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Instruments / Technical Area	Publication Details	Published
I03-Macromolecular Crystallography	Oxidation of human copper chaperone Atox1 and disulfide bond cleavage by cisplatin and glutathione Maria I. Nardella , Antonio Rosato , Benny D. Belviso , Rocco Caliandro , Giovanni Natile , Fabio Arnesano International Journal Of Molecular Sciences 20 DOI: 10.3390/ijms20184390 Diamond Proposal Number(s): [15832] Open Access Show Abstract ▼ Abstract: Cancer cells cope with high oxidative stress levels, characterized by a shift toward the oxidized form (GSSG) of glutathione (GSH) in the redox couple GSSG/2GSH. Under these conditions, the cytosolic copper chaperone Atox1, which delivers Cu(I) to the secretory pathway, gets oxidized, i.e., a disulfide bond is formed between the cysteine residues of the Cu(I)-binding CxxC motif. Switching to the covalently-linked form, sulfur atoms are not able to bind the Cu(I) ion and Atox1 cannot play an antioxidant role. Atox1 has also been implicated in the resistance to platinum chemotherapy. In the presence of excess GSH, the anticancer drug cisplatin binds to Cu(I)-Atox1 but not to the reduced apoprotein. With the aim to investigate the interaction of cisplatin with the disulfide form of the protein, we performed a structural characterization in solution and in the solid state of oxidized human Atox1 and explored its ability to bind cisplatin under conditions mimicking an oxidizing environment. Cisplatin targets a methionine residue of oxidized Atox1; however, in the presence of GSH as reducing agent, the drug binds irreversibly to the protein with ammine ligands trans to Cys12 and Cys15. The results are discussed with reference to the available literature data and a mechanism is proposed connecting platinum drug processing to redox and copper homeostasis.	Sep 2019
I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Crystal structures of the recombinant β-factor XIIa protease with bound Thr-Arg and Pro-Arg substrate mimetics Monika Pathak , Rosa Manna , Chan Li , Bubacarr G. Kaira , Badraldin Kareem Hamad , Benny Danilo Belviso , Camila R. Bonturi , Ingrid Dreveny , Peter M. Fischer , Lodewijk V. Dekker , Maria Luiza Vilela Oliva , Jonas Emsley Acta Crystallographica Section D Structural Biology 75 DOI: 10.1107/S2059798319006910 Diamond Proposal Number(s): [19880] Open Access Show Abstract ▼ Abstract: Coagulation factor XII (FXII) is a key initiator of the contact pathway, which contributes to inflammatory pathways. FXII circulates as a zymogen, which when auto-activated forms factor XIIa (FXIIa). Here, the production of the recombinant FXIIa protease domain (βFXIIaHis) with yields of ∼1–2 mg per litre of insect-cell culture is reported. A second construct utilized an N-terminal maltose-binding protein (MBP) fusion (MBP-βFXIIaHis). Crystal structures were determined of MBP-βFXIIaHis in complex with the inhibitor D-Phe-Pro-Arg chloromethyl ketone (PPACK) and of βFXIIaHis in isolation. The βFXIIaHis structure revealed that the S2 and S1 pockets were occupied by Thr and Arg residues, respectively, from an adjacent molecule in the crystal. The Thr-Arg sequence mimics the P2–P1 FXIIa cleavage-site residues present in the natural substrates prekallikrein and FXII, and Pro-Arg (from PPACK) mimics the factor XI cleavage site. A comparison of the βFXIIaHis structure with the available crystal structure of the zymogen-like FXII protease revealed large conformational changes centred around the S1 pocket and an alternate conformation for the 99-loop, Tyr99 and the S2 pocket. Further comparison with activated protease structures of factors IXa and Xa, which also have the Tyr99 residue, reveals that a more open form of the S2 pocket only occurs in the presence of a substrate mimetic. The FXIIa inhibitors EcTI and infestin-4 have Pro-Arg and Phe-Arg P2–P1 sequences, respectively, and the interactions that these inhibitors make with βFXIIa are also described. These structural studies of βFXIIa provide insight into substrate and inhibitor recognition and establish a scaffold for the structure-guided drug design of novel antithrombotic and anti-inflammatory agents.	Jun 2019
I04-Macromolecular Crystallography	Protein crystallization in ionic-liquid hydrogel composite membranes Benny Danilo Belviso , Rosanna Caliandro , Shabnam Majidi Salehi , Gianluca Di Profio , Rocco Caliandro Crystals 9 DOI: 10.3390/cryst9050253 Diamond Proposal Number(s): [15832] Open Access Show Abstract ▼ Abstract: Protein crystallization is a powerful purification tool. It is the first step for crystallographic structural investigations, and can be preparatory for biotechnological applications. However, crystallizing proteins is challenging and methods to control the crystallization process are needed. Ionic-liquid hydrogel composite membranes (IL-HCMs) have been used here as material capable of supporting protein crystallization and hosting grown crystals. We found that IL-HCMs affect the selection mechanism of glucose isomerase (GI) polymorphs and make GI crystals grow completely immersed into the hydrogel layer. X-ray