Zinc Coordination Sphere Research Articles

Imination of Sterically Unshielded o‐Quinones in the Coordination Sphere of Zinc (II)

Abstracto‐Iminoquinones bearing unshielded chelating OCCN(R) site were synthesized in high yields in the zinc coordination sphere as the neutral complexes. The stability of one‐electron‐reduced forms of these o‐iminoquinones and the distribution of the unpaired electron density in the corresponding radical anion species were studied using cyclic voltammetry and EPR spectroscopy methods

Probing the active site of Class 3 L-asparaginase by mutagenesis. I. Tinkering with the zinc coordination site of ReAV.

ReAV, the inducible Class-3 L-asparaginase from the nitrogen-fixing symbiotic bacterium Rhizobium etli, is an interesting candidate for optimizing its enzymatic potential for antileukemic applications. Since it has no structural similarity to known enzymes with this activity, it may offer completely new ways of approach. Also, as an unrelated protein, it would evade the immunological response elicited by other asparaginases. The crystal structure of ReAV revealed a uniquely assembled protein homodimer with a highly specific C135/K138/C189 zinc binding site in each subunit. It was also shown before that the Zn2+ cation at low and optimal concentration boosts the ReAV activity and improves substrate specificity, which indicates its role in substrate recognition. However, the detailed catalytic mechanism of ReAV is still unknown. In this work, we have applied site-directed mutagenesis coupled with enzymatic assays and X-ray structural analysis to elucidate the role of the residues in the zinc coordination sphere in catalysis. Almost all of the seven ReAV muteins created in this campaign lost the ability to hydrolyze L-asparagine, confirming our predictions about the significance of the selected residues in substrate hydrolysis. We were able to crystallize five of the ReAV mutants and solve their crystal structures, revealing some intriguing changes in the active site area as a result of the mutations. With alanine substitutions of Cys135 or Cys189, the zinc coordination site fell apart and the mutants were unable to bind the Zn2+ cation. Moreover, the absence of Lys138 induced atomic shifts and conformational changes of the neighboring residues from two active-site Ser-Lys tandems. Ser48 from one of the tandems, which is hypothesized to be the catalytic nucleophile, usually changes its hydration pattern in response to the mutations. Taken together, the results provide many useful clues about the catalytic mechanism of the enzyme, allowing one to cautiously postulate a possible enzymatic scenario.

Halogen Bonding in the Decoration of Secondary Coordination Sphere of Zinc(II) and Cadmium(II) Complexes: Catalytic Application in Cycloaddition Reaction of CO2 with Epoxides.

Three new zinc(II) complexes [Zn(H2L3)2(H2O)3] (Zn2), [Zn(H3L2a)(H2O)3]n (Zn3) (H3L2a = 2,4-diiodo-5-(2-(2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)hydrazineyl)isophthalate) and [Zn(HL4)(DMF)(H2O)]n (Zn4) were synthesized by the reaction of Zn(II) salts with 5-(2-(2,4-dioxopentan-3-ylidene)hydrazineyl) isophthalic acid (H3L3), 2,4,6-triiodo-5-(2-(2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)hydrazineyl) isophthalic acid (H5L2) (in the presence of NH2OH·HCl) and 5-(2-(2,4-dioxopentan-3-ylidene)hydrazineyl)-2,4,6-triiodoisophthalic acid (H3L4), respectively. According to the X-ray structural analysis, the intramolecular resonance-assisted hydrogen bond ring remains intact, with N···O distances of 2.562(5) and 2.573(5) Å in Zn2, 2.603(6) Å in Zn3, and 2.563(8) Å in Zn4. In the crystal packing of Zn3, the cooperation of I···O and I···I types of halogen bonds between tectons leads to a one-dimensional supramolecular polymer, while I···O interactions aggregate 1D chains of coordination polymer Zn4. These new complexes (Zn2, Zn3, and Zn4) and known [Zn(H3L1)(H2O)2]n (Zn1) (H3L1 = 5-(2-(2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene) hydrazineyl)isophthalate), {[Zn(H3L1)(H2O)3]·3H2O}n (Zn5), [Cd(H3L1)(H2O)2]n (Cd1), {[Cd(HL3)(H2O)2(DMF)]·H2O}n (Cd2), [Cd(H3L3)]n (Cd-3), {[Cd2(μ-H2O)2(μ-H2L4)2(H2L4)2]·2H2O}n (Cd4), and {[Cd(H3L1)(H2O)3]·4H2O}n (Cd5) were tested as catalysts in the cycloaddition reaction of CO2 with epoxides in the presence of tetrabutylammonium halides as the cocatalyst. The halogen-bonded catalyst Zn4 is the most efficient one in the presence of tetrabutylammonium bromide by affording a high yield (85-99%) of cyclic carbonates under solvent-free conditions after 48 h at 40 bar and 80 °C.

