Peptide Hydrolysis by Metal-Cyclen Complexes and Their Analogues: Insights from Theoretical Studies

In the present DFT study, mechanisms of peptide hydrolysis by Co­(III)- and Cu­(II)-containing complexes of 1,4,7,10-tetraazacyclododecane (cyclen), <b>1-Co</b> and <b>1-Cu</b>, respectively, and 1-oxa-4,7,10-triazacyclododecane (oxacyclen), <b>2-Co</b> and <b>2-Cu</b>, respectively, and their analogues have been investigated. In addition, the effects of the ligand environment, pendant (an organic group containing a recognition site) and metal ion (Co­(III), Cu­(II), Ni­(II), Zn­(II), Cd­(II), and Pd­(II)), on the energetics of this reaction have been elucidated. The reactant of the <b>1-Co</b> complex exists in the <i>syn–anti</i> conformation, while that of <b>1-Cu</b> in the <i>syn–syn</i> form. For both these complexes, stepwise and concerted mechanisms were found to occur with similar barriers. The substitution of one of the nitrogen atoms in the cyclen macrocycle to create oxacyclen should occur at position 10 in the Co­(III) case and at position 4 in the Cu­(II) case. A comparison between the barriers using the common conformation (<i>syn–anti</i>) of <b>1-Co</b> and <b>2-Co</b> showed that both complexes hydrolyze the peptide bond with similar barriers, i.e., 39.8 kcal/mol for the former and 40.1 kcal/mol for the latter. This result is in line with the measured data that suggest that the oxacyclen complex exhibits just four times greater activity than the cyclen complex. The removal of the pendant (−C₂H₅) group in the Co­(III)- and Cu­(II)-cyclen complexes (<b>1</b>′<b>-Co</b> and <b>1</b>′<b>-Cu</b>, respectively) reduced the barriers by 9.3 and 3.0 kcal/mol, respectively. For <b>1</b>′<b>-Co</b>, the barrier of 30.5 kcal/mol is in agreement with the experimental value of 25.9 kcal/mol for the cleavage of myoglobin at pH 9.0 and 50 °C. The reactants of <b>1</b>′<b>-Cu</b>,<b> 1</b>′<b>-Zn</b>,<b> 1</b>′<b>-Pd</b>, and <b>1</b>′<b>-Cd</b> adopt the <i>syn–syn</i> conformation, whereas <b>1</b>′<b>-Ni</b> and <b>1</b>′<b>-Co</b> exist in the <i>syn–anti</i> geometry. The barriers for <b>1</b>′<b>-Ni</b> (triplet spin state), <b>1</b>′<b>-Cu</b> (doublet spin state), <b>1</b>′<b>-Cd</b> (singlet spin state), <b>1</b>′<b>-Co</b> (singlet spin state), and <b>1</b>′<b>-Zn</b> (singlet spin state) are similar, i.e., 27.2, 29.7, 30.5, 30.5, and 31.9 kcal/mol, respectively, and the highest barrier (41.5 kcal/mol) is computed for <b>1</b>′<b>-Pd</b> (singlet spin state)

Formation of Catalytically Active Binuclear Center of Glycerophosphodiesterase: A Molecular Dynamics Study

Glycerophosphodiesterase (GpdQ) is a binuclear metallophosphatase that catalyzes the hydrolytic cleavage of mono-, di-, and triphosphoester bonds of a wide range of critical molecules. Upon substrate binding, this enzyme undergoes a complex transformation from an inactive mononuclear form (<b>E</b>_<b>m</b>, where the metal resides in the α site) to an active binuclear center (<b>E</b>_<b>b</b><b>-S</b>, with metals bound to both the α and β sites) through a mononuclear, substrate-bound intermediate state (<b>E</b>_<b>m</b><b>-S</b>). In this study, all-atom molecular dynamics simulations have been employed to investigate structures and dynamical transformations in this process using eight different variants, i.e., five wild-type and three mutant forms of the enzyme. Additionally, the effects of an actual substrate, bis-(<i>para</i>-nitrophenyl) phosphate (b<i>p</i>NPP), a metal-bridging nucleophilic hydroxyl, and specific first and second coordination shell residues have been investigated. The initial binding of the substrate to <b>E</b>_<b>m</b> enhances the metal binding affinity of the α site and prepares the β site for coordination of the second metal ion. These results are in agreement with stopped-flow fluorescence and calorimetry data. In <b>E</b>_<b>b</b><b>-S</b>, the computed increase in the substrate and metal (both α and β) binding energies is also in line with the experimental data. However, removal of the substrate from this complex is found to cause substantial reduction in binding energies of both α and β metals. The role of the substrate in the creation and stabilization of the active site predicted in this study is supported by the kinetic measurements using both stopped-flow and nuclear magnetic resonance techniques. Importantly, residue Asn80, a ligand of the metal in the β site, exhibits coordination flexibility by acting as a gate in the formation of <b>E</b>_<b>b</b><b>-S</b>, in good agreement with mutagenesis and spectroscopic data

