Recent Theoretical Insights into the Oxidative Degradation of Biopolymers and Plastics by Metalloenzymes.

Molecular modeling techniques have become indispensable in many fields of molecular sciences in which the details related to mechanisms and reactivity need to be studied at an atomistic level. This review article provides a collection of computational modeling works on a topic of enormous interest and urgent relevance: the properties of metalloenzymes involved in the degradation and valorization of natural biopolymers and synthetic plastics on the basis of both circular biofuel production and bioremediation strategies. In particular, we will focus on lytic polysaccharide monooxygenase, laccases, and various heme peroxidases involved in the processing of polysaccharides, lignins, rubbers, and some synthetic polymers. Special attention will be dedicated to the interaction between these enzymes and their substrate studied at different levels of theory, starting from classical molecular docking and molecular dynamics techniques up to techniques based on quantum chemistry.

N-Acetylcysteine Antagonizes NGF Activation of TrkA through Disulfide Bridge Interaction, an Effect Which May Contribute to Its Analgesic Activity.

N-acetylcysteine (NAC), a mucolytic agent and an antidote to acetaminophen intoxication, has been studied in experimental conditions and trials exploring its analgesic activity based on its antioxidant and anti-inflammatory properties. The purpose of this study is to investigate additional mechanisms, namely, the inhibition of nerve growth factor (NGF) and the activation of the Tropomyosin receptor kinase A (TrkA) receptor, which is responsible for nociception. In silico studies were conducted to evaluate dithiothreitol and NAC's interaction with TrkA. We also measured the autophosphorylation of TrkA in SH-SY5Y cells via ELISA to assess NAC's in vitro activity against NGF-induced TrkA activation. The in silico and in vitro tests show that NAC interferes with NGF-induced TrkA activation. In particular, NAC breaks the disulfide-bound Cys 300-345 of TrkA, perturbing the NGF-TrkA interaction and producing a rearrangement of the binding site, inducing a consequent loss of their molecular recognition and spatial reorganization, which are necessary for the induction of the autophosphorylation process. The latter was inhibited by 40% using 20 mM NAC. These findings suggest that NAC could have a role as a TrkA antagonist, an action that may contribute to the activity and use of NAC in various pain states (acute, chronic, nociplastic) sustained by NGF hyperactivity and/or accompanied by spinal cord sensitization.

First-Principles Calculations on Ni,Fe-Containing Carbon Monoxide Dehydrogenases Reveal Key Stereoelectronic Features for Binding and Release of CO2 to/from the C-Cluster.

In view of the depletion of fossil fuel reserves and climatic effects of greenhouse gas emissions, Ni,Fe-containing carbon monoxide dehydrogenase (Ni-CODH) enzymes have attracted increasing interest in recent years for their capability to selectively catalyze the reversible reduction of CO2 to CO (CO2 + 2H+ + 2e- ⇌ CO + H2O). The possibility of converting the greenhouse gas CO2 into useful materials that can be used as synthetic building blocks or, remarkably, as carbon fuels makes Ni-CODH a very promising target for reverse-engineering studies. In this context, in order to provide insights into the chemical principles underlying the biological catalysis of CO2 activation and reduction, quantum mechanics calculations have been carried out in the framework of density functional theory (DFT) on different-sized models of the Ni-CODH active site. With the aim of uncovering which stereoelectronic properties of the active site (known as the C-cluster) are crucial for the efficient binding and release of CO2, different coordination modes of CO2 to different forms and redox states of the C-cluster have been investigated. The results obtained from this study highlight the key role of the protein environment in tuning the reactivity and the geometry of the C-cluster. In particular, the protonation state of His93 is found to be crucial for promoting the binding or the dissociation of CO2. The oxidation state of the C-cluster is also shown to be critical. CO2 binds to Cred2 according to a dissociative mechanism (i.e., CO2 binds to the C-cluster after the release of possible ligands from Feu) when His93 is doubly protonated. CO2 can also bind noncatalytically to Cred1 according to an associative mechanism (i.e., CO2 binding is preceded by the binding of H2O to Feu). Conversely, CO2 dissociates when His93 is singly protonated and the C-cluster is oxidized at least to the Cint redox state.