diffraction studies show that IL ions do not bind to the protein, likely because IL molecules are constrained in the polymeric framework. Our GI crystal structures have been compared with many existing GI crystal structures using multivariate analysis tools, allowing a comprehensive overview of factors determining structural similarities, i.e., temperature variations and external stresses exerted during or after crystal growth, such as dehydration or presence of hydrogel of a different nature. GI crystals grown on IL-HCM fit perfectly in this framework, showing typical features induced by external forces. Overall, protein crystallization by IL-HCMs show potential for biotechnological applications, as it could constitute a natural means for containing crystallized enzymes in working conditions.	May 2019
I04-Macromolecular Crystallography	Optimization of vapor diffusion conditions for anti-CD20 crystallization and scale-up to meso batch Huaiyu Yang , Benny Danilo Belviso , Xiaoyu Li , Wenqian Chen , Teresa Fina Mastropietro , Gianluca Di Profio , Rocco Caliandro , Jerry Y. Y. Heng Crystals 9 DOI: 10.3390/cryst9050230 Diamond Proposal Number(s): [15832] Open Access Show Abstract ▼ Abstract: The crystal form is one of the preferred formulations for biotherapeutics, especially thanks to its ability to ensure high stability of the active ingredient. In addition, crystallization allows the recovery of a very pure drug, thus facilitating the manufacturing process. However, in many cases, crystallization is not trivial, and other formulations, such as the concentrate solution, represent the only choice. This is the case of anti-cluster of differentiation 20 (anti-CD20), which is one of the most sold antibodies for therapeutic uses. Here, we propose a set of optimized crystallization conditions for producing anti-CD20 needle-shaped crystals within 24 h in a very reproducible manner with high yield. High crystallization yield was obtained with high reproducibility using both hanging drop vapor diffusion and meso batch, which is a major step forward toward further scaling up the crystallization of anti-CD20. The influence of anti-CD20 storage conditions and the effect of different ions on the crystallization processes were also assessed. The crystal quality and the high yield allowed the first crystallographic investigation on anti-CD20, which positively confirmed the presence of the antibody in the crystals.	Apr 2019
I04-Macromolecular Crystallography	Hydrogel Composite Membranes Incorporating Iron Oxide Nanoparticles as Topographical Designers for Controlled Heteronucleation of Proteins Shabnam Majidi Salehi , Ana C. Manjua , Benny D. Belviso , Carla A. M. Portugal , Isabel M. Coelhoso , Valentina Mirabelli , Enrica Fontananova , Rocco Caliandro , João G. Crespo , Efrem Curcio , Gianluca Di Profio Crystal Growth & Design DOI: 10.1021/acs.cgd.7b01760 Diamond Proposal Number(s): [15832] Show Abstract ▼ Abstract: In this study, we exploited the possibility of tuning physical–chemical properties of hydrogel composite membranes (HCMs) surfaces, by using iron oxide nanoparticles (NPs) as topographical designers, with the aim of examining the effect of surface topography and wettability on the heterogeneous nucleation of protein crystals. On the basis of roughness and contact angle measurements, it was found that surface structural characteristics, in addition to chemical interactions between the surface and protein molecules, have influence on the heterogeneous nucleation of lysozyme and thermolysin crystals to different extents. We demonstrated that increasing the amount of NPs incorporated in the hydrogel matrix promotes protein nucleation to a higher extent, potentially due to the increase of local solute concentration, arising from the enhanced wetting tendency in the Wenzel regime, and physical confinement at rougher hydrophilic surfaces. An extensive crystallographic analysis suggested the tendency of the growing crystals to incorporate hydrogel materials, which allows inducement of protein conformational states slightly different from those covered by standard crystallization methods. Protein flexibility can be thus sampled by changing the amount of NPs in the HCMs, with negligible influence on the quantity and quality of X-ray diffraction data.	May 2018
I04-Macromolecular Crystallography	Enzyme Crystals and Hydrogel Composite Membranes as New Active Food Packaging Material Valentina Mirabelli , Shabnam Majidi Salehi , Luisa Angiolillo , Benny Danilo Belviso , Amalia Conte , Matteo Alessandro Del Nobile , Gianluca Di Profio , Rocco Caliandro Global Challenges 65 DOI: 10.1002/gch2.201700089 Diamond Proposal Number(s): [15832] Open Access Show Abstract ▼ Abstract: The great antimicrobial and antioxidant potential of enzymes makes them prone to be used as active packaging materials to preserve food from contamination or degradation. Major drawbacks are connected to the use of enzymes freely dispersed in solution, due to reduced protein stability. The immobilization of enzymes on solid supports to create biocatalytic interfaces has instead been proven to increase their stability