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A New FeMo Complex as a Model of Heterobimetallic Assemblies in Natural Systems: Mössbauer and Density Functional Theory Investigations.
Inorg Chem
PUBLISHED: 10-20-2014
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The design of the new FeMo heterobimetallic species [FeMo(CO)5(?(2)-dppe)(?-pdt)] is reported. Mössbauer spectroscopy and density functional theory calculations give deep insight into the electronic and structural properties of this compound.
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Evolutionary analysis identifies an MX2 haplotype associated with natural resistance to HIV-1 infection.
Mol. Biol. Evol.
PUBLISHED: 06-14-2014
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The protein product of the myxovirus resistance 2 (MX2) gene restricts HIV-1 and simian retroviruses. We demonstrate that MX2 evolved adaptively in mammals with distinct sites representing selection targets in distinct branches; selection mainly involved residues in loop 4, previously shown to carry antiviral determinants. Modeling data indicated that positively selected sites form a continuous surface on loop 4, which folds into two antiparallel ?-helices protruding from the stalk domain. A population genetics-phylogenetics approach indicated that the coding region of MX2 mainly evolved under negative selection in the human lineage. Nonetheless, population genetic analyses demonstrated that natural selection operated on MX2 during the recent history of human populations: distinct selective events drove the frequency increase of two haplotypes in the populations of Asian and European ancestry. The Asian haplotype carries a susceptibility allele for melanoma; the European haplotype is tagged by rs2074560, an intronic variant. Analyses performed on three independent European cohorts of HIV-1-exposed seronegative individuals with different geographic origin and distinct exposure route showed that the ancestral (G) allele of rs2074560 protects from HIV-1 infection with a recessive effect (combined P = 1.55 × 10(-4)). The same allele is associated with lower in vitro HIV-1 replication and increases MX2 expression levels in response to IFN-?. Data herein exploit evolutionary information to identify a novel host determinant of HIV-1 infection susceptibility.
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Structural investigation of the cold-adapted acylaminoacyl peptidase from Sporosarcina psychrophila by atomistic simulations and biophysical methods.
Biochim. Biophys. Acta
PUBLISHED: 04-29-2014
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Protein structure and dynamics are crucial for protein function. Thus, the study of conformational properties can be very informative for characterizing new proteins and to rationalize how residue substitutions at specific protein sites affect its dynamics, activity and thermal stability. Here, we investigate the structure and dynamics of the recently isolated cold-adapted acylaminoacyl peptidase from Sporosarcina psychrophila (SpAAP) by the integration of simulations, circular dichroism, mass spectrometry and other experimental data. Our study notes traits of cold-adaptation, such as lysine-to-arginine substitutions and a lack of disulphide bridges. Cold-adapted enzymes are generally characterized by a higher number of glycine residues with respect to their warm-adapted counterparts. Conversely, the SpAAP glycine content is lower than that in the warm-adapted variants. Nevertheless, glycine residues are strategically located in proximity to the functional sites in SpAAP, such as the active site and the linker between the two domains.. In particular, G457 reduces the steric hindrance around the nucleophile elbow. Our results suggest a local weakening of the intramolecular interactions in the cold-adapted enzyme. This study offers a basis for the experimental mutagenesis of SpAAP and related enzymes. The approaches employed in this study may also provide a more general framework to characterize new protein structures in the absence of X-ray or NMR data.
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Quantum mechanical methods for the investigation of metalloproteins and related bioinorganic compounds.
Methods Mol. Biol.
PUBLISHED: 03-19-2014
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It is well known that transition metal ions are often bound to proteins, conveying very specific functional properties. In fact, metalloproteins play crucial biological roles in the transport and activation of small molecules such as H2, O2, and N2, as well as in several other biochemical processes. However, even if the presence of transition metals in the active site of proteins allows a very rich biochemistry, the experimental disclosure of structure-activity relationships in metalloproteins is generally difficult exactly because of the presence of transition metals, which are intrinsically characterized by a very versatile and often elusive chemistry. For this reason, computational methods are becoming very popular tools in the characterization of metalloproteins. In particular, since computing power is becoming less and less expensive, due to the continuous technological development of CPUs, the computational tools suited to investigate metalloproteins are becoming more accessible and therefore more commonly used also in molecular biology and biochemistry laboratories. Here, we present the main procedures and computational methods based on quantum mechanics, which are commonly used to study the structural, electronic, and reactivity properties of metalloproteins and related bioinspired compounds, with a specific focus on the practical and technical aspects that must be generally tackled to properly study such biomolecular systems.
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An evolutionary analysis of antigen processing and presentation across different timescales reveals pervasive selection.
PLoS Genet.
PUBLISHED: 03-01-2014
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The antigenic repertoire presented by MHC molecules is generated by the antigen processing and presentation (APP) pathway. We analyzed the evolutionary history of 45 genes involved in APP at the inter- and intra-species level. Results showed that 11 genes evolved adaptively in mammals. Several positively selected sites involve positions of fundamental importance to the protein function (e.g. the TAP1 peptide-binding domains, the sugar binding interface of langerin, and the CD1D trafficking signal region). In CYBB, all selected sites cluster in two loops protruding into the endosomal lumen; analysis of missense mutations responsible for chronic granulomatous disease (CGD) showed the action of different selective forces on the very same gene region, as most CGD substitutions involve aminoacid positions that are conserved in all mammals. As for ERAP2, different computational methods indicated that positive selection has driven the recurrent appearance of protein-destabilizing variants during mammalian evolution. Application of a population-genetics phylogenetics approach showed that purifying selection represented a major force acting on some APP components (e.g. immunoproteasome subunits and chaperones) and allowed identification of positive selection events in the human lineage. We also investigated the evolutionary history of APP genes in human populations by developing a new approach that uses several different tests to identify the selection target, and that integrates low-coverage whole-genome sequencing data with Sanger sequencing. This analysis revealed that 9 APP genes underwent local adaptation in human populations. Most positive selection targets are located within noncoding regions with regulatory function in myeloid cells or act as expression quantitative trait loci. Conversely, balancing selection targeted nonsynonymous variants in TAP1 and CD207 (langerin). Finally, we suggest that selected variants in PSMB10 and CD207 contribute to human phenotypes. Thus, we used evolutionary information to generate experimentally-testable hypotheses and to provide a list of sites to prioritize in follow-up analyses.
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The oxidative inactivation of FeFe hydrogenase reveals the flexibility of the H-cluster.
Nat Chem
PUBLISHED: 02-11-2014
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Nature is a valuable source of inspiration in the design of catalysts, and various approaches are used to elucidate the mechanism of hydrogenases, the enzymes that oxidize or produce H2. In FeFe hydrogenases, H2 oxidation occurs at the H-cluster, and catalysis involves H2 binding on the vacant coordination site of an iron centre. Here, we show that the reversible oxidative inactivation of this enzyme results from the binding of H2 to coordination positions that are normally blocked by intrinsic CO ligands. This flexibility of the coordination sphere around the reactive iron centre confers on the enzyme the ability to avoid harmful reactions under oxidizing conditions, including exposure to O2. The versatile chemistry of the diiron cluster in the natural system might inspire the design of novel synthetic catalysts for H2 oxidation.
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Disclosure of key stereoelectronic factors for efficient H2 binding and cleavage in the active site of [NiFe]-hydrogenases.
J. Am. Chem. Soc.
PUBLISHED: 01-24-2014
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A comparative analysis of a series of DFT models of [NiFe]-hydrogenases, ranging from minimal NiFe clusters to very large systems including both the first and second coordination sphere of the bimetallic cofactor, was carried out with the aim of unraveling which stereoelectronic properties of the active site of [NiFe]-hydrogenases are crucial for efficient H2 binding and cleavage. H2 binding to the Ni-SIa redox state is energetically favored (by 4.0 kcal mol(-1)) only when H2 binds to Ni, the NiFe metal cluster is in a low spin state, and the Ni cysteine ligands have a peculiar seesaw coordination geometry, which in the enzyme is stabilized by the protein environment. The influence of the Ni coordination geometry on the H2 binding affinity was then quantitatively evaluated and rationalized analyzing frontier molecular orbitals and populations. Several plausible reaction pathways leading to H2 cleavage were also studied. It turned out that a two-step pathway, where H2 cleavage takes place on the Ni-SIa redox state of the enzyme, is characterized by very low reaction barriers and favorable reaction energies. More importantly, the seesaw coordination geometry of Ni was found to be a key feature for facile H2 cleavage. The discovery of the crucial influence of the Ni coordination geometry on H2 binding and activation in the active site of [NiFe]-hydrogenases could be exploited in the design of novel biomimetic synthetic catalysts.