Reactivation of the Ready and Unready Oxidized States of [NiFe]-Hydrogenases: Mechanistic Insights from DFT Calculations.

The apparently simple dihydrogen formation from protons and electrons (2H+ + 2e- ⇄ H2) is one of the most challenging reactions in nature. It is catalyzed by metalloenzymes of amazing complexity, called hydrogenases. A better understanding of the chemistry of these enzymes, especially that of the [NiFe]-hydrogenases subgroup, has important implications for production of H2 as alternative sustainable fuel. In this work, reactivation mechanism of the oxidized and inactive Ni-B and Ni-A states of the [NiFe]-hydrogenases active site has been investigated using density functional theory. Results obtained from this study show that one-electron reduction and protonation of the active site promote the removal of the bridging hydroxide ligand contained in Ni-B and Ni-A. However, this process is sufficient to activate only the Ni-B state. H2 binding to the active site is required to convert Ni-A to the active Ni-SIa state. Here, we also propose a reasonable structure for the spectroscopically well-characterized Ni-SIr and Ni-SU species, formed respectively from the one-electron reduction of Ni-B and Ni-A. Ni-SIr, depending on the pH at which the reaction occurs, features a bridging hydroxide ligand or a water molecule terminally coordinated to the Ni atom, whereas in Ni-SU a water molecule is terminally coordinated to the Fe atom, and the Cys64 residue is oxidized to sulfenate. The sulfenate oxygen atom in the Ni-A state affects the stereoelectronic properties of the binuclear cluster by modifying the coordination geometry of Ni, and consequently, by switching the regiochemistry of H2O and H2 binding from the Ni to the Fe atom. This effect is predicted to be at the origin of the different reactivation kinetics of the oxidized and inactive Ni-B and Ni-A states.

Redox Potentials of Small Inorganic Radicals and Hexa-Aquo Complexes of First-Row Transition Metals in Water: A DFT Study Based on the Grand Canonical Ensemble.

The potentials of redox systems involving nitrogen, oxygen, and metal ions of the first-row transition series have been computed according to the general approach of the grand canonical ensemble, which leads to the equilibrium value of the reduction potential via a (complete) sampling of configuration space at a given temperature. The approach is a single configuration approach in the sense that identical molecular structures are sampled for both the oxidized and reduced species considered in water solution. In this study, the solute and a cluster of 11-12 water molecules are treated explicitly at the same level of theory and embedded in a continuum solvent. The molecular energies are computed in the framework of the density functional theory. Our approach is basically different from the approach based on the ThermoDynamic Cycle involving gas-phase calculations of the electron affinity of the oxidized species, corrected by the differential hydration energy (obtained from continuum solvent models only) between oxidized and reduced forms. The calculated redox potentials are in agreement with the available experimental data much closer than other results so far presented in the literature. Our results are very satisfactory also in the case of the 3+/2+ redox states of the first-row transition metals, i.e., systems with a high positive charge for which enhanced effects of the solvent are expected.

Catalytic Mechanism of Fungal Lytic Polysaccharide Monooxygenases Investigated by First-Principles Calculations.

Lytic polysaccharide monooxygenases (LPMOs) are Cu-containing enzymes that facilitate the degradation of recalcitrant polysaccharides by the oxidative cleavage of glycosidic bonds. They are gaining rapidly increasing attention as key players in biomass conversion, especially for the production of second-generation biofuels. Elucidation of the detailed mechanism of the LPMO reaction is a major step toward the assessment and optimization of LPMO efficacy in industrial biotechnology, paving the way to utilization of sustainable fuel sources. Here, we used density functional theory calculations to study the reaction pathways suggested to date, exploiting a very large active-site model for a fungal AA9 LPMO and using a celloheptaose unit as a substrate mimic. We identify a copper oxyl intermediate as being responsible for H-atom abstraction from the substrate, followed by a rapid, water-assisted hydroxyl rebound, leading to substrate hydroxylation.

Theoretical investigation of aerobic and anaerobic oxidative inactivation of the [NiFe]-hydrogenase active site.

