Structural insights into the C-terminus of the histone-lysine N-methyltransferase NSD3 by small-angle X-ray scattering.

NSD3 is a member of six H3K36-specific histone lysine methyltransferases in metazoans. Its overexpression or mutation is implicated in developmental defects and oncogenesis. Aside from the well-characterized catalytic SET domain, NSD3 has multiple clinically relevant potential chromatin-binding motifs, such as the proline-tryptophan-tryptophan-proline (PWWP), the plant homeodomain (PHD), and the adjacent Cys-His-rich domain located at the C-terminus. The crystal structure of the individual domains is available, and this structural knowledge has allowed the designing of potential inhibitors, but the intrinsic flexibility of larger constructs has hindered the characterization of mutual domain conformations. Here, we report the first structural characterization of the NSD3 C-terminal region comprising the PWWP2, SET, and PHD4 domains, which has been achieved at a low resolution in solution by small-angle X-ray scattering (SAXS) data on two multiple-domain NSD3 constructs complemented with size-exclusion chromatography and advanced computational modeling. Structural models predicted by machine learning have been validated in direct space, by comparison with the SAXS-derived molecular envelope, and in reciprocal space, by reproducing the experimental SAXS profile. Selected models have been refined by SAXS-restrained molecular dynamics. This study shows how SAXS data can be used with advanced computational modeling techniques to achieve a detailed structural characterization and sheds light on how NSD3 domains are interconnected in the C-terminus.

Stretching the chains: the destabilizing impact of Cu2+ and Zn2+ ions on K48-linked diubiquitin.

Ubiquitin signalling and metal homeostasis play key roles in controlling several physiological cellular activities, including protein trafficking and degradation. While some relationships between these two biochemical pathways have started to surface, our knowledge of their interplay remains limited. Here, we employ a variety of techniques, such as circular dichroism, differential scanning calorimetry, pressure perturbation calorimetry, fluorescence emission, SDS-PAGE, and small-angle X-ray scattering (SAXS) to evaluate the impact of Cu2+ and Zn2+ ions on the structure and stability of K48 linked diubiquitin (K48-Ub2), a simple model for polyubiquitin chains. The SAXS analysis results show that the structure of the metal-free protein is similar to that observed when the protein is bound to the E2 conjugating enzyme, lending support to the idea that the structure of unanchored K48-linked ubiquitin chains is sufficient for identification by conjugating enzymes without the need for an induced fit mechanism. Our results indicate that K48-Ub2 can coordinate up to four metal ions with both copper and zinc ions inducing slight changes to the secondary structure of the protein. However, we noted significant distinctions in their impacts on protein stability and overall architecture. Specifically, Cu2+ ions resulted in a destabilization of the protein structure, which facilitated the formation of dimer aggregates. Next, we observed a shift in the conformational dynamics of K48-Ub2 toward less compact and more flexible states upon metal ion binding, with Zn2+ inducing a more significant effect than Cu2+ ions. Our structural modelling study demonstrates that both metal ions induced perturbations in the K48-Ub2 structure, leading to the separation of the two monomers thus inhibiting interactions with E2 enzymes. In conclusion, the findings from this study enhance our comprehension of the mechanisms underlying Ub chains recognition. Moreover, they strengthen the notion that drug discovery initiatives aimed at targeting metal-mediated disruptions in Ub signaling hold great potential for treating a wide range of diseases that stem from abnormal protein accumulation.

Human aromatic amino acid decarboxylase is an asymmetric and flexible enzyme: Implication in aromatic amino acid decarboxylase deficiency.

Human aromatic amino acid decarboxylase (AADC) is a pyridoxal 5'-phosphate-dependent enzyme responsible for the biosynthesis of dopamine and serotonin, essential neurotransmitters involved in motor and cognitive abilities. Mutations in its gene lead to AADC deficiency, a monogenic rare neurometabolic childhood parkinsonism characterized by severe motor and neurodevelopmental symptoms. Here, for the first time, we solved the crystal structure of human holoAADC in the internal aldimine (1.9 Å) and in the external aldimine (2.4 Å) of the substrate analog L-Dopa methylester. In this intermediate, the highly flexible AADC catalytic loop (CL) is captured in a closed state contacting all protein domains. In addition, each active site, composed by residues of both subunits, is connected to the other through weak interactions and a central cavity. By combining crystallographic analyses with all-atom and coarse-grained molecular dynamics simulations, SAXS investigations and limited proteolysis experiments, we realized that the functionally obligate homodimeric AADC enzyme in solution is an elongated, asymmetric molecule, where the fluctuations of the CL are coupled to flexibility at the edge between the N-terminal and C-terminal domains. The structural integrity of this peripheral protein region is essential to catalysis, as assessed by both artificial and 37 AADC deficiency pathogenic variants leading to the interpretation that structural dynamics in protein regions far from the active site is essential for CL flexibility and the acquirement of a correct catalytically competent structure. This could represent the molecular basis for pathogenicity prediction in AADC deficiency.

hERG stereoselective modulation by mexiletine-derived ureas: Molecular docking study, synthesis, and biological evaluation.

