and functional experiments on wild-type and mutated forms of Prx1 from Schistosoma mansoni, a human parasite, we defined the structural events responsible for the moonlighting behavior of 2-Cys Prx. We present the 3D crystal structures of the decameric reduced form and of the double-doughnut shape assembly: the latter is a stacking of two overlying decamers obtained in acidic conditions. Acidification is coupled to both local structural variations localized at the active site and changes in oligomerization to HMW forms, similar to those induced by oxidative stress. Abstract Activation of the holdase form by overoxidation and acidic conditions: probing the mechanism by mutagenesis The first crystallographic structure of the HMW form of a typical 2-Cys Prx 6 , obtained in 100mM PBS/citrate buffer, pH 4.2, 200mM NaCl, 20% PEG8000. Stacking of two decamers D5 point group symmetry Reduced Cys48 Local Unfolding conformation of α2 No electron density for C-terminal region Crystals structure of the peroxidase form, obtained in 100 mM potassium citrate, 20% PEG3350, 20 mM DTT. The active site is in its FF conformation while C48 is reduced. It shares the same basic quaternary structure of typical Prxs, being contributed by an arrangement of homodimers Size exclusion chromatography 1) ( ) reduced, pH = 7.4 ▲ 2) ( ) reduced, pH = 4.2 □ 3) ( ) overoxidized (Cys-SO ● 2 - /SO 3 - ), pH = 7.4 Acidic pH shifts aggregation toward HMW species Temperature-induced aggregation of Citrate Synthase (43°C, pH = 7.0, λ=360nm) ( ) 1mM CS; ■ ( ) 1mM CS + 2 eq. reduced SmPrxI ○ ( ) 1mM CS + 0.5 eq. overoxidized ▲ () 1mM CS + 1 eq. overoxidized □ () 1mM CS + 2 eq. overoxidized ● Overoxidized form has holdase activity Temperature-induced aggregation of Citrate Synthase (43°C, pH = 4.2, λ=360nm) ( ) 0.8 mM CS (ctrl); ▲ (+) 0.8 mM CS +Prx-SO 2 H/SO 3 H-monomer 10 mM (X) 0.8 mM CS +reduced PrxI-monomer 10 mM () □ 20 mM CS ( ) 50 mM CS ● Acidic form has holdase activity weaker than that observed for overoxidized Prx at pH 7.0 Structural superposition of the LMW over the HMW form. Major changes were detected at the active site and the dimer interface, while C-terminus is lacking in the HMW structure References 1. Manson’s tropical diseases. 22nd edn. 2009 GC Cook, AI Zumla. Elsevier. 2. Schistosomiasis: Epidemiology, Diagnosis and Treatment. 2014, Adriana E. Miele. Nova Biomedical. 3. Wood ZA, Schröder E, Robin Harris J, Poole LB. Structure, mechanism and regulation of peroxiredoxins. Trends Biochem Sci. 2003, 28, 32-40. 4. Sayed AA, et al. Redox balance mechanisms in Schistosoma mansoni rely on peroxiredoxins and albumin and implicate peroxiredoxins as novel drug targets. J Biol Chem. 2006, 281, 17001-17010. Cys48 was replaced with: Pro, as helix breaker; Asp, to mimick the sulfinic form of Cys P ; Ser, to mimick the protonation of sulfur Cys P . A C-terminal truncated form was also generated. All the experiments confirmed the hypothesis that chemical modification of Cys P bring to holdase chaperone/HMW form. Crystal structures of the C48D and C48P mutants were also solved 7 . All the mutants displayed chaperonic activity holdase activity strong holdase activity weak holdase activity MW=500kDa MW=250kDa SEC showed a shift of the elution profile toward HMW. TEM experiments demonstrated that C48D, C48S and ∆Cter form long filaments of stacked rings. C48S in the crystal: single decamer; sulfate anion inhibit HMW formation C48P in the crystal: fourty protomers in the asymmetric unit Both overoxidation and protonation bring to HMW formation that displays holdase activity. All changes seems to be triggered by changing in the chemistry of Cys P 200 nm 5. Kumsta C, Jakob U. Redox-regulated chaperones. Biochemistry 2009, 48, 4666-4676. 6. Saccoccia F, et al. Moonlighting by different stressors: crystal structure of the chaperone species of a 2-Cys peroxiredoxin. Structure 2012, 20, 429-439. 7. Angelucci F, Saccoccia F, et al. Switching between the alternative structures and functions of a 2-Cys peroxiredoxin, by site-directed mutagenesis. J Mol Biol. 2013, 425, 4556-4568. 43°C, pH 7.0 se. F1000 Posters: Use Permitted under Creat mmons License. F1000 Posters: Use Permitted under Creative Commons r Creative Commons License. F1000 Posters: Use Permitted under Creative Commons License. ermitted under Creative Commons License. F1000 Posters: Use Permitted under Creative Commons Lice osters: Use Permitted under Creative Commons License. F1000 Posters: Use Permitted under Creative C F1000 Posters: Use Permitted under Creative Commons License. F1000 Posters: Use Permitted un Posters: Use Permitted under Creative Commons License. F1000 Posters: Use Permitted under Creative Commons License. F1000