and efficiency. In this work, it is shown that enzymes crystallized on hydrogel composite membranes (HCMs) can exert an effective antimicrobial action, thus making the composite membrane and crystals biofilm a potential active substrate for food packaging applications. The antimicrobial hen egg white lysozyme is crystallized on the surface of the hydrogel layer of HCMs, and its activity is determined by measuring the decrease in absorbance of Micrococcus lysodeikticus culture incubated with the specimen. The overall catalytic efficiency of the antimicrobial HCMs increases by a factor of 2 compared to the pure enzyme dissolved in solution at the same quantity. Because the enzyme in crystalline form is present in higher concentration and purity than in the solution, both its overall catalytic efficiency and antimicrobial action increase. Moreover, the hydrogel environment allows a better protein stabilization and retention during crystals dissolution.	Jan 2018
I02-Macromolecular Crystallography I03-Macromolecular Crystallography	Oxaliplatin Binding to Human Copper Chaperone Atox1 and Protein Dimerization Benny D. Belviso , Angela Galliani , Alessia Lasorsa , Valentina Mirabelli , Rocco Caliandro , Fabio Arnesano , Giovanni Natile Inorganic Chemistry 55, 6563 - 6573 DOI: 10.1021/acs.inorgchem.6b00750 Diamond Proposal Number(s): [11690] Show Abstract ▼ Abstract: Copper tra ffi cking proteins have been impli- cated in the cellular response to platinum anticancer drugs. We investigated the reaction of the chaperone Atox1 with an activated form of oxaliplatin, the third platinum drug to reach worldwide approval. Unlike cisplatin, which contains mono- dentate ammines, oxaliplatin contains chelated 1,2-diaminocy- clohexane (DACH), which is more resistant to displacement by nucleophiles. In solution, one or two {Pt(DACH) 2+ } moieties bind to the conserved CXXC metal-binding motif of Atox1; in the latter case the two sulfur atoms likely bridging the two platinum units. At longer reaction times, a dimeric species is formed whose composition, Atox1 2 · Pt 2+ 2 , indicates complete loss of the diamine ligands. Such a dimerization process is accompanied by partial unfolding of the protein. Crystallization experiments aiming at the characterization of the monomeric species have a ff orded, instead, a dimeric species resembling that already obtained by Boal and Rosenzweig in a similar reaction performed with cisplatin. However, while in the latter case there was only one Pt-binding site (0.4 occupancy) made of four sulfur atoms of the CXXC motifs of the two Atox1 chains in a tetrahedral arrangement, we found, in addition, a secondary Pt-binding site involving Cys41 of the B chain (0.25 occupancy). Moreover, both platinum atoms have lost their diamines. Thus, there appears to be little relationship between what is observed in solution and what is formed in the solid state. Since full occupancy of the tetrahedral cavity is a common feature of all Atox1 dimeric structures obtained with other metal ions (Cu + , Cd 2+ , and Hg 2+ ), we propose that in the case of platinum, where the occupancy is only 0.4, the remaining cavities are occupied by Cu + ions. Experimental evidence is reported in support of the latter hypothesis. Our proposal represents a meeting point between the initial proposal of Boal and Rosenzweig (0.4 Pt occupancy) and the reinterpretation of the original crystallographic data put forward by Shabalin et al. (1 Cu occupancy), and could apply to other cases.	Jul 2016
I24-Microfocus Macromolecular Crystallography	How a β-d-Glucoside Side Chain Enhances Binding Affinity to Thrombin of Inhibitors Bearing 2-Chlorothiophene as P1 Moiety: Crystallography, Fragment Deconstruction Study, and Evaluation of Antithrombotic Properties Benny D. Belviso , Rocco Caliandro , Modesto De Candia , Giorgia Zaetta , Gianfranco Lopopolo , Francesca Incampo , Mario Colucci , Cosimo D. Altomare Journal Of Medicinal Chemistry 57 (20), 8563 - 8575 DOI: 10.1021/jm5010754 PMID: 25268757 Show Abstract ▼ Abstract: The β-d-glucose-containing compound 3, bearing 2-chlorothiophene and 1-isopropylpiperidine moieties as binders of the S1 and S4 pockets, respectively, proved to be potent competitive inhibitor of factor Xa (fXa, Ki = 0.090 nM) and thrombin (fIIa, Ki = 100 nM). The potency of 3 increases, over the parent compound 1, against fIIa (110-fold), much more than against fXa (7-fold). Experimental deconstruction of 3 into smaller fragments revealed a binding cooperativity of the P3/P4 and propylene-linked β-d-glucose fragments, stronger in fIIa (15.5 kJ·mol–1) than in fXa (2.8 kJ·mol–1). The crystal structure of human fIIa in complex with 3 revealed a binding mode including a strong H-bond network between the glucose O1′, O3′, and O5′ and two critical residues, namely R221a and K224, belonging to the Na+-binding site which may allosterically perturb the specificity sites. The potential of 3 as antithrombotic agent was supported by its ability to inhibit thrombin generation and to stimulate fibrinolysis at submicromolar concentration.	Oct 2014

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