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Inhibitors of the Cdc34 acidic loop: A computational investigation integrating molecular dynamics, virtual screening and docking approaches.
FEBS Open Bio
PUBLISHED: 01-01-2014
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Among the different classes of enzymes involved in the ubiquitin pathway, E2 ubiquitin-conjugating enzymes occupy a central role in the ubiquitination cascade. Cdc34-like E2 enzymes are characterized by a 12-14 residue insertion in the proximity of the catalytic site, known as the acidic loop. Cdc34 ubiquitin-charging activity is regulated by CK2-dependent phosphorylation and the regulatory mechanism involves the acidic loop. Indeed, the phosphorylation stabilizes the loop in an open conformation that is competent for ubiquitin charging. Cdc34 is associated with a variety of diseases, such as hepatocellular carcinomas and prostatic adenocarcinomas. In light of its role, the discovery of potential inhibitory compounds would provide the mean to effectively modulate its activity. Here, we carried out a computational study based on molecular dynamics, virtual screening and docking to identify potential inhibitory compounds of Cdc34, modulating the acidic loop conformation. The molecules identified in this study have been designed to act as molecular hinges that can bind the acidic loop in its closed conformation, thus inhibiting the Cdc34-mediated ubiquitination cascade at the ubiquitin-charging step. In particular, we proposed a pharmacophore model featuring two amino groups in the central part of the model and two lateral aromatic chains, which respectively establish electrostatic interactions with the acidic loop (Asp 108 and Glu 109) and a hydrogen bond with Ser 139, which is one of the key residues for Cdc34 activity.
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Bromoperoxidase activity of amavadin dissected: a DFT investigation.
Chem. Commun. (Camb.)
PUBLISHED: 11-14-2013
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Bromoperoxidase catalytic activity exerted by oxidated amavadin [V(HIDPA)2](-) (HIDPA = 2,2-(hydroxyimino) dipropionate) in mono- and bis-protonated forms has been investigated by DFT. Possible reaction pathways for formation of peroxido/hydroperoxido complexes and subsequent bromide oxidation have been systematically dissected. The effect of increasing [H(+)] on catalytically active species and on halogenide oxidation has been also studied. Similarly to vanadium haloperoxidase (VHPO), the results point to a hydroperoxido amavadin adduct as the most reactive species toward bromide oxidation. However, comparison of the reactivity of amavadin and VHPO reveals also crucial differences in the catalytic mechanism of such natural V complexes.
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Crystallographic Characterization of a Fully Rotated, Basic Diiron Dithiolate: Model for the Hred State?
Chemistry
PUBLISHED: 08-27-2013
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A lucky break: A combination of steric pressure and electron asymmetry has provided the first example of a diiron dithiolate that is both rotated and basic. The present work establishes the feasibility of a hydride free rotated structure for Hred state of the enzyme.
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New Fe(I) -Fe(I) Complex Featuring a Rotated Conformation Related to [2?Fe]H Subsite of the [Fe-Fe] Hydrogenase.
Chemistry
PUBLISHED: 08-23-2013
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Rotated geometry: The first example of a dinuclear iron(I)-iron(I) complex featuring a fully rotated geometry related to the active site of [Fe-Fe] hydrogenase is reported.
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Excited state properties of diiron dithiolate hydrides: implications in the unsensitized photocatalysis of H2 evolution.
Inorg Chem
PUBLISHED: 08-16-2013
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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)(?-H)(CO)4dppv](+) (1(+), dppv = cis-1,2-C2H2(PPh2)2; pdt = 1,3-propanedithiolate), as well as the symmetric species [Fe2(pdt)(?-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(?+) and Fe-H(?-) 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.
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RIG-I-Like Receptors Evolved Adaptively in Mammals, with Parallel Evolution at LGP2 and RIG-I.
J. Mol. Biol.
PUBLISHED: 07-16-2013
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RIG-I-like receptors (RLRs) are nucleic acid sensors that activate antiviral innate immune response. These molecules recognize diverse non-self RNA substrates and are antagonized by several viral inhibitors. We performed an evolutionary analysis of RLR genes (RIG-I, MDA5, and LGP2) in mammals. Results indicated that purifying selection had a dominant role in driving the evolution of RLRs. However, application of maximum-likelihood analyses identified several positions that evolved adaptively. Positively selected sites are located in all domains of MDA5 and RIG-I, whereas in LGP2 they are confined to the helicase domain. In both MDA5 and RIG-I, the linkers separating the caspase activation and recruitment domain and the helicase domain represented preferential targets of positive selection. Independent selective events in RIG-I and LGP2 targeted the corresponding site (Asp421 and Asp179, respectively) within a protruding ?-helix that grips the V-shaped structure formed by the pincer. Most of the positively selected sites in MDA5 are in regions unique to this RLR, including a characteristic insertion within the helicase domain. Additional selected sites are located at the contact interface between MDA5 monomers, in spatial proximity to a positively selected human polymorphism (Arg843His) and immediately external to the parainfluenza virus 5 V protein binding region. Structural analyses suggested that the positively selected His834 residue is involved in parainfluenza virus 5 V protein binding. Data herein suggest that RLRs have been engaged in host-virus genetic conflict leading to diversifying selection and indicate parallel evolution at the same site in RIG-I and LGP2, a position likely to be of central importance in antiviral responses.
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Uncovering a dynamically formed substrate access tunnel in carbon monoxide dehydrogenase/acetyl-CoA synthase.
J. Am. Chem. Soc.
PUBLISHED: 06-13-2013
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The transport of small ligands to active sites of proteins is the basis of vital processes in biology such as enzymatic catalysis and cell signaling, but also of more destructive ones including enzyme inhibition and oxidative damage. Here, we show how a diffusion-reaction model solved by means of molecular dynamics and density functional theory calculations provides novel insight into the transport of small ligands in proteins. In particular, we unravel the existence of an elusive, dynamically formed gas channel, which CO2 takes to diffuse from the solvent to the active site (C-cluster) of the bifunctional multisubunit enzyme complex carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS). Two cavities forming this channel are temporarily created by protein fluctuations and are not apparent in the X-ray structures. The ligand transport is controlled by two residues at the end of this tunnel, His113 and His116, and occurs on the same time scale on which chemical binding to the active site takes place (0.1-1 ms), resulting in an overall binding rate on the second time scale. We find that upon reduction of CO2 to CO, the newly formed Fe-hydroxy ligand greatly strengthens the hydrogen-bond network, preventing CO from exiting the protein through the same way that CO2 takes to enter the protein. This is the basis for directional transport of CO from the production site (C-cluster of CODH subunit) to the utilization site (A-cluster of ACS subunit). In view of these results, a general picture emerges of how large proteins guide small ligands toward their active sites.
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A 175 million year history of T cell regulatory molecules reveals widespread selection, with adaptive evolution of disease alleles.
Immunity
PUBLISHED: 04-23-2013
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T cell activation plays a central role in immune response and in the maintenance of self-tolerance. We analyzed the evolutionary history of T cell regulatory molecules. Nine genes involved in triggering T cell activation or in regulating the ensuing response evolved adaptively in mammals. Several positively selected sites overlap with positions interacting with the binding partner or with cellular components. Population genetic analysis in humans revealed a complex scenario of local (FASLG, CD40LG, HAVCR2) and worldwide (FAS, ICOSLG) adaptation and H. sapiens-to-Neandertal gene flow (gene transfer between populations). Disease variants in these genes are preferential targets of pathogen-driven selection, and a Crohns disease risk polymorphism targeted by bacterial-driven selection modulates the expression of ICOSLG in response to a bacterial superantigen. Therefore, we used evolutionary information to generate experimentally testable hypotheses concerning the function of specific genetic variants and indicate that adaptation to infection underlies the maintenance of autoimmune risk alleles.