The extraordinary capability of [NiFe]-hydrogenases to catalyse the reversible interconversion of protons and electrons into dihydrogen (H2) has stimulated numerous experimental and theoretical studies addressing the direct utilization of these enzymes in H2 production processes. Unfortunately, the introduction of these natural H2-catalysts in biotechnological applications is limited by their inhibition under oxidising (aerobic and anaerobic) conditions. With the aim of contributing to overcome this limitation, we studied the oxidative inactivation mechanism of [NiFe]-hydrogenases by performing Density Functional Theory (DFT) calculations on a very large model of their active site in which all the amino acids forming the first and second coordination spheres of the NiFe cluster have been explicitly included. We identified an O2 molecule and two H2O molecules as sources of the two oxygen atoms that are inserted at the active site of the inactive forms of the enzyme (Ni-A and Ni-B) under aerobic and anaerobic conditions, respectively. Furthermore, our results support the experimental evidence that the Ni-A-to-Ni-B ratio strongly depends on the number of reducing equivalents available for the process and on the oxidizing conditions under which the reaction takes place.

Excited state properties of diiron dithiolate hydrides: implications in the unsensitized photocatalysis of H2 evolution.

Density functional theory (DFT) and time-dependent DFT (TDDFT) have been used to investigate how visible light photons can excite an asymmetrically substituted diiron hydride, [Fe2(pdt)(µ-H)(CO)4dppv](+) (1(+), dppv = cis-1,2-C2H2(PPh2)2; pdt = 1,3-propanedithiolate), as well as the symmetric species [Fe2(pdt)(µ-H)(CO)4(PMe3)2](+) (2(+)), which are the first photocatalysts of proton reduction operating without employing sensitizers (Wang, W.; Rauchfuss, T. B.; Bertini, L.; Zampella, G.; J. Am. Chem. Soc., 2012, 134, 4525). Theoretical results illustrate that the peculiar reactivity associated to the excited states of 1(+) and 2(+) is compatible with three different scenarios: (i) it can arise from the movement of the hydride ligand from fully bridging to semibridging/terminal coordination, which is expected to be more reactive toward protons; (ii) reactivity could be related to cleavage of a Fe-S bond, which implies formation of a transient Fe penta-coordinate species that would trigger a facile turnstile hydride isomerization, if lifetime excitation is long enough; (iii) also in line with a Fe-S bond cleavage is the possibility that after excited state decay, a highly basic S center is protonated so that a species simultaneously containing S-H(δ+) and Fe-H(δ-) moieties is formed and, once reduced by a suitable electron donor, it can readily afford H2 plus an unprotonated form of the FeFe complex. This last possibility is consistent with (31)P NMR and IR solution data. All the three possibilities are compatible with the capability of 1(+) and 2(+) to perform photocatalysis of hydrogen evolving reaction (HER) without sensitizer. Moreover, even though it turned out difficult to discriminate among the three scenarios, especially because of the lack of experimental excitation lifetimes, it is worth underscoring that all of the three pathways represent a novelty regarding diiron carbonyl photoreactivity, which is usually associated with CO loss. Results provide also a rationale to the experimental observations which showed that the simultaneous presence of donor ligands (dppv in the case of 1(+)) and a H ligand in the coordination environment of diiron complexes is a key factor to prevent CO photodissociation and catalyze HER. Finally, the comparison of photoexcitation behavior of 1(+) and 2(+) allows a sort of generalization about the functioning of such hydride species.

An acidic loop and cognate phosphorylation sites define a molecular switch that modulates ubiquitin charging activity in Cdc34-like enzymes.

E2 ubiquitin-conjugating enzymes are crucial mediators of protein ubiquitination, which strongly influence the ultimate fate of the target substrates. Recently, it has been shown that the activity of several enzymes of the ubiquitination pathway is finely tuned by phosphorylation, an ubiquitous mechanism for cellular regulation, which modulates protein conformation. In this contribution, we provide the first rationale, at the molecular level, of the regulatory mechanism mediated by casein kinase 2 (CK2) phosphorylation of E2 Cdc34-like enzymes. In particular, we identify two co-evolving signature elements in one of the larger families of E2 enzymes: an acidic insertion in ß4α2 loop in the proximity of the catalytic cysteine and two conserved key serine residues within the catalytic domain, which are phosphorylated by CK2. Our investigations, using yeast Cdc34 as a model, through 2.5 µs molecular dynamics simulations and biochemical assays, define these two elements as an important phosphorylation-controlled switch that modulates opening and closing of the catalytic cleft. The mechanism relies on electrostatic repulsions between a conserved serine phosphorylated by CK2 and the acidic residues of the ß4α2 loop, promoting E2 ubiquitin charging activity. Our investigation identifies a new and unexpected pivotal role for the acidic loop, providing the first evidence that this loop is crucial not only for downstream events related to ubiquitin chain assembly, but is also mandatory for the modulation of an upstream crucial step of the ubiquitin pathway: the ubiquitin charging in the E2 catalytic cleft.