Mechanistic Insights into Metal (Pd²⁺, Co²⁺, and Zn²⁺)−β-Cyclodextrin Catalyzed Peptide Hydrolysis: A QM/MM Approach

In this study, mechanistic insights into the hydrolysis of an extremely stable tertiary peptide bond (Ser–Pro) in the Ser-Pro-Phe sequence by an artificial enzyme, metal (Pd²⁺, Co²⁺, or Zn²⁺)−β-cyclodextrin (CD) complex, have been provided. In particular, the exact reaction mechanism, the location of CD (number of −CH₂ groups downstream from the metal center), conformation of CD (primary or secondary rim of CD facing the substrate), the number of CD (one or two), and the optimum metal ion (Pd²⁺, Co²⁺, or Zn²⁺) have been suggested using a state-of-the-art hybrid quantum mechanics/molecular mechanics (QM/MM: B3LYP/Amber) approach. The QM/MM calculations suggest that the internal delivery mechanism is the most energetically feasible for the peptide hydrolysis. The inclusion of a CD ring at two CH₂ groups downstream from the metal center can provide 3 × 10⁵ times acceleration in the activity, while the replacement of Pd²⁺ with Co²⁺ enhances the rate activity another 3.7 × 10⁴ times

Hydrocarbons Depending on the Chain Length and Head Group Adopt Different Conformations within a Water-Soluble Nanocapsule: ¹H NMR and Molecular Dynamics Studies

In this study we have examined the conformational preference of phenyl-substituted hydrocarbons (alkanes, alkenes, and alkynes) of different chain lengths included within a confined space provided by a molecular capsule made of two host cavitands known by the trivial name “octa acid” (OA). One- and two-dimensional ¹H NMR experiments and molecular dynamics (MD) simulations were employed to probe the location and conformation of hydrocarbons within the OA capsule. In general, small hydrocarbons adopted a linear conformation while longer ones preferred a folded conformation. In addition, the extent of folding and the location of the end groups (methyl and phenyl) were dependent on the group (H₂C–CH₂, HCCH, and CC) adjacent to the phenyl group. In addition, the rotational mobility of the hydrocarbons within the capsule varied; for example, while phenylated alkanes tumbled freely, phenylated alkenes and alkynes resisted such a motion at room temperature. Combined NMR and MD simulation studies have confirmed that molecules could adopt conformations within confined spaces different from that in solution, opening opportunities to modulate chemical behavior of guest molecules

Investigating Polyoxometalate–Protein Interactions at Chemically Distinct Binding Sites

In this study, a combined molecular docking (rigid and flexible) and all-atom molecular dynamics simulations technique have been employed to investigate interactions of 1:1 Zr-containing Keggin polyoxometalate (ZrK) with four chemically distinct cleavage sites [Arg114–Leu115 (site 1), Ala257–Asp258 (site 2), Lys313–Asp314 (site 3), and Cys392–Glu393 (site 4)] of human serum albumin (HSA). The ZrK–HSA complexations were analyzed using electrostatic potentials, the chemical nature of amino acid residues, binding free energies, and secondary structures as parameters. They suggested that ZrK binds in a rather distinct manner to different cleavage sites, and its association was dominated by hydrogen bonding, both direct and solvent mediated, and electrostatic interactions, as suggested experimentally. The computed binding free interaction energies (−57.5, −24.2, −50.8, and −91.2 kJ/mol for sites 1, 2, 3, and 4, respectively) predicted the existence of one major binding site (site 4) and three minor binding sites (site 1, site 2, and site 3). The strong exothermicity of the binding was also supported by isothermal calorimetry experiments. Additionally, the binding of ZrK did not alter the overall α-helical secondary structure of HSA, which was in line with experimental observation. Furthermore, hydrolysis of the peptide bonds of the substrate was found to retain its overall structure. These results have provided a deeper understanding of the complex ZrK interactions with proteins, and they will lead to the design of the next generation of catalytically active polyoxometalates with improved hydrolytic activities