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Does the environment around the H-cluster allow coordination of the pendant amine to the catalytic iron center in [FeFe] hydrogenases? Answers from theory.
J. Biol. Inorg. Chem.
PUBLISHED: 04-17-2013
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[FeFe] hydrogenases are H2-evolving enzymes that feature a diiron cluster in their active site (the [2Fe]H cluster). One of the iron atoms has a vacant coordination site that directly interacts with H2, thus favoring its splitting in cooperation with the secondary amine group of a neighboring, flexible azadithiolate ligand. The vacant site is also the primary target of the inhibitor O2. The [2Fe]H cluster can span various redox states. The active-ready form (Hox) attains the Fe(II)Fe(I) state. States more oxidized than Hox were shown to be inactive and/or resistant to O2. In this work, we used density functional theory to evaluate whether azadithiolate-to-iron coordination is involved in oxidative inhibition and protection against O2, a hypothesis supported by recent results on biomimetic compounds. Our study shows that Fe-N(azadithiolate) bond formation is favored for an Fe(II)Fe(II) active-site model which disregards explicit treatment of the surrounding protein matrix, in line with the case of the corresponding Fe(II)Fe(II) synthetic system. However, the study of density functional theory models with explicit inclusion of the amino acid environment around the [2Fe]H cluster indicates that the protein matrix prevents the formation of such a bond. Our results suggest that mechanisms other than the binding of the azadithiolate nitrogen protect the active site from oxygen in the so-called H ox (inact) state.
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A conserved loop in polynucleotide phosphorylase (PNPase) essential for both RNA and ADP/phosphate binding.
Biochimie
PUBLISHED: 01-21-2013
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Polynucleotide phosphorylase (PNPase) reversibly catalyzes RNA phosphorolysis and polymerization of nucleoside diphosphates. Its homotrimeric structure forms a central channel where RNA is accommodated. Each protomer core is formed by two paralogous RNase PH domains: PNPase1, whose function is largely unknown, hosts a conserved FFRR loop interacting with RNA, whereas PNPase2 bears the putative catalytic site, ?20 ? away from the FFRR loop. To date, little is known regarding PNPase catalytic mechanism. We analyzed the kinetic properties of two Escherichia coli PNPase mutants in the FFRR loop (R79A and R80A), which exhibited a dramatic increase in Km for ADP/Pi binding, but not for poly(A), suggesting that the two residues may be essential for binding ADP and Pi. However, both mutants were severely impaired in shifting RNA electrophoretic mobility, implying that the two arginines contribute also to RNA binding. Additional interactions between RNA and other PNPase domains (such as KH and S1) may preserve the enzymatic activity in R79A and R80A mutants. Inspection of enzyme structure showed that PNPase has evolved a long-range acting hydrogen bonding network that connects the FFRR loop with the catalytic site via the F380 residue. This hypothesis was supported by mutation analysis. Phylogenetic analysis of PNPase domains and RNase PH suggests that such network is a unique feature of PNPase1 domain, which coevolved with the paralogous PNPase2 domain.
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Reciprocal influence of protein domains in the cold-adapted acyl aminoacyl peptidase from Sporosarcina psychrophila.
PLoS ONE
PUBLISHED: 01-07-2013
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Acyl aminoacyl peptidases are two-domain proteins composed by a C-terminal catalytic ?/?-hydrolase domain and by an N-terminal ?-propeller domain connected through a structural element that is at the N-terminus in sequence but participates in the 3D structure of the C-domain. We investigated about the structural and functional interplay between the two domains and the bridge structure (in this case a single helix named ?1-helix) in the cold-adapted enzyme from Sporosarcina psychrophila (SpAAP) using both protein variants in which entire domains were deleted and proteins carrying substitutions in the ?1-helix. We found that in this enzyme the inter-domain connection dramatically affects the stability of both the whole enzyme and the ?-propeller. The ?1-helix is required for the stability of the intact protein, as in other enzymes of the same family; however in this psychrophilic enzyme only, it destabilizes the isolated ?-propeller. A single charged residue (E10) in the ?1-helix plays a major role for the stability of the whole structure. Overall, a strict interaction of the SpAAP domains seems to be mandatory for the preservation of their reciprocal structural integrity and may witness their co-evolution.
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Oxidatively induced reactivity of [Fe2(CO)4(?2-dppe)(?-pdt)]: an electrochemical and theoretical study of the structure change and ligand binding processes.
Inorg Chem
PUBLISHED: 11-22-2011
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The one-electron oxidation of the diiron complex [Fe(2)(CO)(4)(?(2)-dppe)(?-pdt)] (1) (dppe = Ph(2)PCH(2)CH(2)PPh(2); pdt = S(CH(2))(3)S) has been investigated in the absence and in the presence of P(OMe)(3), by both electrochemical and theoretical methods, to shed light on the mechanism and the location of the oxidatively induced structure change. While cyclic voltammetric experiments did not allow to discriminate between a two-step (EC) and a concerted, quasi-reversible (QR) process, density functional theory (DFT) calculations favor the first option. When P(OMe)(3) is present, the one-electron oxidation produces singly and doubly substituted cations, [Fe(2)(CO)(4-n){P(OMe)(3)}(n)(?(2)-dppe)(?-pdt)](+) (n = 1: 2(+); n = 2: 3(+)) following mechanisms that were investigated in detail by DFT. Although the most stable isomer of 1(+) and 2(+) (and 3(+)) show a rotated Fe(dppe) center, binding of P(OMe)(3) occurs at the neighboring iron center of both 1(+) and 2(+). The neutral compound 3 was obtained by controlled-potential reduction of the corresponding cation, while 2 was quantitatively produced by reaction of 3 with CO. The CO dependent conversion of 3 into 2 as well as the 2(+) ? 3(+) interconversion were examined by DFT.
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Mechanistic and physiological implications of the interplay among iron-sulfur clusters in [FeFe]-hydrogenases. A QM/MM perspective.
J. Am. Chem. Soc.
PUBLISHED: 10-26-2011
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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.
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Stereochemistry of electrophilic attack at 34e? dimetallic complexes: the case of diiron dithiolato carbonyls + MeS?.
Chem. Commun. (Camb.)
PUBLISHED: 07-22-2011
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Experimental and computational experiments show that the electrophile MeS(+) attacks a single Fe center in Fe(2)(propanedithiolate)(CO)(4)(PMe(3))(2) followed by isomerization of this terminal thiolato complex to the corresponding ?-SMe derivative.
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A theoretical study on the enhancement of functionally relevant electron transfers in biomimetic models of [FeFe]-hydrogenases.
Inorg Chem
PUBLISHED: 07-05-2011
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Recent advances aimed at modeling the chemistry of the active site of [FeFe]-hydrogenases (the H-cluster, composed by a catalytic Fe(2)S(2) subcluster and an Fe(4)S(4) portion) have led to the synthesis of binuclear coordination compounds containing a noninnocent organophosphine ligand [2,3-bis(diphenylphosphino)maleic anhydride, bma] that is able to undergo monoelectron reduction, analogously to the tetranuclear Fe(4)S(4) subcluster portion of the H-cluster. However, such a synthetic model was shown to feature negligible electronic communication between the noninnocent ligand and the remaining portion of the cluster, at variance with the enzyme active site. Here, we report a theoretical investigation that shows why the electron transfer observed in the enzyme upon protonation of the catalytic Fe(2)S(2) subsite cannot take place in the bma-containing cluster. In addition, we show that targeted modifications of the bma ligand are sufficient to restore the electronic communication within the model, such that electron density can be more easily withdrawn from the noninnocent ligand, as a result of protonation of the iron centers. Similar results were also obtained with a ligand derived from cobaltocene. The relevance of our findings is discussed from the perspective of biomimetic reproduction of proton reduction to yield molecular hydrogen.