Mechanistic and physiological implications of the interplay among iron-sulfur clusters in [FeFe]-hydrogenases. A QM/MM perspective.

Key stereoelectronic properties of Desulfovibrio desulfuricans [FeFe]-hydrogenase (DdH) were investigated by quantum mechanical description of its complete inorganic core, which includes a Fe(6)S(6) active site (the H-cluster), as well as two ancillary Fe(4)S(4) assemblies (the F and F' clusters). The partially oxidized, active-ready form of DdH is able to efficiently bind dihydrogen, thus starting H(2) oxidation catalysis. The calculations allow us to unambiguously assign a mixed Fe(II)Fe(I) state to the catalytic core of the active-ready enzyme and show that H(2) uptake exerts subtle, yet crucial influences on the redox properties of DdH. In fact, H(2) binding can promote electron transfer from the H-cluster to the solvent-exposed F'-cluster, thanks to a 50% decrease of the energy gap between the HOMO (that is localized on the H-cluster) and the LUMO (which is centered on the F'-cluster). Our results also indicate that the binding of the redox partners of DdH in proximity of its F'-cluster can trigger one-electron oxidation of the H(2)-bound enzyme, a process that is expected to have an important role in H(2) activation. Our findings are analyzed not only from a mechanistic perspective, but also in consideration of the physiological role of DdH. In fact, this enzyme is known to be able to catalyze both the oxidation and the evolution of H(2), depending on the cellular metabolic requirements. Hints for the design of targeted mutations that could lead to the enhancement of the oxidizing properties of DdH are proposed and discussed.

Isocyanide in biochemistry? A theoretical investigation of the electronic effects and energetics of cyanide ligand protonation in [FeFe]-hydrogenases.

The presence of Fe-bound cyanide ligands in the active site of the proton-reducing enzymes [FeFe]-hydrogenases has led to the hypothesis that such Brønsted-Lowry bases could be protonated during the catalytic cycle, thus implying that hydrogen isocyanide (HNC) might have a relevant role in such crucial microbial metabolic paths. We present a hybrid quantum mechanical/molecular mechanical (QM/MM) study of the energetics of CN(-) protonation in the enzyme, and of the effects that cyanide protonation can have on [FeFe]-hydrogenase active sites. A detailed analysis of the electronic properties of the models and of the energy profile associated with H(2) evolution clearly shows that such protonation is dysfunctional for the catalytic process. However, the inclusion of the protein matrix surrounding the active site in our QM/MM models allowed us to demonstrate that the amino acid environment was finely selected through evolution, specifically to lower the Brønsted-Lowry basicity of the cyanide ligands. In fact, the conserved hydrogen-bonding network formed by these ligands and the neighboring amino acid residues is able to impede CN(-) protonation, as shown by the fact that the isocyanide forms of [FeFe]-hydrogenases do not correspond to stationary points on the enzyme QM/MM potential-energy surface.

Probing the effects of one-electron reduction and protonation on the electronic properties of the Fe-S clusters in the active-ready form of [FeFe]-hydrogenases. A QM/MM investigation.