Role of Labile Methanol on Bio-inspired Catalytic Activity of Zn(II) based Compound: An Experimental and Theoretical Investigation.

Two Zn(II) based compounds, [Zn2L1(OAc)3(MeOH)] (1) and [Zn2L2(OAc)3]n (2) have been reported where HL1 is (E)-4-Bromo-2-methoxy-6-(((2-morpholino ethyl)imino) methyl)phenol and HL2 is (E)-4-Bromo-2-methoxy-6-(((2-(piperazine-1-yle)ethyl)imino)methyl) phenol. SCXRD analysis unveils vivid change in structural arrangements and dimensionality from 1 to 2 due to change in coordinated atom from oxygen to nitrogen of the ligands. SCXRD study shows that compound 1 is dinuclear but compound 2 has a 1-dimensional polymeric structure having helical chain. Structural diversity greatly influences the catalytic activity. Compound 1 acts as excellent catalyst for conversion of 3, 5-di-tert-butyl catechol (3, 5-DTBC) to 3, 5-di-tert-butylbenzoquinone (3, 5-DTBQ) with the turnover number (kcat) value of 34.94 sec-1. Further, compound 1 reveals phosphatase like activity for conversion of disodium salt of (4-nitrophenyl)-phosphate hexahydrate to p-nitrophenolate with the kcat value of 24.64 sec-1. Amusingly, compound 2 does not show any catalytic activity. To correlate this distinctly different catalytic behavior of two compounds, DFT calculation was carried out. The calculation reveals that detachment of coordinated methanol from coordination sphere of zinc in compound 1 is energytically favourable which creates room for substrate binding, resulting high catalytic activity. Whereas in compound 2, detachment of piperazine or Zn-O of -COOH group are energetically unfavouable, resulting no catalytic activity.

Understanding and Expanding Zinc Cation/Amine Frustrated Lewis Pair Catalyzed C-H Borylation.

[(NacNac)Zn(DMT)][B(C6F5)4], 1, (NacNac = {(2,6- i Pr2H3C6)N(CH3)C}2CH), DMT = N,N-dimethyl-4-toluidine), was synthesized via two routes starting from either (NacNac)ZnEt or (NacNac)ZnH. Complex 1 is an effective (pre)catalyst for the C-H borylation of (hetero)arenes using catecholborane (CatBH) with H2 the only byproduct. The scope included weakly activated substrates such as 2-bromothiophene and benzothiophene. Computational studies elucidated a plausible reaction mechanism that has an overall free energy span of 22.4 kcal/mol (for N-methylindole borylation), consistent with experimental observations. The calculated mechanism starting from 1 proceeds via the displacement of DMT by CatBH to form [(NacNac)Zn(CatBH)]+, D, in which CatBH binds via an oxygen to zinc which makes the boron center much more electrophilic based on the energy of the CatB-based LUMO. Combinations of D and DMT act as a frustrated Lewis pair (FLP) to effect C-H borylation in a stepwise process via an arenium cation that is deprotonated by DMT. Subsequent B-H/[H-DMT]+ dehydrocoupling and displacement from the coordination sphere of zinc of CatBAr by CatBH closes the cycle. The calculations also revealed a possible catalyst decomposition pathway involving hydride transfer from boron to zinc to form (NacNac)ZnH which reacts with CatBH to ultimately form Zn(0). In addition, the key rate-limiting transition states all involve the base, thus fine-tuning of the steric and electronic parameters of the base enabled a further minor enhancement in the C-H borylation activity of the system. Outlining the mechanism for all steps of this FLP-mediated process will facilitate the development of other main group FLP catalysts for C-H borylation and other transformations.