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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.
Chemphyschem
PUBLISHED: 06-29-2011
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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.
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DFT characterization of key intermediates in thiols oxidation catalyzed by amavadin.
Dalton Trans
PUBLISHED: 05-31-2011
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Amavadin is an unusual octa-coordinated V(IV) complex isolated from Amanita muscaria mushrooms. The outer-sphere catalytic properties of such a complex toward several oxidation reactions are well known. Nevertheless, a remarkable example exists, in which the V(V) (d(0)) oxidized form of amavadin is able to electro-catalyze the oxidation of some thiols to the corresponding disulfides through an inner-sphere mechanism (Guedes da Silva et al. J. Am. Chem. Soc.1996, 118, 7568-7573.) The reaction mechanism implies the formation of an amavadin-substrate intermediate, whose half-life is about 0.3 s. By means of Density Functional Theory (DFT) computations and Quantum Theory of Atoms in Molecules (QTAIM) analysis of the electron density, we have first characterized the stereoelectronic features of the V(IV) (inactive) and V(V) (active) states of amavadin. Then, the formation of the V(V) complex with methyl mercaptoacetate (MMA), which has been chosen as a prototypical substrate, has been characterized both thermodynamically and kinetically. DFT results reveal that protonation of V(V) amavadin at a carboxylate oxygen not directly involved in the V coordination, favors MMA binding into the first coordination sphere of vanadium, by substitution of the amavadin carboxylate oxygen with that of the substrate and formation of an S-HO hydrogen bond interaction. The latter interaction can promote SH deprotonation and binding of the thiolate group to vanadium. The kinetic and thermodynamic feasibility of the V(V)-MMA intermediates formation is in agreement, along with electrochemical experimental data, also with the biological role exerted by amavadin. Finally, the presence of an ester functional group as an essential requisite for MMA oxidation has been rationalized.
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Molecular dynamics of mesophilic-like mutants of a cold-adapted enzyme: insights into distal effects induced by the mutations.
PLoS ONE
PUBLISHED: 04-25-2011
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Networks and clusters of intramolecular interactions, as well as their "communication" across the three-dimensional architecture have a prominent role in determining protein stability and function. Special attention has been dedicated to their role in thermal adaptation. In the present contribution, seven previously experimentally characterized mutants of a cold-adapted ?-amylase, featuring mesophilic-like behavior, have been investigated by multiple molecular dynamics simulations, essential dynamics and analyses of correlated motions and electrostatic interactions. Our data elucidate the molecular mechanisms underlying the ability of single and multiple mutations to globally modulate dynamic properties of the cold-adapted ?-amylase, including both local and complex unpredictable distal effects. Our investigation also shows, in agreement with the experimental data, that the conversion of the cold-adapted enzyme in a warm-adapted variant cannot be completely achieved by the introduction of few mutations, also providing the rationale behind these effects. Moreover, pivotal residues, which are likely to mediate the effects induced by the mutations, have been identified from our analyses, as well as a group of suitable candidates for protein engineering. In fact, a subset of residues here identified (as an isoleucine, or networks of mesophilic-like salt bridges in the proximity of the catalytic site) should be considered, in experimental studies, to get a more efficient modification of the features of the cold-adapted enzyme.
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An acidic loop and cognate phosphorylation sites define a molecular switch that modulates ubiquitin charging activity in Cdc34-like enzymes.
PLoS Comput. Biol.
PUBLISHED: 04-01-2011
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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 ?4?2 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 ?4?2 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.
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Targeting intermediates of [FeFe]-hydrogenase by CO and CN vibrational signatures.
Inorg Chem
PUBLISHED: 03-28-2011
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In this work, we employ density functional theory to assign vibrational signatures of [FeFe]-hydrogenase intermediates to molecular structures. For this purpose, we perform an exhaustive analysis of structures and harmonic vibrations of a series of CN and CO containing model clusters of the [FeFe]-hydrogenase enzyme active site considering also different charges, counterions, and solvents. The pure density functional BP86 in combination with a triple-? polarized basis set produce reliable molecular structures as well as harmonic vibrations. Calculated CN and CO stretching vibrations are analyzed separately. Scaled vibrational frequencies are then applied to assign intermediates in [FeFe]-hydrogenases reaction cycle. The results nicely complement the previous studies of Darensbourg and Hall, and Zilberman et al. The infrared spectrum of the H(ox) form is in very good agreement with the calculated spectrum of the Fe(I)Fe(II) model complex featuring a free coordination site at the distal Fe atom, as well as, with the calculated spectra of the complexes in which H(2) or H(2)O are coordinated at this site. The spectrum of H(red) measured from Desulfovibrio desulfuricans is compatible with a mixture of a Fe(I)Fe(I) species with all terminal COs, and a Fe(I)Fe(I) species with protonated dtma ligand, while the spectrum of H(red) recently measured from Chlamydomonas reinhardtii is compatible with a mixture of a Fe(I)Fe(I) species with a bridged CO, and a Fe(II)Fe(II) species with a terminal hydride bound to the Fe atom.
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Compaction properties of an intrinsically disordered protein: Sic1 and its kinase-inhibitor domain.
Biophys. J.
PUBLISHED: 02-04-2011
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IDPs in their unbound state can transiently acquire secondary and tertiary structure. Describing such intrinsic structure is important to understand the transition between free and bound state, leading to supramolecular complexes with physiological interactors. IDP structure is highly dynamic and, therefore, difficult to study by conventional techniques. This work focuses on conformational analysis of the KID fragment of the Sic1 protein, an IDP with a key regulatory role in the cell-cycle of Saccharomyces cerevisiae. FT-IR spectroscopy, ESI-MS, and IM measurements are used to capture dynamic and short-lived conformational states, probing both secondary and tertiary protein structure. The results indicate that the isolated Sic1 KID retains dynamic helical structure and populates collapsed states of different compactness. A metastable, highly compact species is detected. Comparison between the fragment and the full-length protein suggests that chain length is crucial to the stabilization of compact states of this IDP. The two proteins are compared by a length-independent compaction index.
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CO disrupts the reduced H-cluster of FeFe hydrogenase. A combined DFT and protein film voltammetry study.
J. Am. Chem. Soc.
PUBLISHED: 01-27-2011
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Carbon monoxide is often described as a competitive inhibitor of FeFe hydrogenases, and it is used for probing H(2) binding to synthetic or in silico models of the active site H-cluster. Yet it does not always behave as a simple inhibitor. Using an original approach which combines accurate electrochemical measurements and theoretical calculations, we elucidate the mechanism by which, under certain conditions, CO binding can cause permanent damage to the H-cluster. Like in the case of oxygen inhibition, the reaction with CO engages the entire H-cluster, rather than only the Fe(2) subsite.
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Isocyanide in biochemistry? A theoretical investigation of the electronic effects and energetics of cyanide ligand protonation in [FeFe]-hydrogenases.
Chemistry
PUBLISHED: 01-12-2011
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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.
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DFT characterization of the reaction pathways for terminal- to ?-hydride isomerisation in synthetic models of the [FeFe]-hydrogenase active site.
Chem. Commun. (Camb.)
PUBLISHED: 10-18-2010
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The mechanism of terminal- to ?-hydride isomerisation in models of synthetic complexes resembling the [FeFe]-hydrogenase active site has been elucidated by DFT calculations, revealing that Ray-Dutt reaction pathways are generally favoured, and providing some clues for the rational design of novel synthetic catalysts to produce H(2).
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Investigation of Streptomyces antibioticus tyrosinase reactivity toward chlorophenols.
Arch. Biochem. Biophys.