A QM/MM investigation of the active-ready (H(ox)) form of [FeFe]-hydrogenase from D. desulfuricans, in which the electronic properties of all Fe-S clusters (H, F and F') have been simultaneously described using DFT, was carried out with the aim of disclosing a possible interplay between the H-cluster and the accessory iron-sulfur clusters in the initial steps of the catalytic process leading to H(2) formation. It turned out that one-electron addition to the active-ready form leads to reduction of the F'-cluster and not of the H-cluster. Protonation of the H-cluster in H(ox) is unlikely, and in any case it would not trigger electron transfer from the accessory Fe(4)S(4) clusters to the active site. Instead, one-electron reduction and protonation of the active-ready form trigger electron transfer within the protein, a key event in the catalytic cycle. In particular, protonation of the H-cluster after one-electron reduction of the enzyme lowers the energy of the lowest unoccupied molecular orbitals localized on the H-cluster to such an extent that a long-range electron transfer from the F'-cluster towards the H-cluster itself is allowed.

NGF and heart: Is there a role in heart disease?

The review emphasizes the role of NGF, the most representative member of the neurotrophins family, in cardiac physiopathology with a particular focus on healing and sprouting processes occurring after tissue damage. Cardiac and circulating NGF levels dramatically increase following myocardial injury (MI). A very early rise of this neurotrophin is indeed observed soon after MI (hours). Such a rise may lead to sympathetic nerve sprouting which may underlie the later genesis of arrhythmias but may also favor the healing process. At later times (months after), when heart failure develops, the opposite is detected and NGF tissue levels are below the normal range, an event that may in turn participate to defective innervation and cardiac failure. Through a careful analysis of preclinical and clinical studies, this review proposes that time is the key variable when studying these opposite changes in NGF expression observed following MI and attempting to interpret and correlate them with cardiac physiopathology. The examination of the results leads to the speculation that NGF modulation may be a pharmacological target for interventions in specific stages of heart dysfunction following MI.

Functionally relevant interplay between the Fe(4)S(4) cluster and CN(-) ligands in the active site of [FeFe]-hydrogenases.

[FeFe]-hydrogenases are highly efficient H(2)-evolving metalloenzymes that include cyanides and carbonyls in the active site. The latter is an Fe(6)S(6) cluster (the so-called H-cluster) that can be subdivided into a binuclear portion carrying the CO and CN(-) groups and a tetranuclear subcluster. The fundamental role of cyanide ligands in increasing the basicity of the H-cluster has been highlighted previously. Here a more subtle but crucial role played by the two CN(-) ligands in the active site of [FeFe]-hydrogenases is disclosed. In fact, QM/MM calculations on all-atom models of the enzyme from Desulfovibrio desulfuricans show that the cyanide groups fine-tune the electronic and redox properties of the active site, affecting both the protonation regiochemistry and electron transfer between the two subclusters of the H-cluster. Despite the crucial role of cyanides in the protein active site, the currently available bioinspired electrocatalysts generally lack CN(-) groups in order to avoid competition between the latter and the catalytic metal centers for proton binding. In this respect, we show that a targeted inclusion of phosphine ligands in hexanuclear biomimetic clusters may restore the electronic and redox features of the wild-type H-cluster.

Forced unbinding of GPR17 ligands from wild type and R255I mutant receptor models through a computational approach.

BACKGROUND: GPR17 is a hybrid G-protein-coupled receptor (GPCR) activated by two unrelated ligand families, extracellular nucleotides and cysteinyl-leukotrienes (cysteinyl-LTs), and involved in brain damage and repair. Its exploitment as a target for novel neuro-reparative strategies depends on the elucidation of the molecular determinants driving binding of purinergic and leukotrienic ligands. Here, we applied docking and molecular dynamics simulations (MD) to analyse the binding and the forced unbinding of two GPR17 ligands (the endogenous purinergic agonist UDP and the leukotriene receptor antagonist pranlukast from both the wild-type (WT) receptor and a mutant model, where a basic residue hypothesized to be crucial for nucleotide binding had been mutated (R255I) to Ile. RESULTS: MD suggested that GPR17 nucleotide binding pocket is enclosed between the helical bundle and extracellular loop (EL) 2. The driving interaction involves R255 and the UDP phosphate moiety. To support this hypothesis, steered MD experiments showed that the energy required to unbind UDP is higher for the WT receptor than for R255I. Three potential binding sites for pranlukast where instead found and analysed. In one of its preferential docking conformations, pranlukast tetrazole group is close to R255 and phenyl rings are placed into a subpocket highly conserved among GPCRs. Pulling forces developed to break polar and aromatic interactions of pranlukast were comparable. No differences between the WT receptor and the R255I receptor were found for the unbinding of pranlukast. CONCLUSIONS: These data thus suggest that, in contrast to which has been hypothesized for nucleotides, the lack of the R255 residue doesn't affect the binding of pranlukast a crucial role for R255 in binding of nucleotides to GPR17. Aromatic interactions are instead likely to play a predominant role in the recognition of pranlukast, suggesting that two different binding subsites are present on GPR17.