Long QT syndrome (LQTS) is a disorder of cardiac electrophysiology resulting in life-threatening arrhythmias; nowadays, only a few drugs are available for the management of LQTS. Focusing our attention on LQT2, one of the most common subtypes of LQTS caused by mutations in the human ether-à-go-go-related gene (hERG), in the present work, the stereoselectivity of the recently discovered mexiletine-derived urea 8 was investigated on the hERG potassium channel. According to preliminary in silico predictions, in vitro studies revealed a stereoselective behavior, with the meso form showing the greatest hERG opening activity. In addition, functional studies on guinea pig isolated left atria, aorta, and ileum demonstrated that 8 does not present any cardiac or intestinal liability in our ex vivo studies. Due to its overall profile, (R,S)-8 paves the way for the design and development of a new series of compounds potentially useful in the treatment of both congenital and drug-induced forms of LQTS.

Unveiling the Influence of Hydrated Deep Eutectic Solvents on the Dynamics of Water-Soluble Proteins.

Deep eutectic solvents (DESs) are mixtures of two or more pure compounds (e.g., Lewis or Brønsted acids and bases, anionic and/or cationic species) in a well-defined stoichiometric proportion, with a melting point lower to that of an ideal liquid mixture. These neoteric solvents are highly tunable through varying the structure or relative ratio of parent components and have been evaluated as solvents able to improve biomolecules' performance, specifically their stability and biocatalytic properties. Inspired by a recent crystallographic study, we have explored through molecular dynamics (MD) simulations the dynamic properties of two different proteins (hen egg-white lysozyme and the human VH antibody fragment HEL4) in a (20% w/w) hydrated solution of choline chloride-glycerol (1:2). We have developed proper force fields to account for DES, protein, and DES-protein interactions, which have been calibrated using pair distribution function measurements of pure DES solutions. MD results show that the presence of DES quenches the protein motion, increasing the rigidity of the overall protein structure. Specific interactions among DES components and protein residues, such as those between choline ions and two Tryptophan residues of lysozyme, may amplify the protein-DES interactions and lead to protein crystallization in the presence of hydrated DES. These findings open new horizons to improve or achieve control on protein properties by a proper choice of hydrated DESs used as solvents.

Structure-based drug design of potential inhibitors of FBXW8, the substrate recognition component of Cullin-RING ligase 7.

FBXW8 plays an irreplaceable role in the substrate recognition of ubiquitin-dependent proteolysis, which further regulates cell cycle progression and signal transduction. However, the abnormal expression of FBXW8 triggers malignancy, inflammation, and autophagy irregulation. FBXW8 is considered as an effective therapeutic target for Cullin-RING ligase 7 (CRL7)-related cancers. Still, the lack of selective inhibitors hinders further therapeutic development and limits the exploration of its biological mechanism. This study constructed an integrated protocol that combines pharmacophore modeling, structure-based virtual screening, and Molecular Dynamic Simulation. It was then used as a screening query to identify hit compounds targeted at the substrate recognition site of FBXW8 from a large-scale compound database including 120 million compounds. Then, ten lead compounds were retrieved by using molecular docking analysis and ADMET prediction. Finally, MD simulations were performed to validate the binding stability of selected drug candidates. The result indicated that three newly obtained compounds, namely ZINC96179876, ZINC72174069, and ZINC97730272, might be potent FBXW8 inhibitors against CRL7-related cancers such as endometrial cancer.

Crystal Structure of the Human Copper Chaperone ATOX1 Bound to Zinc Ion.

The bioavailability of copper (Cu) in human cells may depend on a complex interplay with zinc (Zn) ions. We investigated the ability of the Zn ion to target the human Cu-chaperone Atox1, a small cytosolic protein capable of anchoring Cu(I), by a conserved surface-exposed Cys-X-X-Cys (CXXC) motif, and deliver it to Cu-transporting ATPases in the trans-Golgi network. The crystal structure of Atox1 loaded with Zn displays the metal ion bridging the CXXC motifs of two Atox1 molecules in a homodimer. The identity and location of the Zn ion were confirmed through the anomalous scattering of the metal by collecting X-ray diffraction data near the Zn K-edge. Furthermore, soaking experiments of the Zn-loaded Atox1 crystals with a strong chelating agent, such as EDTA, caused only limited removal of the metal ion from the tetrahedral coordination cage, suggesting a potential role of Atox1 in Zn metabolism and, more generally, that Cu and Zn transport mechanisms could be interlocked in human cells.