PUBLISHED: 07-07-2010
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Tyrosinase (Ty) is a copper-containing enzyme ubiquitously distributed in nature. In recent years, Ty has attracted interest as a potential detoxifying agent for xenobiotic compounds with phenolic structure. Among these, chlorophenols are particularly relevant pollutants, commonly found in waste waters. The activity of Streptomyces antibioticus tyrosinase toward isomeric monochlorophenols was studied. Tyrosinase oxidizes both 3- and 4-chlorophenol to the same product, 4-chloro-1,2-ortho-quinone, which subsequently undergoes a nucleophilic substitution reaction at the chlorine atom by excess phenol to give the corresponding phenol-quinone adduct. By contrast, 2-chlorophenol is not reactive and acts as a competitive inhibitor. Docking calculations suggest that the substrates point to one of the copper atoms of the dinuclear center (copper B) and appear to interact preferentially with one of the two coordinated oxygen atoms. The approach of the substrate toward the active site is favored by a ?-stacking interaction with one of the copper-coordinated histidines (His194) and by a hydrogen bonding interaction with the O1 oxygen. With this study, we provide the first characterization of the early intermediates in the biotechnologically relevant reaction of Ty with chlorophenols. Additionally, combining experimental evidences with molecular modeling simulations, we propose a detailed reaction scheme for Ty-mediated oxidation of monochlorophenols.
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Electrocatalytic dihydrogen evolution mechanism of [Fe2(CO)4(kappa(2)-Ph2PCH2CH2PPh2)(mu-S(CH2)3S)] and related models of the [FeFe]-hydrogenases active site: a DFT investigation.
Dalton Trans
PUBLISHED: 07-01-2010
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A DFT study of protonation thermodynamics in H(2)-evolving biomimetic catalysts related to [FeFe]-hydrogenases active site is presented here. Taking as a reference system the electrocatalytic dihydrogen evolution mechanism recently proposed for the synthetic assembly [Fe(2)(CO)(4)(kappa(2)-Ph(2)PCH(2)CH(2)PPh(2))(mu-S(CH(2))(3)S)] (a, which is able to release H(2) after having undergone monoelectron reduction steps and three sequential protonation reactions), we show how the reduction of model complexes to oxidation states lower than those observed in [FeFe]-hydrogenases cofactor leads to a protonation regiochemistry that has no counterpart in the enzymatic mechanism of H(2) production. In particular, double protonation of the metal centers turned out to be disfavored in a by up to 12.5 kcal mol(-1) with respect to alternative protonation paths; as for the regiochemistry of triple protonation, the formation of eta(2)-H(2) adducts is disfavored by at least approximately 25 kcal mol(-1). Structural analysis of the theoretical models also revealed that over-reduction of synthetic complexes, though necessary for observing H(2) evolution from the currently available biomimetic electrocatalysts, can generally impair their structural integrity. Possible approaches for the modulation of protonation regiochemistry are then proposed; in particular, it turned out that a targeted use of sigma-donating ligands showing low basicity can favor double protonation of iron centers.
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Near native-state conformational landscape of psychrophilic and mesophilic enzymes: probing the folding funnel model.
J Phys Chem B
PUBLISHED: 06-04-2010
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In recent years, increased interest has been directed to the study of enzyme adaptation to low temperatures. In particular, a peculiar folding funnel model was proposed for the free energy landscape of a psychrophilic alpha-amylase and other cold-adapted enzymes. In the present contribution, the comparison between the near native-state dynamics and conformational landscape in the essential subspace of different cold-adapted enzymes with their mesophilic counterparts, as obtained by more than 0.1 micros molecular dynamics simulations at different temperatures, allows the folding funnel model to be probed. Common characteristics were highlighted in the near native-state dynamics of psychrophilic enzymes belonging to different enzymatic families when compared to the mesophilic counterparts. According to the model, a cold-adapted enzyme in its native-state consists of a large population of conformations which can easily interconvert and result in high structural flexibility.
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Investigation on the protonation of a trisubstituted [Fe(2)(CO)(3)(PPh(3))(kappa(2)-phen)(mu-pdt)] complex: rotated versus unrotated intermediate pathways.
Inorg Chem
PUBLISHED: 05-07-2010
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The substitution of PPh(3) for a carbonyl group at the {Fe(CO)(3)} moiety in [Fe(2)(CO)(4)(kappa(2)-phen)(mu-pdt)] results in the formation of the trisubstituted complex [Fe(2)(CO)(3)(PPh(3))(kappa(2)-phen)(mu-pdt)] (2). Unlike its tetracarbonyl precursor, the protonation of 2 at low temperature does not afford any apparent transient terminal hydride species. Hydride formation for [Fe(2)(CO)(3)(L)(kappa(2)-phen)(mu-pdt)] (L = PPh(3), CO) species is also studied by density functional theory calculations, which show that activation barriers to give terminal and bridging hydrides can be remarkably close for this class of organometallic compounds.
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Functionally relevant interplay between the Fe(4)S(4) cluster and CN(-) ligands in the active site of [FeFe]-hydrogenases.
J. Am. Chem. Soc.
PUBLISHED: 03-23-2010
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[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.
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Quantum refinement of [FeFe] hydrogenase indicates a dithiomethylamine ligand.
J. Am. Chem. Soc.
PUBLISHED: 03-17-2010
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The active site of the [FeFe] hydrogenases contains two Fe ions bound to one Cys ligand, three CO molecules, two CN(-) ions, and a dithiolate ligand. The nature of the last of these has been much discussed, and it has been suggested that it contains C, N, or O as the bridgehead atom. Most experimental studies indicate a N atom, whereas a recent density functional theory (DFT) study of a crystal structure indicated an O atom. Here, we performed quantum refinement on the same crystal structure with five different models of the dithiolate ligand X(CH(2)S(-))(2), with X = CH(2), NH(2)(+), NH (two conformations), or O; we found that structures with a N bridgehead atom actually provide the best fit to the raw crystallographic data. Quantum refinement is standard crystallographic refinement in which the molecular mechanics force field normally used to supplement the experimental raw data to give a more chemical structure is replaced by more accurate DFT calculations for the active site. Thereby, we obtain structures that are an ideal compromise between DFT and crystallography.
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C-type natriuretic peptide: Structural studies, fragment synthesis, and preliminary biological evaluation in human osteosarcoma cell lines.
Biopolymers
PUBLISHED: 03-13-2010
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Natriuretic peptides (NP) are a family of structurally related but genetically distinct hormones/paracrine factors that regulate blood volume, blood pressure, ventricular hypertrophy, pulmonary hypertension, fat metabolism, and long bone growth. In this work we present computational structural analysis of the three human NP in solution, the synthesis and preliminary biological assays of a short fragment of CNP, I(14)GSM(17), together with one small mimetic, GGSM. Synthetic peptides IGSM, GGSM, and full length CNP were preliminary tested for their ability to influence cell growth of three human osteosarcoma cell lines. Synthetic peptides were shown to successfully mimic the biological activity of the full length natural peptide: their effects, although different upon the cell types used, are in accordance with the current literature, designating a possible role for CNP, and its derivatives, in skeletogenesis.
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Molecular dynamics investigation of cyclic natriuretic peptides: dynamic properties reflect peptide activity.
J. Mol. Graph. Model.
PUBLISHED: 03-01-2010
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Natriuretic peptides (NPs) are a family of structurally related hormone/paracrine factors (ANP, BNP and CNP), which mediate a broad array of physiological effects by interacting with specific guanylyl cyclase receptors (NPR) and have promising therapeutic and clinical applications. NPs are specific for different NPRs and share a common ring structure in which a disulfide bond between two conserved cysteine residues is formed. Residues within the cyclic loop are largely responsible for receptor selectivity. Structural features of free NPs in solution have not been investigated in details even if their characterization would be very useful in order to identify important aspects related to NPs function and receptor selectivity. In light of the above scenario, we carried out a 0.1 micros molecular dynamics investigation of NPs with the aim of providing a high-resolution atomistic view of specific of their conformational ensemble in solution. Our results clearly indicate that NP receptor-bound conformations are not stable solution structure and that induced-fit mechanisms are involved in the formation of NP-NPR complexes. Moreover, in agreement with the current view on strictly relationship between protein dynamics and protein function and activity, it turns out that differences in activity and NPR specificity of CNP and ANP/BNP might be correlated to different amino acid composition of the cyclic loop, propensity to form beta-sheet structures, flexibility patterns, dynamics properties and free conformations explored during the simulations.