Unveiling how stereoelectronic factors affect kinetics and thermodynamics of protonation regiochemistry in [FeFe] hydrogenase synthetic models: a DFT investigation.

The DFT investigation of protonation regiochemistry for a series of [Fe(I)](2)(edt)(PR)(x)(CO)(6-x)] complexes differing for steric and electronic properties of ligands has allowed the disclosure of several key relations between the structure of the complexes and reactivity toward acids, from both a thermodynamics and kinetics perspective. The phosphine/CO ratio strongly affects both the thermodynamics and kinetics of protonation. In particular, with the exception of dppv complexes, in which steric factors become more important, the presence of phosphines, which are better electron donors than CO ligands, leads to lower reaction barriers. The presence of bulky phosphine ligands, which severely hinder the accessibility to the Fe-Fe bond, is a crucial factor responsible for kinetic preference of terminal- versus mu-protonation in symmetric complexes. The investigation of asymmetric models allowed us to rationalize why protonation takes place preferentially on the less electron-rich iron atom, i. e., the iron atom coordinated by the largest number of CO ligands. Importantly, the presence of at least one electron-donor ligand on the protonating Fe atom is fundamental to allow facile terminal protonation, suggesting that one of the reasons for the presence of CN(-) ligands in the enzyme might be related to the facile formation of catalytically relevant terminally protonated species. Finally, it was found that poorly reacting mu-H Fe(II)Fe(II) species are always thermodynamically more stable than corresponding terminal-hydride forms, indicating that one of the main challenges for the development of efficient synthetic catalysts inspired to the [FeFe] hydrogenase active site will be the design of complexes that undergo terminal protonation but cannot interconvert to the corresponding mu-H forms.

DFT/TDDFT exploration of the potential energy surfaces of the ground state and excited states of Fe2(S2C3H6)(CO)6: a simple functional model of the [FeFe] hydrogenase active site.

Fe(2)(S(2)C(3)H(6))(CO)(6) (a) is a simple model of the [FeFe] hydrogenase catalytic site. The topology of the potential energy surface (PES) of this complex, of its cationic and anionic species (a(+) and a(-)), and of its lowest triplet state was studied using density functional theory (DFT) with BP86 and B3LYP functionals, while selected low- and high-lying singlet excited states were studied with the time-dependent density functional theory (TDDFT). The global minima of a and a(-) PESs are characterized by an all-terminal CO ligand arrangement, while the two rotated forms are transition states (TS). On the contrary, for the a(+) and lowest triplet state PES, the three forms considered are local minima, and the syn rotated form is the global minimum. The relative stability of the rotated forms and the all-terminal CO form on the a, a(+), and a(-) PESs is discussed in light of the Quantum Theory of Atoms in Molecules (QTAIM) analysis of the electron density. By comparing the Fe-Fe bond features of the three forms for each PES, we found that the global minimum structure is characterized by the shortest Fe-Fe bond distance and highest electron density at the Fe-Fe critical point. This approach gave evidence that in the a rotated forms, the weak Fe-C(mu) interaction between the Fe atom of the unrotated Fe(CO)(3) and the C atom of the semibridged CO is formed to the detriment of the Fe-Fe bond interaction. These results suggest that the stabilization of the rotated forms on the cationic PES might be due to the formation of the weak Fe-C(mu) interaction minimizing the weakening of the Fe-Fe bond. The low-lying and lowest triplet excited-state PES investigated are characterized by the stabilization of the rotated forms over the all-terminal CO ligand arrangement. On the first singlet 1(1)A'' excited-state PES, an Fe(CO)(3) semirotated structure is the lowest-energy stationary point, while the exploration of the 1(1)A' and 2(1)A'' singlet excited PESs evidences the stabilization of the rotated over the all-terminal CO forms. Singlet excited-state optimized geometry results are compared with excited-state nuclear distortions recently obtained from resonance Raman excitation profiles. Finally, the results of the exploration of the 6(1)A' and 9(1)A' high-lying excited PESs are discussed in light of the recent ultraviolet photolysis experiments on a.