Regulation of skeletal muscle AMP deaminase. Carbethoxylation of His-51 belonging to the zinc coordination sphere of the rabbit enzyme promotes its desensitization towards the inhibition by ATP

BackgroundSkeletal muscle AMP deaminase (AMPD1) regulates the concentration of adenine nucleotides during muscle contraction. We previously provided evidence that rabbit AMPD1 is composed by two HPRG 73 kDa subunits and two 85 kDa catalytic subunits with a dinuclear zinc site with an average of two histidine residues at each metal site. AMPD1 is mainly expressed in fast twitching fibers and is inhibited by ATP. The limited trypsinization of the 95-residue N-terminal domain of rabbit AMPD1 desensitizes the enzyme towards ATP inhibition at the optimal pH 6.5, but not at pH 7.1. MethodsThe modified residues of rabbit AMPD1 after incubation with radioactive diethyl pyrocarbonate ([14C]DEP) causing the desensitization to inhibition by ATP at pH 7.1 have been identified by sequence analysis and MS analysis of the radioactive peptides liberated from the carbethoxylated enzyme by limited proteolysis with trypsin. ResultsThe study confirms the presence of a dinuclear zinc site in rabbit AMPD1 and shows that carbethoxylation of His-51 at the N-terminus of the catalytic subunit removes the inhibition of the enzyme by ATP at pH 7.1. ConclusionsThe desensitization to ATP is due to the modification of His-51 of the Zn2 coordination sphere which is transduced in a conformational change of the enzyme C-terminus, where an ATP-binding site has been localized. General significanceThe progress in the study of the complex regulation of rabbit AMPD1 that shares an identical amino acid sequence with the human enzyme is important in relation to the role of the enzyme during mammalian evolution.

The change of conditions does not affect Ros87 downhill folding mechanism

Downhill folding has been defined as a unique thermodynamic process involving a conformations ensemble that progressively loses structure with the decrease of protein stability. Downhill folders are estimated to be rather rare in nature as they miss an energetically substantial folding barrier that can protect against aggregation and proteolysis. We have previously demonstrated that the prokaryotic zinc finger protein Ros87 shows a bipartite folding/unfolding process in which a metal binding intermediate converts to the native structure through a delicate barrier-less downhill transition. Significant variation in folding scenarios can be detected within protein families with high sequence identity and very similar folds and for the same sequence by varying conditions. For this reason, we here show, by means of DSC, CD and NMR, that also in different pH and ionic strength conditions Ros87 retains its partly downhill folding scenario demonstrating that, at least in metallo-proteins, the downhill mechanism can be found under a much wider range of conditions and coupled to other different transitions. We also show that mutations of Ros87 zinc coordination sphere produces a different folding scenario demonstrating that the organization of the metal ion core is determinant in the folding process of this family of proteins.

Fluorescence pH-dependent sensing of Zn(II) by a tripodal ligand. A comparative X-ray and DFT study

A new complex was obtained starting from a pyridyl/phenolic/benzothiazole functionalized ligand (BPAP) with zinc acetate. The structural and photoluminescence properties of the complex were investigated by X-ray diffraction and confirmed by DFT calculations. By comparing the results with previous studies, we found that the mode of binding of the sensor was influenced by the pH of the medium, changing the zinc coordination sphere. In a basic aqueous solution, the metal coordinates two neutral nitrogen atoms and two negative sites, the phenate oxygen and the deprotonate amide nitrogen and a water molecule. In this condition, the ligand was found to behave as a more sensitive naked eye fluorescence chemosensor towards Zn2+, showing blue photoluminescence with a lower detection limit of 375 nM.

Temperature effect on the structure and conformational fluctuations in two zinc knuckles from the mouse mammary tumor virus

Zinc fingers are small protein domains in which zinc plays a structural role, contributing to the stability of the zinc–peptide complex. Zinc fingers are structurally diverse and are present in proteins that perform a broad range of functions in various cellular processes, such as replication and repair, transcription and translation, metabolism and signaling, cell proliferation, and apoptosis. Zinc fingers typically function as interaction modules and bind to a wide variety of compounds, such as nucleic acids, proteins, and small molecules. In this study, we investigated the structural properties, in solution, of the proximal and distal zinc knuckles of the nucleocapsid (NC) protein from the mouse mammary tumor virus (MMTV) (MMTV NC). For this purpose, we performed a series of molecular dynamics simulations in aqueous solution at 300 K, 333 K, and 348 K. The temperature effect was evaluated in terms of root mean square deviation of the backbone atoms and root mean square fluctuation of the coordinating residue atoms. The stability of the zinc coordination sphere was analyzed based upon the time profile of the interatomic distances between the zinc ions and the chelator atoms. The results indicate that the hydrophobic character of the proximal zinc finger is dominant at 333 K. The low mobility of the coordinating residues suggests that the strong electrostatic effect exerted by the zinc ion on its coordinating residues is not influenced by the increase in temperature. The evolution of the structural parameters of the coordination sphere of the distal zinc finger at 300 K gives us a reasonable picture of the unfolding pathway, as proposed by Bombarda and coworkers (Bombarda et al., 2005), which can predict the binding order of the four conserved ligand-binding residues. Our results support the conclusion that the structural features can vary significantly between the two zinc knuckles of MMTV NC.