Structural Characterization of the Full-Length Anti-CD20 Antibody Rituximab.

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.

Structural Insights into the Vapochromic Behavior of Pt- and Pd-Based Compounds.

Anionic complexes having vapochromic behavior are investigated: [K(H2O)][M(ppy)(CN)2], [K(H2O)][M(bzq)(CN)2], and [Li(H2O)n][Pt(bzq)(CN)2], where ppy = 2-phenylpyridinate, bzq = 7,8-benzoquinolate, and M = Pt(II) or Pd(II). These hydrated potassium/lithium salts exhibit a change in color upon being heated to 380 K, and they transform back into the original color upon absorption of water molecules from the environment. The challenging characterization of their structure in the vapochromic transition has been carried out by combining several experimental techniques, despite the availability of partially ordered and/or impure crystalline material. Room-temperature single-crystal and powder X-ray diffraction investigation revealed that [K(H2O)][Pt(ppy)(CN)2] crystallizes in the Pbca space group and is isostructural to [K(H2O)][Pd(ppy)(CN)2]. Variable-temperature powder X-ray diffraction allowed the color transition to be related to changes in the diffraction pattern and the decrease in sample crystallinity. Water loss, monitored by thermogravimetric analysis, occurs in two stages, well separated for potassium Pt compounds and strongly overlapped for potassium Pd compounds. The local structure of potassium compounds was monitored by in situ pair distribution function (PDF) measurements, which highlighted changes in the intermolecular distances due to a rearrangement of the crystal packing upon vapochromic transition. A reaction coordinate describing the structural changes was extracted for each compound by multivariate analysis applied to PDF data. It contributed to the study of the kinetics of the structural changes related to the vapochromic transition, revealing its dependence on the transition metal ion. Instead, the ligand influences the critical temperature, higher for ppy than for bzq, and the inclination of the molecular planes with respect to the unit cell planes, higher for bzq than for ppy. The first stage of water loss triggers a unit cell contraction, determined by the increase in the b axis length and the decrease in the a (for ppy) or c (for bzq) axis lengths. Consequent interplane distance variations and in-plane roto-translations weaken the π-stacking of the room-temperature structure and modify the distances and angles of Pt(II)/Pd(II) chains. The curve describing the intermolecular Pt(II)/Pd(II) distances as a function of temperature, validated by X-ray absorption spectroscopy, was found to reproduce the coordinate reaction determined by the model-free analysis.

Three-dimensional structure of human cyclooxygenase (hCOX)-1.

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.

Chiral Separation, X-ray Structure, and Biological Evaluation of a Potent and Reversible Dual Binding Site AChE Inhibitor.

Acetylcholinesterase (AChE) inhibitors (AChEIs) still remain the leading therapeutic options for the symptomatic treatment of cognitive deficits associated with mild-to-moderate Alzheimer's disease. The search for new AChEIs benefits from well-established knowledge of the molecular interactions of selective AChEIs, such as donepezil and related dual binding site inhibitors. Starting from a previously disclosed coumarin-based inhibitor (±)-cis-1, active as racemate in the nanomolar range toward AChE, we proceeded on a double track by (i) achieving chiral resolution of the enantiomers of 1 by HPLC and (ii) preparing two close achiral analogues of 1, i.e., compounds 4 and 6. An eudismic ratio as high as 20 was observed for the (-) enantiomer of cis-1. The X-ray crystal structure of the complex between the (-)-cis-1 eutomer (coded as MC1420) and T. californica AChE was determined at 2.8 Å, and docking calculation results suggested that the eutomer in (1R,3S) absolute configuration should be energetically more favored in binding the enzyme than the eutomer in (1S,3R) configuration. The achiral analogues 4 and 6 were less effective in inhibiting AChE compared to (±)-cis-1, but interestingly butylamide 4 emerged as a potent inhibitor of butyrylcholinesterase (BChE).

Crystal structures of the recombinant ß-factor XIIa protease with bound Thr-Arg and Pro-Arg substrate mimetics.