Effects of Ligand Environment in Zr(IV) Assisted Peptide Hydrolysis

In this DFT study, activities of 11 different N₂O₄, N₂O₃, and NO₂ core containing Zr­(IV) complexes, 4,13-diaza-18-crown-6 (<b>I′</b>_<b>N2O4</b>), 1,4,10-trioxa-7,13-diazacyclopentadecane (<b>I′</b>_<b>N2O3</b>), and 2-(2-methoxy)­ethanol (<b>I′</b>_<b>NO2</b>), respectively, and their analogues in peptide hydrolysis have been investigated. Based on the experimental information, these molecules were created by altering protonation states (singly protonated, doubly protonated, or doubly deprotonated) and number of their ligands. The energetics of the <b>I′</b>_<b>N2O4</b>, and <b>I′</b>_<b>NO2</b> and their analogues predicted that both stepwise and concerted mechanisms occurred either with similar barriers, or the latter was more favorable than the former. They also showed that the doubly deprotonated form hydrolyzed the peptide bond with substantially lower barriers than the barriers for other protonation states. For NO₂ core possessing complexes, Zr-(NO₂)­(OH^H)­(H₂O/OH)_<i>n</i> for <i>n</i> = 1–3, the hydroxyl group containing molecules were found to be more reactive than their water ligand possessing counterparts. The barriers for these complexes reduced with an increase in the coordination number (6–8) of the Zr­(IV) ion. Among all 11 molecules, the NO₂ core possessing and two hydroxyl group containing <b>I′</b>_{<b>DNO2–2H</b>} complex was found to be the most reactive complex with a barrier of 28.9 kcal/mol. Furthermore, barriers of 27.5, 28.9, and 32.0 kcal/mol for hydrolysis of Gly-Glu (negative), Gly-Gly (neutral), and Gly-Lys (positive) substrates, respectively, by this complex were in agreement with experiments. The activities of these complexes were explained in terms of basicity of their ligand environment and nucleophilicity of the Zr­(IV) center using metal–ligand distances, charge on the metal ion, and the metal–nucleophile distance as parameters. These results provide a deeper understanding of the functioning of these complexes and will help design Zr­(IV)-based synthetic metallopeptidases

Dimerization of the Full-Length Alzheimer Amyloid β-Peptide (Aβ42) in Explicit Aqueous Solution: A Molecular Dynamics Study

In this study, the mechanism of dimerization of the full-length Alzheimer amyloid beta (Aβ42) peptide and structural properties of the three most stable dimers have been elucidated through 0.8 μs classical molecular dynamics (MD) simulations. The Aβ42 dimer has been reported to be the smallest neurotoxic species that adversely affects both memory and synaptic plasticity. On the basis of interactions between the distinct regions of the Aβ42 monomer, 10 different starting configurations were developed from their native folded structures. However, only six of them were found to form dimers and among them the three most stable (<b>X</b>^<b>P</b>, <b>C</b>–<b>C</b>^<b>AP</b>, and <b>N</b>–<b>N</b>^<b>P</b>) were chosen for the detailed analysis. The structural properties of these dimers were compared with the available experimental and theoretical data. The MD simulations show that hydrophobic regions of both monomers play critical roles in the dimerization process. The high content of the α-helical structure in all the dimers is in line with its experimentally proposed role in the oligomerization. The formation of a zipper-like structure in <b>X</b>^<b>P</b> is also in accordance with its existence in the aggregates of several short amyloidogenic peptides. The computed values of translational (<i>D</i>_T) and rotational (<i>D</i>_R) diffusion constants of 0.63 × 10^–6 cm²/s and 0.035 ns^–1, respectively, for this dimer are supported by the corresponding values of the Aβ42 monomer. These simulations have also elucidated several other key structural properties of these peptides. This information will be very useful to design small molecules for the inhibition and disruption of the critical Aβ42 dimers