Crystal Chemistry of Zinc Quinaldinate Complexes with Pyridine-Based Ligands

Substitution of methanol in [Zn(quin)2(CH3OH)2] (quin− denotes an anionic form of quinoline-2-carboxylic acid, also known as quinaldinic acid) with pyridine (Py) or its substituted derivatives, 3,5-lutidine (3,5-Lut), nicotinamide (Nia), 3-hydroxypyridine (3-Py-OH), 3-hydroxymethylpyridine (3-Hmpy), 4-hydroxypyridine (4-Py-OH) and 4-hydroxymethylpyridine (4-Hmpy), afforded a series of novel heteroleptic complexes with compositions [Zn(quin)2(Py)2] (1), [Zn(quin)2(3,5-Lut)2] (2), [Zn(quin)2(Nia)2]·2CH3CN (3), [Zn(quin)2(3-Py-OH)2] (4), [Zn(quin)2(3-Hmpy)2] (5), [Zn(quin)2(4-Pyridone)] (6) (4-Pyridone = a keto tautomer of 4-hydroxypyridine), and [Zn(quin)2(4-Hmpy)2] (7). In all reactions, the {Zn(quin)2} structural fragment with quinaldinate ions bound in a bidentate chelating manner retained its structural integrity. With the exception of [Zn(quin)2(4-Pyridone)] (6), all complexes feature a six-numbered coordination environment of metal ion that may be described as a distorted octahedron. The arrangement of ligands is trans. The coordination sphere of zinc(II) in the 4-pyridone complex consists of only three ligands, two quinaldinates, and one secondary ligand. The metal ion thereby attains a five-numbered coordination environment that is best described as a distorted square-pyramid (τ parameter equals 0.39). The influence of substituents on the pyridine-based ligand over intermolecular interactions in the solid state is investigated. Since pyridine and 3,5-lutidine are not able to form hydrogen-bonding interactions, the solid state structures of their complexes, [Zn(quin)2(Py)2] (1) and [Zn(quin)2(3,5-Lut)2] (2), are governed by π···π stacking, C–H∙∙∙π, and C–H∙∙∙O intermolecular interactions. With other pyridine ligands possessing amide or hydroxyl functional groups, the connectivity patterns in the crystal structures of their complexes are governed by hydrogen bonding interactions. Thermal decomposition studies of novel complexes have shown the formation of zinc oxide as the end product.

X-ray Absorption Spectroscopy Combined with Time-Dependent Density Functional Theory Elucidates Differential Substitution Pathways of Au(I) and Au(III) with Zinc Fingers.

A combination of two elements' (Au, Zn) X-ray absorption spectroscopy (XAS) and time-dependent density functional theory (TD-DFT) allowed the elucidation of differential substitution pathways of Au(I) and Au(III) compounds reacting with biologically relevant zinc fingers (ZnFs). Gold L3-edge XAS probed the interaction of gold and the C-terminal Cys2HisCys finger of the HIV-1 nucleocapsid protein NCp7, and the Cys2His2 human transcription factor Sp1. The use of model compounds helped assign oxidation states and the identity of the gold-bound ligands. The computational studies accurately reproduced the experimental XAS spectra and allowed the proposition of structural models for the interaction products at early time points. The direct electrophilic attack on the ZnF by the highly thiophilic Au(I) resulted in a linear P-Au-Cys coordination sphere after zinc ejection whereas for the Sp1, loss of PEt3 results in linear Cys-Au-Cys or Cys-Au-His arrangements. Reactions with Au(III) compounds, on the other hand, showed multiple binding modes. Prompt reaction between [AuCl(dien)]2+ and [Au(dien)(DMAP)]3+ with Sp1 showed a partially reduced Au center and a final linear His-Au-His coordination. Differently, in the presence of NCp7, [AuCl(dien)]2+ readily reduces to Au(I) and changes from square-planar to linear geometry with Cys-Au-His coordination, while [Au(dien)(DMAP)]3+ initially maintains its Au(III) oxidation state and square-planar geometry and the same first coordination sphere. The latter is the first observation of a "noncovalent" interaction of a Au(III) complex with a zinc finger and confirms early hypotheses that stabilization of Au(III) occurs with N-donor ligands. Modification of the zinc coordination sphere, suggesting full or partial zinc ejection, is observed in all cases, and for [Au(dien)(DMAP)]3+ this represents a novel mechanism for nucleocapsid inactivation. The combination of XAS and TD-DFT presents the first direct experimental observation that not only compound reactivity, but also ZnF core specificity, can be modulated on the basis of the coordination sphere of Au(III) compounds.