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Structure-activity studies on arylamides and arysulfonamides Ras inhibitors.
Curr Cancer Drug Targets
PUBLISHED: 01-22-2010
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This paper reports the synthesis of a panel of small molecules with arylamides and arylsulfonamides groups and their biological activity in inhibiting nucleotide exchange on human Ras. The design of these molecules was guided by experimental and molecular modelling data previously collected on similar compounds. Aim of this work is the validation of the hypothesis that a phenyl hydroxylamine group linked to a second aromatic moiety generates a pharmacophore capable to interact with Ras and to inhibit its activation. In vitro experiments on purified human Ras clearly show that the presence of an aromatic hydroxylamine and a sulfonamide group in the same molecule is a necessary condition for Ras binding and nucleotide exchange inhibition. The inhibitor potency is lower in molecules in which either the hydroxylamine has been replaced by other functional groups or the sulfonamide has been replaced by an amide. In the case both these moieties, the hydroxylamine and sulfonamide are absent, inactive compounds are obtained.
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Dynamic properties of a psychrophilic alpha-amylase in comparison with a mesophilic homologue.
J Phys Chem B
PUBLISHED: 09-25-2009
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The cold-active, chloride-dependent alpha-amylase from Pseudoalteromonas haloplanktis (AHA) is one of the best characterized psychrophilic enzymes, and shares high sequence and structural similarity with its mesophilic porcine counterpart (PPA). An atomic detail comparative analysis was carried out by performing more than 60 ns of multiple-replica explicit-solvent molecular dynamics simulations on the two enzymes in order to characterize the differences in ensemble properties and dynamics in solution between the two homologues. We find in both enzymes high flexibility clusters in the surroundings of the substrate-binding groove, primarily involving the long loops that protrude from the main domains barrel structure. These loops are longer in PPA and extend further away from the core of the barrel, where the active site is located: essential fluctuations in PPA mainly affect the highly solvent-accessible portions of these loops, whereas AHA is characterized by greater flexibility in the immediate surroundings of the active site. Furthermore, detailed analysis of active-site dynamics has revealed that elements previously identified through X-ray crystallography as involved in substrate binding in both enzymes undergo concerted motions that may be linked to catalysis.
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Isomerization of the hydride complexes [HFe2(SR)2(PR3)(x)(CO)(6-x)]+ (x = 2, 3, 4) relevant to the active site models for the [FeFe]-hydrogenases.
Dalton Trans
PUBLISHED: 09-16-2009
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The stepwise formation of bridging (mu-) hydrides of diiron dithiolates is discussed with attention on the pathway for protonation and subsequent isomerizations. Our evidence is consistent with protonations occurring at a single Fe center, followed by isomerization to a series of mu-hydrides. Protonation of Fe(2)(edt)(CO)(4)(dppv) (1) gave a single mu-hydride with dppv spanning apical and basal sites, which isomerized at higher temperatures to place the dppv into a dibasal position. Protonation of Fe(2)(pdt)(CO)(4)(dppv) (2) followed an isomerization pathway similar to that for [1H](+), except that a pair of isomeric terminal hydrides were observed initially, resulting from protonation at the Fe(CO)(3) or Fe(CO)(dppv) site. The first observable product from low temperature protonation of the tris-phosphine Fe(2)(edt)(CO)(3)(PMe(3))(dppv) (3) was a single mu-hydride wherein PMe(3) is apical and the dppv ligand spans apical and basal sites. Upon warming, this isomer converted fully but in a stepwise manner to a mixture of three other isomeric hydrides. Protonation of Fe(2)(pdt)(CO)(3)(PMe(3))(dppv) (4) proceeded similarly to the edt analogue 3, however a terminal hydride was observed, albeit only briefly and at very low temperatures (-90 degrees C). Low-temperature protonation of the bis-chelates Fe(2)(xdt)(CO)(2)(dppv)(2) produced exclusively the terminal hydrides [HFe(2)(xdt)(mu-CO)(CO)(dppv)(2)](+) (xdt = edt and pdt), which subsequently isomerized to a pair of mu-hydrides. At room temperature these (dppv)(2) derivatives convert to an equilibrium of two isomers, one C(2)-symmetric and the other C(s)-symmetric. The stability of the terminal hydrides correlates with the (C(2)-isomer)/(C(s)-isomer) equilibrium ratio, which reflects the size of the dithiolate. The isomerization was found to be unaffected by the presence of excess acid, by solvent polarity, and the presence of D(2)O. This isomerization mechanism is proposed to be intramolecular, involving a 120 degrees rotation of the HFeL(3) subunit to an unobserved terminal basal hydride as the rate-determining step. The observed stability of the hydrides was supported by DFT calculations, which also highlight the instability of the basal terminal hydrides. Isomerization of the mu-hydride isomers occurs on alternating FeL(3) via 120 degree rotations without generating D(2)O-exchangeable intermediates.
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Unveiling how stereoelectronic factors affect kinetics and thermodynamics of protonation regiochemistry in [FeFe] hydrogenase synthetic models: a DFT investigation.
J. Am. Chem. Soc.
PUBLISHED: 07-23-2009
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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.
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Structure prediction and functional analysis of KdsD, an enzyme involved in lipopolysaccharide biosynthesis.
Biochem. Biophys. Res. Commun.
PUBLISHED: 07-21-2009
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Lipopolysaccharide is an essential component of the outer membrane of Gram-negative bacteria and consists of three elements: lipid A, the core oligosaccharide and the O-antigen. The inner core region is highly conserved and contains at least one residue of 3-deoxy-D-manno-octulosonate (Kdo). The first committed step of Kdo biosynthesis is the aldol-keto isomerisation of d-ribulose 5-phosphate to d-arabinose 5-phosphate catalyzed by arabinose 5-phosphate isomerase encoded in Escherichia coli by the kdsD gene. KdsD contains an N-terminal sugar isomerase (SIS) domain commonly found in phosphosugar isomerases but its three-dimensional structure is unknown. The structure of the KdsD SIS domain has been predicted by homology modeling using the hypothetical 3etn protein as a template. Moreover by sequence alignments, comparison with other sugar isomerases structurally related to KdsD, and site-directed mutagenesis we implicated four residues in KdsD activity or substrate recognition. A possible role of these residues in the catalysis is discussed.
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Selective cytotoxicity of a bicyclic Ras inhibitor in cancer cells expressing K-Ras(G13D).
Biochem. Biophys. Res. Commun.
PUBLISHED: 06-11-2009
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Mutation of RAS genes is a critical event in the pathogenesis of different human tumors and in some developmental disorders. Here we present an arabinose-derived bicyclic compound displaying selective cytotoxicity in human colorectal cancer cells expressing K-Ras(G13D), that shows high intrinsic nucleotide exchange rate. We characterize binding of bicyclic compounds by docking and NMR experiments and their inhibitory activity on GEF-mediated nucleotide exchange on wild-type and mutant Ras proteins. We demonstrate that the in vitro inhibition of Ras nucleotide exchange depends on the molar ratio between Ras and its GEF activator, suggesting that the observed in vivo selective effect may depend on biochemical parameters and actual intracellular concentration of the Ras protein and its regulators.
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Relevance of metal ions for lipase stability: structural rearrangements induced in the Burkholderia glumae lipase by calcium depletion.
J. Struct. Biol.
PUBLISHED: 05-19-2009
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We have studied the accessibility of the structural calcium ion in the Burkholderia glumae lipase and the consequences of its removal on the protein conformation by different biophysical techniques (circular dichroism, fluorimetry, and mass spectrometry) and by molecular-dynamics simulations. We show that, in the native protein, calcium is not accessible unless specific flexible loops are displaced, for example, by a temperature increase. Such movements concern the whole calcium-binding pocket and particularly the environment of the coordinating aspartate residue 241. As a consequence of metal depletion the protein unfolds irreversibly and undergoes aggregation. The removal of the metal ion causes major structural transitions and leads to an increase in beta-structure, in particular in protein regions that are largely unstructured in the native protein and encompass the calcium coordination residues.
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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.
J Phys Chem A
PUBLISHED: 04-22-2009
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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.