Unraveling the Photoluminescence Response of Light-Switching Ruthenium(II) Complexes Bound to Amyloid‑β

Photoluminescent molecules are widely used for real-time monitoring of peptide aggregation. In this Article, we detail both experimental and computational modeling to elucidate the interaction between [Ru­(bpy)₂dppz]²⁺ and amyloid-β (Aβ_1–40) aggregates. The transition from monomeric to fibrillar Aβ is of interest in the study of Alzheimer’s disease. Concentration-dependent experiments allowed the determination of a dissociation constant of 2.1 μM, while Job plots provided a binding stoichiometry of 2.6 Aβ monomers per [Ru­(bpy)₂dppz]²⁺. Our computational approach that combines molecular docking (both rigid and flexible) and all-atom molecular dynamics (MD) simulations predicts that the hydrophobic cleft between Val18 and Phe20 is a plausible binding site, which could also explain the increase in photoluminescence of [Ru­(bpy)₂dppz]²⁺ upon binding. This binding site is parallel to the fibril axis, in marked contrast to the binding site of these complexes in DNA (perpendicular to the DNA axis). Other binding sites may exist at the edges of the Aβ fibril, but they are actually of low abundance in an Aβ fibril several micrometers long. The assignment of the binding site was confirmed by binding studies in an Aβ fragment (Aβ_25–35) that lacked the amino acids necessary to form the binding site. The agreement between the experimental and computational work is remarkable and provides a general model that can be used for studying the interaction of amyloid-binding molecules to Aβ

Candidoses invasives en réanimation : données épidémiologiques, élaboration d’un score prédictif et mise au point de PCR pour le diagnostic

Patients in intensive care units (ICU) are at very high risk of invasive candidiasis associated with high mortality rate. Candida species are the third cause of septicemia. Clinical signs lack of specificity and blood cultures lack of sensitivity, and therefore the diagnosis remains a challenge. In order to improve the identification of patients with invasive candidiasis, predictive rules, biomarkers and PCR have been developed. The first part of this work describes the evolution over a ten years period in one ICU in Candida species distribution, susceptibility to antifungal drugs and consumption of antifungal agents. Changes in antifungal drug consumption were observed but they were not associated with significant changes in fungal ecology or with the emergence of resistant species. In a second part, we present a prospective, observational and bicentric study performed in 435 non-neutropenic patients in ICU. Several variables (risk factors of invasive candidiasis, Candida colonization, mannan antigen and anti-mannan antibodies) were analyzed and a predictive score of invasive candidiasis has been developed. Finally, the last part presents the development of Candida real-time PCR in blood, as well as the evaluation of a digital PCR.Les patients de réanimation sont des patients à très haut risque de survenue de candidoses invasives associées à une importante mortalité. Les espèces du genre Candida sont retrouvées en troisième position des agents infectieux les plus fréquemment isolés au cours des septicémies. Le diagnostic reste difficile en raison d’une clinique aspécifique et d’une sensibilité médiocre des hémocultures. Des scores prédictifs, des biomarqueurs ou encore des PCR ont été développés de manière à améliorer le diagnostic et l’identification des patients à risque. Dans ce travail, la première partie présente les données de l’évolution de l’écologie fongique, des candidoses invasives, des prescriptions d’antifongiques et des sensibilités aux antifongiques sur une période de dix ans dans un service de réanimation. Au cours de cette période, les changements observés dans la prescription d’antifongiques n’ont pas entrainé de modifications significatives de l’écologie fongique ni d’apparition de résistances. Dans une deuxième partie, nous présentons les résultats d’une étude prospective observationnelle bicentrique réalisée chez 435 patients non neutropéniques de réanimation. L’analyse de plusieurs variables (facteurs de risque de candidose invasive, colonisation à Candida sp., dosages d’antigène mannane et d’anticorps anti-mannane) a permis l’élaboration d’un score prédictif de survenue de candidose invasive. Finalement, la dernière partie du travail présente la mise au point de PCR Candida en temps réel dans le sang ainsi qu’une évaluation de la technologie de digital PCR

Snapshots of insulin dimers from MD simulations.

<p>The β-sheet character of each structure is shown in parentheses.</p