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 (ßFXIIaHis) 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-ßFXIIaHis). Crystal structures were determined of MBP-ßFXIIaHis in complex with the inhibitor D-Phe-Pro-Arg chloromethyl ketone (PPACK) and of ßFXIIaHis in isolation. The ßFXIIaHis 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 ßFXIIaHis 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 ßFXIIa are also described. These structural studies of ßFXIIa provide insight into substrate and inhibitor recognition and establish a scaffold for the structure-guided drug design of novel antithrombotic and anti-inflammatory agents.

Interaction between the photosynthetic anoxygenic microorganism Rhodobacter sphaeroides and soluble gold compounds. From toxicity to gold nanoparticle synthesis.

Biological processes using microorganisms for nanoparticle synthesis are appealing as eco-friendly nanofactories. The response of the photosynthetic bacterium Rhodobacter sphaeroides to gold exposure and its reducing capability of Au(III) to produce stable gold nanoparticles (AuNPs), using metabolically active bacteria and quiescent biomass, is reported in this study. In the former case, bacterial cells were grown in presence of gold chloride at physiological pH. Gold exposure was found to cause a significant increase of the lag-phase duration at concentrations higher than 10 µM, suggesting the involvement of a resistance mechanism activated by Au(III). Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy/Energy Dispersive X-ray Spectrometry (SEM/EDS) analysis of bacterial cells confirmed the extracellular formation of AuNPs. Further studies were carried out on metabolically quiescent biomass incubated with gold chloride solution. The biosynthesized AuNPs were spherical in shape with an average size of 10 ±â€¯3 nm, as analysed by Transmission Electron Microscopy (TEM). The nanoparticles were hydrophilic and stable against aggregation for several months. In order to identify the functional groups responsible for the reduction and stabilization of nanoparticles, AuNPs were analysed by Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectroscopy, X-ray Photoelectron Spectroscopy (XPS), X-ray Fluorescence Spectrometry (XRF) and X-ray Absorption Spectroscopy (XAS) measurements. The obtained results indicate that gold ions bind to functional groups of cell membrane and are subsequently reduced by reducing sugars to gold nanoparticles and capped by a protein/peptide coat. Gold nanoparticles demonstrated to be efficient homogeneous catalysts in the degradation of nitroaromatic compounds.

Principal component analysis for automatic extraction of solid-state kinetics from combined in situ experiments.

Solid-state reactivity is often studied by in situ experiments with a multi-technique approach, where complementarity of different probes is exploited. In situ data are usually analysed using a complex protocol: first the reaction model most suited to describe the specific solid-state reaction is chosen, second the reaction coordinate is obtained from the data, the order of reaction is then calculated by applying a specific kinetic equation, and finally kinetic parameters are obtained with an Arrhenius plot. The approach is both time consuming and subject to errors due to the arbitrariness of extraction of the reaction coordinate, typically from individual peak intensity variations during the reaction. In addition, application of the different kinetic equations to obtain the best fitting one is tedious and no general method to select the best model with an unbiased approach is available. Here we propose a new procedure based on principal component analysis to get kinetic information from in situ data, which simplifies and speeds up the process of kinetic parameter calculation from a three- to a two- or even a one-step form, reaching a high degree of automation and the ability to manage the huge amount of data produced by in situ multi-technique experiments. The new approach treats data as a whole, without biases introduced by manual methods of obtaining the reaction coordinate by peak intensity evaluation from individual patterns typical of the traditional approach. The procedure is described in its theoretical framework and applied to the formation of a molecular complex, monitored by in situ X-ray powder diffraction and Raman measurements.

PqsL uses reduced flavin to produce 2-hydroxylaminobenzoylacetate, a preferred PqsBC substrate in alkyl quinolone biosynthesis in Pseudomonas aeruginosa.