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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.
Angew. Chem. Int. Ed. Engl.
PUBLISHED: 04-08-2009
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Natures recipe: A theoretical study analyzes how the environment of the [FeFe] hydrogenases 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.
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Free-energy landscape, principal component analysis, and structural clustering to identify representative conformations from molecular dynamics simulations: the myoglobin case.
J. Mol. Graph. Model.
PUBLISHED: 01-27-2009
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Several molecular dynamics (MD) simulations were used to sample conformations in the neighborhood of the native structure of holo-myoglobin (holo-Mb), collecting trajectories spanning 0.22 micros at 300 K. Principal component (PCA) and free-energy landscape (FEL) analyses, integrated by cluster analysis, which was performed considering the position and structures of the individual helices of the globin fold, were carried out. The coherence between the different structural clusters and the basins of the FEL, together with the convergence of parameters derived by PCA indicates that an accurate description of the Mb conformational space around the native state was achieved by multiple MD trajectories spanning at least 0.14 micros. The integration of FEL, PCA, and structural clustering was shown to be a very useful approach to gain an overall view of the conformational landscape accessible to a protein and to identify representative protein substates. This method could be also used to investigate the conformational and dynamical properties of Mb apo-, mutant, or delete versions, in which greater conformational variability is expected and, therefore identification of representative substates from the simulations is relevant to disclose structure-function relationship.
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Intramolecular interactions stabilizing compact conformations of the intrinsically disordered kinase-inhibitor domain of Sic1: a molecular dynamics investigation.
Front Physiol
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Cyclin-dependent kinase inhibitors (CKIs) are key regulatory proteins of the eukaryotic cell cycle, which modulate cyclin-dependent kinase (Cdk) activity. CKIs perform their inhibitory effect by the formation of ternary complexes with a target kinase and its cognate cyclin. These regulators generally belong to the class of intrinsically disordered proteins (IDPs), which lack a well-defined and organized three-dimensional (3D) structure in their free state, undergoing folding upon binding to specific partners. Unbound IDPs are not merely random-coil structures, but can present intrinsically folded structural units (IFSUs) and collapsed conformations. These structural features can be relevant to protein function in vivo. The yeast CKI Sic1 is a 284-amino acid IDP that binds to Cdk1 in complex with the Clb5,6 cyclins, preventing phosphorylation of G1 substrates and, therefore, entrance to the S phase. Sic1 degradation, triggered by multiple phosphorylation events, promotes cell-cycle progression. Previous experimental studies pointed out a propensity of Sic1 and its isolated domains to populate both extended and compact conformations. The present contribution provides models for compact conformations of the Sic1 kinase-inhibitory domain (KID) by all-atom molecular dynamics (MD) simulations in explicit solvent and in the absence of interactors. The results are integrated by spectroscopic and spectrometric data. Helical IFSUs are identified, along with networks of intramolecular interactions. The results identify a group of putative hub residues and networks of electrostatic interactions, which are likely to be involved in the stabilization of the globular states.
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Terminal vs bridging hydrides of diiron dithiolates: protonation of Fe2(dithiolate)(CO)2(PMe3)4.
J. Am. Chem. Soc.
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This investigation examines the protonation of diiron dithiolates, exploiting the new family of exceptionally electron-rich complexes Fe(2)(xdt)(CO)(2)(PMe(3))(4), where xdt is edt (ethanedithiolate, 1), pdt (propanedithiolate, 2), and adt (2-aza-1,3-propanedithiolate, 3), prepared by the photochemical substitution of the corresponding hexacarbonyls. Compounds 1-3 oxidize near -950 mV vs Fc(+/0). Crystallographic analyses confirm that 1 and 2 adopt C(2)-symmetric structures (Fe-Fe = 2.616 and 2.625 Å, respectively). Low-temperature protonation of 1 afforded exclusively [?-H1](+), establishing the non-intermediacy of the terminal hydride ([t-H1](+)). At higher temperatures, protonation afforded mainly [t-H1](+). The temperature dependence of the ratio [t-H1](+)/[?-H1](+) indicates that the barriers for the two protonation pathways differ by ?4 kcal/mol. Low-temperature (31)P{(1)H} NMR measurements indicate that the protonation of 2 proceeds by an intermediate, proposed to be the S-protonated dithiolate [Fe(2)(Hpdt)(CO)(2)(PMe(3))(4)](+) ([S-H2](+)). This intermediate converts to [t-H2](+) and [?-H2](+) by first-order and second-order processes, respectively. DFT calculations support transient protonation at sulfur and the proposal that the S-protonated species (e.g., [S-H2](+)) rearranges to the terminal hydride intramolecularly via a low-energy pathway. Protonation of 3 affords exclusively terminal hydrides, regardless of the acid or conditions, to give [t-H3](+), which isomerizes to [t-H3](+), wherein all PMe(3) ligands are basal.
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Loop 7 of E2 enzymes: an ancestral conserved functional motif involved in the E2-mediated steps of the ubiquitination cascade.
PLoS ONE
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The ubiquitin (Ub) system controls almost every aspect of eukaryotic cell biology. Protein ubiquitination depends on the sequential action of three classes of enzymes (E1, E2 and E3). E2 Ub-conjugating enzymes have a central role in the ubiquitination pathway, interacting with both E1 and E3, and influencing the ultimate fate of the substrates. Several E2s are characterized by an extended acidic insertion in loop 7 (L7), which if mutated is known to impair the proper E2-related functions. In the present contribution, we show that acidic loop is a conserved ancestral motif in E2s, relying on the presence of alternate hydrophobic and acidic residues. Moreover, the dynamic properties of a subset of family 3 E2s, as well as their binary and ternary complexes with Ub and the cognate E3, have been investigated. Here we provide a model of L7 role in the different steps of the ubiquitination cascade of family 3 E2s. The L7 hydrophobic residues turned out to be the main determinant for the stabilization of the E2 inactive conformations by a tight network of interactions in the catalytic cleft. Moreover, phosphorylation is known from previous studies to promote E2 competent conformations for Ub charging, inducing electrostatic repulsion and acting on the L7 acidic residues. Here we show that these active conformations are stabilized by a network of hydrophobic interactions between L7 and L4, the latter being a conserved interface for E3-recruitment in several E2s. In the successive steps, L7 conserved acidic residues also provide an interaction interface for both Ub and the Rbx1 RING subdomain of the cognate E3. Our data therefore suggest a crucial role for L7 of family 3 E2s in all the E2-mediated steps of the ubiquitination cascade. Its different functions are exploited thank to its conserved hydrophobic and acidic residues in a finely orchestrate mechanism.
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Electrochemical and theoretical investigations of the role of the appended base on the reduction of protons by [Fe2(CO)4(?2-PNP(R)(?-S(CH2)3S] (PNP(R) ={Ph2PCH2}2NR, R=Me, Ph).
Chemistry
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The behavior of [Fe(2)(CO)(4)(?(2)-PNP(R))(?-pdt)] (PNP(R) =(Ph(2)PCH(2))(2)NR, R=Me (1), Ph (2); pdt=S(CH(2))(3)S) in the presence of acids is investigated experimentally and theoretically (using density functional theory) in order to determine the mechanisms of the proton reduction steps supported by these complexes, and to assess the role of the PNP(R) appended base in these processes for different redox states of the metal centers. The nature of the R substituent of the nitrogen base does not substantially affect the course of the protonation of the neutral complex by CF(3)SO(3)H or CH(3)SO(3)H; the cation with a bridging hydride ligand, 1 ?H(+) (R=Me) or 2 ?H(+) (R=Ph) is obtained rapidly. Only 1 ?H(+) can be protonated at the nitrogen atom of the PNP chelate by HBF(4)·Et(2)O or CF(3)SO(3)H, which results in a positive shift of the proton reduction by approximately 0.15 V. The theoretical study demonstrates that in this process, dihydrogen can be released from a ?(2)-H(2) species in the Fe(I)Fe(II) state. When R=Ph, the bridging hydride cation 2 ?H(+) cannot be protonated at the amine function by HBF(4)·Et(2)O or CF(3)SO(3)H, and protonation at the N atom of the one-electron reduced analogue is also less favored than that of a S atom of the partially de-coordinated dithiolate bridge. In this situation, proton reduction occurs at the potential of the bridging hydride cation, 2 ?H(+). The rate constants of the overall proton reduction processes are small for both complexes 1 and 2 (k(obs) ?4-7 s(-1)) because of the slow intramolecular proton migration and H(2) release steps identified by the theoretical study.