High-affinity metal binding by the Escherichia coli [NiFe]-hydrogenase accessory protein HypB is selectively modulated by SlyD.

[NiFe]-hydrogenase, which catalyzes the reversible conversion between hydrogen gas and protons, is a vital component of the metabolism of many pathogens. Maturation of [NiFe]-hydrogenase requires selective nickel insertion that is completed, in part, by the metallochaperones SlyD and HypB. Escherichia coli HypB binds nickel with sub-picomolar affinity, and the formation of the HypB-SlyD complex activates nickel release from the high-affinity site (HAS) of HypB. In this study, the metal selectivity of this process was investigated. Biochemical experiments revealed that the HAS of full length HypB can bind stoichiometric zinc. Moreover, in contrast to the acceleration of metal release observed with nickel-loaded HypB, SlyD blocks the release of zinc from the HypB HAS. X-ray absorption spectroscopy (XAS) demonstrated that SlyD does not impact the primary coordination sphere of nickel or zinc bound to the HAS of HypB. Instead, computational modeling and XAS of HypB loaded with nickel or zinc indicated that zinc binds to HypB with a different coordination sphere than nickel. The data suggested that Glu9, which is not a nickel ligand, directly coordinates zinc. These results were confirmed through the characterization of E9A-HypB, which afforded weakened zinc affinity compared to wild-type HypB but similar nickel affinity. This mutant HypB fully supports the production of [NiFe]-hydrogenase in E. coli. Altogether, these results are consistent with the model that the HAS of HypB functions as a nickel site during [NiFe]-hydrogenase enzyme maturation and that the metal selectivity is controlled by activation of metal release by SlyD.

Calcium Complexes Having Different Amidinate Ligands ‐ Synthesis and Structural Diversity

AbstractA one‐pot reaction of α‐diimine ligand 1,4‐disubstituted diazabutadienes (DAD) with potassium benzyl and anhydrous calcium iodide in 1:1:1 molar ratio afforded unprecedented 3‐phenylprop‐2‐ene‐di‐amido calcium compound [κ2‐(THF)4Ca{DippNC(=CHPh)CH2NDipp}] (1) (Dipp = 2,6‐diisopropylphenyl) in good yield. The solid‐state structure of the compound 1 revealed the formation of dianionic 3‐phenylprop‐2‐ene‐di‐amido ligand having an exocyclic olefinic bond derived from neutral α‐diimine fragment. However, analogous reactions with three different carbodiimides (RN=C=NR; R=Cy, iPr and tBu) with alkyl potassium and anhydrous calcium diiodide yielded corresponding homoleptic calcium compounds with amidinato ligand [κ2‐(THF)2Ca{RN=C(CH2Ph)NR}2] [R=Cy (2), iPr (3) and tBu (4)]. A separate reaction of DAD ligand, LiCH2SiMe3 and anhydrous ZnCl2 in diethylether solvent produced tri‐coordinated zinc compound [κ2‐ {DippN=C(CH2SiMe3)CH2NDipp}Zn[κ1‐{DippN=C(CH2SiMe3)CH2N‐Dipp}] (5) having amidinato moieties in the zinc coordination sphere in high yield. Molecular structures of compounds 2–5 in their solid states were also established.