Influence of the [2Fe]H subcluster environment on the properties of key intermediates in the catalytic cycle of [FeFe] hydrogenases: hints for the rational design of synthetic catalysts.

Nature's recipe: A theoretical study analyzes how the environment of the [FeFe] hydrogenase's catalytic cofactor affects its chemical properties, particularly the relative stability of complexes with bridging and terminal hydride ligands (see picture; Fe teal, S yellow, C green, N blue, O red, H gray). The results help to elucidate key rules for the design of bioinspired synthetic catalysts for H(2) production.

GPR17: molecular modeling and dynamics studies of the 3-D structure and purinergic ligand binding features in comparison with P2Y receptors.

BACKGROUND: GPR17 is a G-protein-coupled receptor located at intermediate phylogenetic position between two distinct receptor families: the P2Y and CysLT receptors for extracellular nucleotides and cysteinyl-LTs, respectively. We previously showed that GPR17 can indeed respond to both classes of endogenous ligands and to synthetic compounds active at the above receptor families, thus representing the first fully characterized non-peptide "hybrid" GPCR. In a rat brain focal ischemia model, the selective in vivo knock down of GPR17 by anti-sense technology or P2Y/CysLT antagonists reduced progression of ischemic damage, thus highlighting GPR17 as a novel therapeutic target for stroke. Elucidation of the structure of GPR17 and of ligand binding mechanisms are the necessary steps to obtain selective and potent drugs for this new potential target. On this basis, a 3-D molecular model of GPR17 embedded in a solvated phospholipid bilayer and refined by molecular dynamics simulations has been the first aim of this study. To explore the binding mode of the "purinergic" component of the receptor, the endogenous agonist UDP and two P2Y receptor antagonists demonstrated to be active on GPR17 (MRS2179 and cangrelor) were then modeled on the receptor. RESULTS: Molecular dynamics simulations suggest that GPR17 nucleotide binding pocket is similar to that described for the other P2Y receptors, although only one of the three basic residues that have been typically involved in ligand recognition is conserved (Arg255). The binding pocket is enclosed between the helical bundle and covered at the top by EL2. Driving interactions are H-bonds and salt bridges between the 6.55 and 6.52 residues and the phosphate moieties of the ligands. An "accessory" binding site in a region formed by the EL2, EL3 and the Nt was also found. CONCLUSION: Nucleotide binding to GPR17 occurs on the same receptor regions identified for already known P2Y receptors. Agonist/antagonist binding mode are similar, but not identical. An accessory external binding site could guide small ligands to the deeper principal binding site in a multi-step mechanism of activation. The nucleotide binding pocket appears to be unable to allocate the leukotrienic type ligands in the same effective way.

Validation of protein models by a neural network approach.

BACKGROUND: The development and improvement of reliable computational methods designed to evaluate the quality of protein models is relevant in the context of protein structure refinement, which has been recently identified as one of the bottlenecks limiting the quality and usefulness of protein structure prediction. RESULTS: In this contribution, we present a computational method (Artificial Intelligence Decoys Evaluator: AIDE) which is able to consistently discriminate between correct and incorrect protein models. In particular, the method is based on neural networks that use as input 15 structural parameters, which include energy, solvent accessible surface, hydrophobic contacts and secondary structure content. The results obtained with AIDE on a set of decoy structures were evaluated using statistical indicators such as Pearson correlation coefficients, Znat, fraction enrichment, as well as ROC plots. It turned out that AIDE performances are comparable and often complementary to available state-of-the-art learning-based methods. CONCLUSION: In light of the results obtained with AIDE, as well as its comparison with available learning-based methods, it can be concluded that AIDE can be successfully used to evaluate the quality of protein structures. The use of AIDE in combination with other evaluation tools is expected to further enhance protein refinement efforts.