Alkyl hydroxyquinoline N-oxides (AQNOs) are antibiotic compounds produced by the opportunistic bacterial pathogen Pseudomonas aeruginosa They are products of the alkyl quinolone (AQ) biosynthetic pathway, which also generates the quorum-sensing molecules 2-heptyl-4(1H)-quinolone (HHQ) and 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS). Although the enzymatic synthesis of HHQ and PQS had been elucidated, the route by which AQNOs are synthesized remained elusive. Here, we report on PqsL, the key enzyme for AQNO production, which structurally resembles class A flavoprotein monooxygenases such as p-hydroxybenzoate 3-hydroxylase (pHBH) and 3-hydroxybenzoate 6-hydroxylase. However, we found that unlike related enzymes, PqsL hydroxylates a primary aromatic amine group, and it does not use NAD(P)H as cosubstrate, but unexpectedly required reduced flavin as electron donor. We also observed that PqsL is active toward 2-aminobenzoylacetate (2-ABA), the central intermediate of the AQ pathway, and forms the unstable compound 2-hydroxylaminobenzoylacetate, which was preferred over 2-ABA as substrate of the downstream enzyme PqsBC. In vitro reconstitution of the PqsL/PqsBC reaction was feasible by using the FAD reductase HpaC, and we noted that the AQ:AQNO ratio is increased in an hpaC-deletion mutant of P. aeruginosa PAO1 compared with the ratio in the WT strain. A structural comparison with pHBH, the model enzyme of class A flavoprotein monooxygenases, revealed that structural features associated with NAD(P)H binding are missing in PqsL. Our study completes the AQNO biosynthetic pathway in P. aeruginosa, indicating that PqsL produces the unstable product 2-hydroxylaminobenzoylacetate from 2-ABA and depends on free reduced flavin as electron donor instead of NAD(P)H.

Improved multivariate analysis for fast and selective monitoring of structural dynamics by in situ X-ray powder diffraction.

The development of two solid-state reactions, Xe absorption into MFI and molecular complex formation, where samples are affected by changes of crystal lattice due to temperature or pressure variation was structurally monitored through in situ or in operando X-ray powder diffraction experiments. Consequent variations of the peak positions prevent collective analysis of measured patterns, aiming at investigating structural changes occurring within the crystal cell. Moreover, an intrinsic and variable error in peak position is unavoidable when using the Bragg-Brentano geometry and, in some cases (sticky, bulky, aggregate samples) the sample mounting can increase the error within a dataset. Here we present a general multivariate analysis method to process in a fast and automatic way in situ XRPD data collected on charge transfer complexes and porous materials, with the capacity of disentangling peak shifts from intensity and shape variations in diffraction signals, thus allowing an efficient separation of the contribution of crystal lattice changes from structural changes. The peak shift correction allowed an improved PCA analysis that turned out to be more sensible than the traditional single pattern Rietveld analysis. The developed algorithms allowed, with respect to the traditional approach, the location of two new Xe positions into MFI with a better interpretation of the experimental data, while a much faster and more efficient recovery of the reaction coordinate was achieved in the molecular complex formation reaction.

Enzyme Crystals and Hydrogel Composite Membranes as New Active Food Packaging Material.

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.

Structural Determinants in the Binding of BB2 Receptor Ligands: In Silico, X-Ray and NMR Studies in PD176252 Analogues.

BACKGROUND: The mammalian bombesin receptor family comprises three G proteincoupled receptors: the neuromedin B receptor, the gastrin-releasing peptide receptor (BB2), and the bombesin receptor subtype 3. BB2 receptor plays a role in gastrointestinal functions; however, at present the role of this subtype in physiological and pathological conditions is unknown due to the lack of specific binders for all subclasses of bombesin receptors. RESULTS: Here, we present a study focused on the properties of the peptoid bombesin antagonist called PD176252, and other structural analogues with the aim to elucidate causes of their different affinity towards the BB2 receptor. CONCLUSION: By means of computational techniques, based on QSAR, docking and homology building, supported by experimental data (X-ray diffraction and NMR spectroscopy) fresh insights on binding modes of this class of biological targets were achieved.

Structure of matrix metalloproteinase-3 with a platinum-based inhibitor.

An X-ray investigation has been performed with the aim of characterizing the binding sites of a platinum-based inhibitor (K[PtCl3(DMSO)]) of matrix metalloproteinase-3 (stromelysin-1). The platinum complex targets His224 in the S1' specificity loop, representing the first step in the selective inhibition process (PDB ID code 4JA1).

Computational studies on a new cationic peroxidase isoenzyme from artichoke leaves.

Previously we presented the purification, biochemical characterization, and cloning of a cationic peroxidase isoenzyme (CysPrx) from artichoke (Cynara cardunculus subsp scolymus (L.) Hegi) leaves. The protein was shown to have some interesting properties, suggesting that CysPrx could be a considered as a potential candidate for industrial application. In addition, from the CysPrx sequence, two full-lengh cDNAs: CysPrx1 and CysPrx2, differing for three amino acids, were isolated. A three-dimensional model was predicted from CysPrx1 by homology modeling, using two different computational tools. Herein we discuss the roles of particular amino acid residues and structural motifs or regions of both deduced sequences with the aim to find new understandings between the new plant peroxidase isoenzymes and their physiological substrates. Additionally, the obtained information may lead to new methods for improving the stability of the enzyme in several processes of biotechnological interest for peroxidase applications.