Metal Organic Nanocapsules as Two-Dimensional Network Building Blocks

Metal–organic frameworks (MOFs) are a class of porous materials with a wide variety of applications, including molecular adsorption and separation. Recently, the first MOF based on the zinc-seamed pyrogallol[4]arene nanocapsule as a secondary building unit was reported. The zinc-seamed nanocapsules are linked together with 4,4′-bipyridine, which is a divergent ligand commonly used in the synthesis of MOFs. In an effort to identify other likely candidates for nanocapsular linking, electronic structure calculations were performed to determine energetic and geometric properties of (Zn(C2O2H3)2)1,2Y model complexes, which have been shown previously to reliably model the zinc coordination sphere found in the nanocapsules. Here, Y represents one of 17 divergent ligands with N, S, or O electron-donating atoms. MOFs were synthesized and characterized from two of the ligands suggested as most suitable for further experimental study.

Amidinate Ligands in Zinc coordination sphere: Synthesis and structural diversity

A one-pot reaction involving neosilyllithium and three different carbodiimides (RN=C=NR, R=cyclohexyl, isopropyl and tert-butyl) in diethyl ether, followed by the addition of anhydrous ZnCl2, afforded, in high yield, corresponding homoleptic zinc amidinate complexes having the molecular formulae [Zn{CyN =C(CH2SiMe3)NCy}2] (1), [Zn{ iPrN =C(CH2SiMe3)N iPr}2] (2) and [Zn{ tBuN =C(CH2SiMe3) NtBu}2] (3), respectively, and amidinato moieties in the zinc coordination sphere. Solid state structures of complexes 1-3 are reported thereafter - all the three complexes are isostructural, and each of them consists of two four-membered metallacycles. We report the syntheses and structural characterization of three homoleptic zinc amidinate complexes derived from one pot reaction of carbodiimides (RN=C=NR, R = cyclohexyl, isopropyl, tert-butyl), neosilyllithium and anhydrous zinc chloride in diethyl ether at ambient temperature 25 °C.

Chirality sensing with stereodynamic biphenolate zinc complexes.

Two bidentate ligands consisting of a fluxional polyarylacetylene framework with terminal phenol groups were synthesized. Reaction with diethylzinc gives stereodynamic complexes that undergo distinct asymmetric transformation of the first kind upon binding of chiral amines and amino alcohols. The substrate-to-ligand chirality imprinting at the zinc coordination sphere results in characteristic circular dichroism signals that can be used for direct enantiomeric excess (ee) analysis. This chemosensing approach bears potential for high-throughput ee screening with small sample amounts and reduced solvent waste compared to traditional high-performance liquid chromatography methods.

Crystal structure of a mixed-ligand dinuclear Ba-Zn complex with 2-meth-oxy-ethanol having tri-phenyl-acetate and chloride bridges.

The dinuclear barium-zinc complex, μ-chlorido-1:2κ(2) Cl:Cl-chlorido-2κCl-bis-(2-meth-oxy-ethanol-1κO)bis-(2-meth-oxy-ethanol-1κ(2) O,O')bis-(μ-tri-phenyl-acetato-1:2κ(2) O:O')bariumzinc, [BaZn(C20H15O2)2Cl2(C3H8O2)4], has been synthesized by the reaction of barium tri-phenyl-acetate, anhydrous zinc chloride and 2-meth-oxy-ethanol in the presence of toluene. The barium and zinc metal cations in the dinuclear complex are linked via one chloride anion and carboxyl-ate O atoms of the tri-phenyl-acetate ligands, giving a Ba⋯Zn separation of 3.9335 (11) Å. The irregular nine-coordinate BaO8Cl coordination centres comprise eight O-atom donors, six of them from 2-meth-oxy-ethanol ligands (four from two bidentate O,O'-chelate inter-actions and two from monodentate inter-actions), two from bridging tri-phenyl-acetate ligands and one from a bridging Cl donor. The distorted tetra-hedral coordination sphere of zinc comprises two O-atom donors from the tri-phenyl-acetate ligands and two Cl donors (one bridging and one terminal). In the crystal, O-H⋯Cl, O-H⋯O and C-H⋯Cl inter-molecular inter-actions form a layered structure, lying parallel to (001).

Polyol specificity of recombinant Arabidopsis thaliana sorbitol dehydrogenase studied by enzyme kinetics and in silico modeling.

