B21-High Throughput SAXS
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Diamond Proposal Number(s):
[15832]
Open Access
Abstract: Rituximab, a murine–human chimera, is the first monoclonal antibody (mAb) developed as a therapeutic agent to target CD20 protein. Its Fab domain and its interaction with CD20 have been extensively studied and high-resolution atomic models obtained by X-ray diffraction or cryo-electron microscopy are available. However, the structure of the full-length antibody is still missing as the inherent protein flexibility hampers the formation of well-diffracting crystals and the reconstruction of 3D microscope images. The global structure of rituximab from its dilute solution is here elucidated by small-angle X-ray scattering (SAXS). The limited data resolution achievable by this technique has been compensated by intensive computational modelling that led to develop a new and effective procedure to characterize the average mAb conformation as well as that of the single domains. SAXS data indicated that rituximab adopts an asymmetric average conformation in solution, with a radius of gyration and a maximum linear dimension of 52 Å and 197 Å, respectively. The asymmetry is mainly due to an uneven arrangement of the two Fab units with respect to the central stem (the Fc domain) and reflects in a different conformation of the individual units. As a result, the Fab elbow angle, which is a crucial determinant for antigen recognition and binding, was found to be larger (169°) in the more distant Fab unit than that in the less distant one (143°). The whole flexibility of the antibody has been found to strongly depend on the relative inter-domain orientations, with one of the Fab arms playing a major role. The average structure and the amount of flexibility has been studied in the presence of different buffers and additives, and monitored at increasing temperature, up to the complete unfolding of the antibody. Overall, the structural characterization of rituximab can help in designing next-generation anti-CD20 antibodies and finding more efficient routes for rituximab production at industrial level.
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Apr 2022
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I03-Macromolecular Crystallography
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Isabella
Bolognino
,
Antonio
Carrieri
,
Rosa
Purgatorio
,
Marco
Catto
,
Rocco
Caliandro
,
Benedetta
Carrozzini
,
Benny Danilo
Belviso
,
Maria
Majellaro
,
Eddy
Sotelo
,
Saverio
Cellamare
,
Cosimo Damiano
Altomare
Diamond Proposal Number(s):
[15832]
Open Access
Abstract: The enantiomeric separation of 15 racemic 4-aryl-3,4-dihydropyrimidin-2(1H)-one (DHP) alkoxycarbonyl esters, some of which proved to be highly active as A2B adenosine receptor antagonists, was carried out by HPLC on ChirobioticTM TAG, a chiral stationary phase (CSP) bearing teicoplanin aglycone (TAG) as the chiral selector. The racemic compounds were separated under polar organic (PO) conditions. Preliminarily, the same selectands were investigated on three different Pirkle-type CSPs in normal-phase (NP) conditions. A baseline separation was successfully obtained on TAG-based CSPs for the majority of compounds, some of which achieved high enantioselectivity ratios (α > 2) in contrast with the smaller α values (1–1.5) and the lack of baseline resolution observed with the Pirkle-type CSPs. In particular, the racemic tetrazole-fused DHP ester derivatives, namely compounds 8 and 9, were separated on TAG-based HPLC columns with noteworthy α values (8.8 and 6.0, respectively), demonstrating the potential of the method for preparative purposes. A competition experiment, carried out with a racemic analyte (6) by adding N-acetyl-d-alanine (NADA) to the mobile phase, suggested that H-bonding interactions involved in the recognition of the natural dipeptide ligand d-Ala-d-Ala into the TAG cleft should be critical for enantioselective recognition of 4-aryl DHPs by TAG. The X-ray crystal structure of TAG was elucidated at a 0.77 Å resolution, whereas the calculation of molecular descriptors of size, polar, and H-bond interactions, were complemented with molecular docking and molecular dynamics calculations, shedding light on repulsive (steric effects) and attractive (H-bond—polar and apolar) interactions between 4-aryl DHP selectands and TAG chiral selectors.
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Dec 2021
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[15832, 21741]
Abstract: Supramolecular structure and properties of deep eutectic solvents (DESs) are known to be highly affected by the addition of water, and their use as solvents for poorly water-soluble macromolecules is being actively investigated. We report the first experimental investigation of protein crystallization in DESs. Different hydrophilic and hydrophobic eutectic mixtures, hydrated at different levels, have been screened as crystallization media. DESs were added to the solution containing the precipitant and the buffer required to crystallize three test proteins, and we observed that the volume ratio between DES and the corresponding solution is a key parameter for the crystallization process. Successful crystallization was achieved for the hen-egg white lysozyme when using choline chloride:urea, choline chloride:glycerol, and choline chloride:glutamic acid eutectic mixtures at a 1:2 molar ratio. High-resolution X-ray diffraction experiments disclosed the possibility to study the intriguing supramolecular network of the molecular complexes formed between protein and DES in the presence of water molecules. Individual DES components have been found to systematically occupy specific protein sites populated by solvent-exposed aromatic residues. Weak interactions between DES components, possibly mediated by water molecules, which resulted in being frozen in the ordered solvent surrounding the protein units in the crystal lattice, were reconstructed at atomic resolution. DESs were found to have a negligible effect on the protein conformation and its flexibility in the solid state. On the other hand, DESs greatly reduced solvent evaporation from the crystallization drop, thereby increasing the dissolution time of the protein crystals. Finally, DESs were found to serve as local modulators of the ordered solvent, and this resulted in a significant change of the protein solubility. In addition, we found that protein crystallization was sped up by tuning DES hydration. This enables the employment of these environmentally responsible solvents to improve biotechnological processes at the industrial level.
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Jun 2021
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[15832]
Open Access
Abstract: The beneficial effects of Cyclooxygenases (COX) inhibitors on human health have been known for thousands of years. Nevertheless, COXs, particularly COX-1, have been linked to a plethora of human diseases such as cancer, heart failure, neurological and neurodegenerative diseases only recently. COXs catalyze the first step in the biosynthesis of prostaglandins (PGs) and are among the most important mediators of inflammation. All published structural work on COX-1 deals with the ovine isoenzyme, which is easier to produce in milligram-quantities than the human enzyme and crystallizes readily. Here, we report the long-sought structure of the human cyclooxygenase-1 (hCOX-1) that we refined to an R/Rfree of 20.82/26.37, at 3.36 Å resolution. hCOX-1 structure provides a detailed picture of the enzyme active site and the residues crucial for inhibitor/substrate binding and catalytic activity. We compared hCOX-1 crystal structure with the ovine COX-1 and human COX-2 structures by using metrics based on Cartesian coordinates, backbone dihedral angles, and solvent accessibility coupled with multivariate methods. Differences and similarities among structures are discussed, with emphasis on the motifs responsible for the diversification of the various enzymes (primary structure, stability, catalytic activity, and specificity). The structure of hCOX-1 represents an essential step towards the development of new and more selective COX-1 inhibitors of enhanced therapeutic potential.
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Feb 2021
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[15832]
Open Access
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.
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Sep 2019
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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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
Diamond Proposal Number(s):
[19880]
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.
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Jun 2019
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[15832]
Open Access
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.
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May 2019
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[15832]
Open Access
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.
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Apr 2019
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I04-Macromolecular Crystallography
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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
Diamond Proposal Number(s):
[15832]
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.
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May 2018
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[15832]
Open Access
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.
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Jan 2018
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