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Evidence for the formation of a Mo-H intermediate in the catalytic cycle of formate dehydrogenase.
Inorg Chem
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DFT/BP86/TZVP and DFT/B3LYP/TZVP have been used to investigate systematically the reaction pathways associated with the H-transfer step, which is the rate-determining step of the reaction HCOO(-) ? CO(2) + H(+) + 2e(-), as catalyzed by metalloenzyme formate dehydrogenase (FDH). Actually, the energetics associated with the transfer from formate to all H (proton or hydride) acceptors that are present within the FDH active site have been sampled. This study points to a viable intimate mechanism in which the metal center mediates H transfer from formate to the final acceptor, i.e. a selenocysteine residue. The Mo-based reaction pathway, consisting of a ?-H elimination to metal with concerted decarboxylation, turned out to be favored over previously proposed routes in which proton transfer occurs directly from HCOO(-) to selenocysteine. The proposed reaction pathway is reminiscent of the key step of metal-based catalysis of the water-gas shift reaction.
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The reactions of pyridinyl thioesters with triiron dodecacarbonyl: their novel diiron carbonyl complexes and mechanistic investigations.
Dalton Trans
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Reaction of Fe(3)(CO)(12) with pyridinyl thioester ligand PyCH(2)SCOCH(3) (L(1), Py = pyridin-2-yl) produced complex, [Fe(2)(?-COCH(3))(?-SCH(2)Py)(CO)(5)] (1) (PyCH(2)S = pyridin-2-ylmethanethiolate). When complex 1 reacted with PPh(3), a monosubstituted complex, [Fe(2)(?-COCH(3))(?-SCH(2)Py)(CO)(4)PPh(3)] (2), was derived. Reaction of the same precursor with analogous thioester ligand PyCH(2)SCOPy (L(2)) generated three novel diiron complexes, [Fe(2)(?-Py)(?-SCH(2)Py)(CO)(5)] (3), [Fe(2)(?-Py)(?-SCH(2)Py)(CO)(5)] (4), and [Fe(2)(?-Py)(?-SCH(2)Py)(CO)(6)] (5). Complexes 3 and 4 are structural isomers. Complex 5 could be converted into complex 4 but the conversion from complex 5 to the isomer 3 was not observed. All the five complexes were fully characterised using FTIR, NMR, and other techniques. Their structures were determined using X-ray single crystal diffraction analysis. The oxidative formation of complexes 1, 3, 4, and 5 involved C-S and/or C-C bonds cleavages. To probe possible mechanisms for these cleavages, DFT calculations were performed. From the calculations, viable reaction pathways leading to the formation of all the isolated products were delineated. The results of the theoretic calculations also allowed rationalisation of the experimental observations.
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Paths of long-range communication in the E2 enzymes of family 3: a molecular dynamics investigation.
Phys Chem Chem Phys
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Molecular dynamics (MD) simulations have the ability to help reveal the relationship between protein structure, dynamics and function. Here, we describe MD simulations of the representative members of family 3 of E2 enzymes that we performed and analyzed with the aim of providing a quantitative description of the functional dynamics in this biologically important set of proteins. In particular, we combined a description of the protein as a network of interacting residues with the dynamical cross-correlation method to characterize the correlated motions observed in the simulations. This approach enabled us to detect communication between distal residues in these enzymes, and thus to reliably define all the likely intramolecular pathways of communication. We observed functionally relevant differences between the closed and open conformations of the enzyme, and identified the critical residues involved in the long-range communication paths. Our results highlight how molecular simulations can be used to aid in providing atomic-level details to communication paths within proteins.
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Speciation of copper-peptide complexes in water solution using DFTB and DFT approaches: case of the [Cu(HGGG)(Py)] complex.
J Phys Chem B
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The DFTB and DFT methods are applied to the study of different forms of the [Cu(HGGG)(Py)] complex in water, with the aim of identifying the most stable isomer. The DFTB calculations were possible thanks to a careful parametrization of the atom-atom repulsive energy terms for Cu-H, Cu-C, Cu-N, and Cu-O. The speciation process is carried out by computing different DFTB-steered molecular dynamics (SMD) trajectories, each of which ends in a well-defined different form. The last frame of each trajectory is subjected to geometry optimization at both DFTB and DFT levels, leading to a different isomer. From the corresponding energy values, a rank of relative stability of the isomers can be established. The computational protocol developed here is of general applicability to other metal-peptide systems and represents a new powerful tool for the study of speciation of metal-containing systems in water solution, particularly useful when the full characterization of the compound cannot be carried out on the basis of experimental results only.
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C-Terminal acidic domain of ubiquitin-conjugating enzymes: a multi-functional conserved intrinsically disordered domain in family 3 of E2 enzymes.
J. Struct. Biol.
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E2 ubiquitin-conjugating enzymes are key elements of the ubiquitin (Ub) pathway, since they influence processivity and topology of the Ub chain assembly and, as a consequence, the fate of the target substrates. E2s are multi-domain proteins, with accessory N-terminal or C-terminal domains that can contribute to the specificity for the cognate Ub-like molecules, or even the E3. In this context, the thorough structural characterization of E2 accessory domains is mandatory, in particular when they are associated to specific functions. We here provide, by computational and comparative studies, the first evidence of an acidic domain (AD) conserved in the E2 sub-family 3R. It is an intrinsically disordered domain, in which elements for Ub or E3 recognition are maintained. This conserved acidic domain (AD) shows propensity for ?-helix structures (185-192 and 204-218) in the proximity of the sites for interaction with the Ub or the cognate E3. Moreover, our results also suggest that AD can explore conformations with tertiary contacts mainly driven by aromatic and hydrophobic interactions, in absence of its interaction partners. The globular states are likely to be regulated by multiple phosphorylation events, which can trigger conformational changes toward more extended conformations, as judged by MD simulations of the phospho-variants. The extended conformations, in turn, promote the accessibility of the interaction sites for Ub and the E3. We also trace a parallel between this new and natively unfolded structural motif for Ub-recognition and the natively folded ubiquitin associated domain (UBA) typical of family 1 of E2 enzymes, which includes Ubc1. In fact, according to our calculations, Ubc1 maps at the interface between the space of the natively unfolded and folded proteins, as well as it shares common features with the acidic domain of family 3 members.
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The importance of stereochemically active lone pairs for influencing Pb(II) and As(III) protein binding.
Chemistry
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The toxicity of heavy metals, which is associated with the high affinity of the metals for thiolate rich proteins, constitutes a problem worldwide. However, despite this tremendous toxicity concern, the binding mode of As(III) and Pb(II) to proteins is poorly understood. To clarify the requirements for toxic metal binding to metalloregulatory sensor proteins such as As(III) in ArsR/ArsD and Pb(II) in PbrR or replacing Zn(II) in ?-aminolevulinc acid dehydratase (ALAD), we have employed computational and experimental methods examining the binding of these heavy metals to designed peptide models. The computational results show that the mode of coordination of As(III) and Pb(II) is greatly influenced by the steric bulk within the second coordination environment of the metal. The proposed basis of this selectivity is the large size of the ion and, most important, the influence of the stereochemically active lone pair in hemidirected complexes of the metal ion as being crucial. The experimental data show that switching a bulky leucine layer above the metal binding site by a smaller alanine residue enhances the Pb(II) ?binding affinity by a factor of five, thus supporting experimentally the hypothesis of lone pair steric hindrance. These complementary approaches demonstrate the potential importance of a stereochemically active lone pair as a metal recognition mode in proteins and, specifically, how the second coordination sphere environment affects the affinity and selectivity of protein targets by certain toxic ions.
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