Polyols are enzymatically-produced plant compounds which can act as compatible solutes during periods of abiotic stress. Nicotinamide adenine dinucleotide+-dependent SORBITOL DEHYDROGENASE (SDH, E. C. 1.1.1.14) from Arabidopsis thaliana L. sorbitol dehydrogenase (AtSDH) is capable of oxidizing several polyols including sorbitol, ribitol, and xylitol. In the present study, enzymatic assays using recombinant AtSDH demonstrated a higher specificity constant for xylitol compared to sorbitol and ribitol, all of which are C2 (S) and C4 (R) polyols. Enzyme activity was reduced by preincubation with ethylenediaminetetraacetic acid, indicating a requirement for zinc ions. In humans, it has been proposed that sorbitol becomes part of a pentahedric coordination sphere of the catalytic zinc during the reaction mechanism. In order to determine the validity of this pentahedric coordination model in a plant SDH, homology modeling, and Molecular Dynamics simulations of AtSDH ternary complexes with the three polyols were performed using crystal structures of human and Bemisia argentifolii (Genn.) (Hemiptera: Aleyrodidae) SDHs as scaffolds. The results indicate that the differences in interaction with structural water molecules correlate very well with the observed enzymatic parameters, validate the proposed pentahedric coordination of the catalytic zinc ion in a plant SDH, and provide an explanation for why AtSDH shows a preference for polyols with a chirality of C2 (S) and C4 (R).

Towards understanding the molecular recognition process in prokaryotic zinc-finger domain.

Eukaryotic Cys2His2 zinc finger domain is one of the most common and important structural motifs involved in protein-DNA interaction. The recognition motif is characterized by the tetrahedral coordination of a zinc ion by conserved cysteine and histidine residues. We have characterized the prokaryotic Cys2His2 zinc finger motif, included in the DNA binding region (Ros87) of Ros protein from Agrobacterium tumefaciens, demonstrating that, although possessing a similar zinc coordination sphere, this domain presents significant differences from its eukaryotic counterpart. Furthermore, basic residues flanking the zinc binding region on either side have been demonstrated, by Electrophoretic Mobility Shift Assay (EMSA) experiments, to be essential for Ros DNA binding. In spite of this wealth of knowledge, the structural details of the mechanism through which the prokaryotic zinc fingers recognize their target genes are still unclear. Here, to gain insights into the molecular DNA recognition process of prokaryotic zinc finger domains we applied a strategy in which we performed molecular docking studies using a combination of Nuclear Magnetic Resonance (NMR) and Molecular Dynamics (MD) simulations data. The results demonstrate that the MD ensemble provides a reasonable picture of Ros87 backbone dynamics in solution. The Ros87-DNA model indicates that the interaction involves the first two residue of the first α-helix, and several residues located in the basic regions flanking the zinc finger domain. Interestingly, the prokaryotic zinc finger domain, mainly with the C-terminal tail that is wrapped around the DNA, binds a more extended recognition site than the eukaryotic counterpart. Our analysis demonstrates that the introduction of the protein flexibility in docking studies can improve, in terms of accuracy, the quality of the obtained models and could be particularly useful for protein showing high conformational heterogeneity as well as for computational drug design applications.

Deciphering the zinc coordination properties of the prokaryotic zinc finger domain: The solution structure characterization of Ros87 H42A functional mutant

The zinc coordination sphere in prokaryotic zinc finger domain is extremely versatile and influences the stability and the folding property of the domain. Of a particular interest is the fourth zinc coordinating position, which is frequently occupied by two successive histidines, both able to coordinate the metal ion. To clarify their structural and functional role we report the NMR solution structure and the dynamics behavior of Ros87 H42A, which is a functional mutant of Ros87, the DNA binding domain of the Ros protein containing a prokaryotic Cys2His2 zinc finger domain. The structural analysis indicates that reducing the spacer among the two coordinating histidines from 4 (among His37 and His42) amino acids to 3 (among His37 and His41) increases the helicity of the first α-helix. At the same time, the second helix appears more mobile in the μs-ms timescale and the hydrophobic core is reduced. These data explain the high frequency of three-residue His spacers in the eukaryotic zinc finger domain and their absence in the prokaryotic counterpart. Furthermore, the structural comparison shows that the second coordination position is more sensitive to H42A mutation with respect to the first and the third position, providing the rationale of the high variability of the second and the fourth zinc coordinating position in Ros homologs, which adopt different metal coordination but preserve similar tertiary structures and DNA binding activities. Finally, H/D exchange measurements and NMR thermal unfolding analysis indicate that this mutant likely unfolds via a different mechanism with respect to the wild-type.