Interactions of Truncated Menaquinones in Lipid Monolayers and Bilayers.

Menaquinones (MK) are hydrophobic molecules that consist of a naphthoquinone headgroup and a repeating isoprenyl side chain and are cofactors used in bacterial electron transport systems to generate cellular energy. We have previously demonstrated that the folded conformation of truncated MK homologues, MK-1 and MK-2, in both solution and reverse micelle microemulsions depended on environment. There is little information on how MKs associate with phospholipids in a model membrane system and how MKs affect phospholipid organization. In this manuscript, we used a combination of Langmuir monolayer studies and molecular dynamics (MD) simulations to probe these questions on truncated MK homologues, MK-1 through MK-4 within a model membrane. We observed that truncated MKs reside farther away from the interfacial water than ubiquinones are are located closer to the phospholipid tails. We also observed that phospholipid packing does not change at physiological pressure in the presence of truncated MKs, though a difference in phospholipid packing has been observed in the presence of ubiquinones. We found through MD simulations that for truncated MKs, the folded conformation varied, but MKs location and association with the bilayer remained unchanged at physiological conditions regardless of side chain length. Combined, this manuscript provides fundamental information, both experimental and computational, on the location, association, and conformation of truncated MK homologues in model membrane environments relevant to bacterial energy production.

Escape of a Small Molecule from Inside T4 Lysozyme by Multiple Pathways.

The T4 lysozyme L99A mutant is often used as a model system to study small-molecule binding to proteins, but pathways for ligand entry and exit from the buried binding site and the associated protein conformational changes have not been fully resolved. Here, molecular dynamics simulations were employed to model benzene exit from its binding cavity using the weighted ensemble (WE) approach to enhance sampling of low-probability unbinding trajectories. Independent WE simulations revealed four pathways for benzene exit, which correspond to transient tunnels spontaneously formed in previous simulations of apo T4 lysozyme. Thus, benzene unbinding occurs through multiple pathways partially created by intrinsic protein structural fluctuations. Motions of several α-helices and side chains were involved in ligand escape from metastable microstates. WE simulations also provided preliminary estimates of rate constants for each exit pathway. These results complement previous works and provide a semiquantitative characterization of pathway heterogeneity for binding of small molecules to proteins.

Partition, orientation and mobility of ubiquinones in a lipid bilayer.

Ubiquinone is the universal mobile charge carrier involved in biological electron transfer processes. Its redox properties and biological function depend on the molecular partition and lateral diffusion over biological membranes. However, ubiquinone localization and dynamics within lipid bilayers are long debated and still uncertain. Here we present molecular dynamics simulations of several ubiquinone homologs with variable isoprenoid tail lengths complexed to phosphatidylcholine bilayers. Initially, a new force-field parametrization for ubiquinone is derived from and compared to high level quantum chemical data. Free energy profiles for ubiquinone insertion in the lipid bilayer are obtained with the new force-field. The profiles allow for the determination of the equilibrium location of ubiquinone in the membrane as well as for the validation of the simulation model by direct comparison with experimental partition coefficients. A detailed analysis of structural properties and interactions shows that the ubiquinone polar head group is localized at the water-bilayer interface at the same depth of the lipid glycerol groups and oriented normal to the membrane plane. Both the localization and orientation of ubiquinone head groups do not change significantly when increasing the number of isoprenoid units. The isoprenoid tail is extended and packed with the lipid acyl chains. For ubiquinones with long tails, the terminal isoprenoid units have high flexibility. Calculated ubiquinone diffusion coefficients are similar to that found for the phosphatidylcholine lipid. These results may have further implications for the mechanisms of ubiquinone transport and binding to respiratory and photosynthetic protein complexes.

Ferric-Thiolate Bond Dissociation Studied with Electronic Structure Calculations.

The stability and reactivity of iron-sulfur clusters are fundamental properties for the biological function of these prosthetic groups. Here, we investigate the ferric-thiolate bond dissociation of model iron-sulfur tetrahedral complexes with high-level ab initio multiconfigurational electronic structure calculations. We find that the reaction mechanism is homolytic with a spin-crossing from the sextet state in the reactant to quartet state in the product. We also compare several density functionals and semiempirical configuration interaction with the high-level ab initio results to find an accurate but computationally more efficient method to describe the reaction. The functionals M06 and those based on the OPTX exchange functional show the best performance and may reasonably describe the various electron correlation effects involved in ferric-thiolate bond dissociation.

Ligand-receptor affinities computed by an adapted linear interaction model for continuum electrostatics and by protein conformational averaging.

Accurate calculations of free energies involved in small-molecule binding to a receptor are challenging. Interactions between ligand, receptor, and solvent molecules have to be described precisely, and a large number of conformational microstates has to be sampled, particularly for ligand binding to a flexible protein. Linear interaction energy models are computationally efficient methods that have found considerable success in the prediction of binding free energies. Here, we parametrize a linear interaction model for implicit solvation with coefficients adapted by ligand and binding site relative polarities in order to predict ligand binding free energies. Results obtained for a diverse series of ligands suggest that the model has good predictive power and transferability. We also apply implicit ligand theory and propose approximations to average contributions of multiple ligand-receptor poses built from a protein conformational ensemble and find that exponential averages require proper energy discrimination between plausible binding poses and false-positives (i.e., decoys). The linear interaction model and the averaging procedures presented can be applied independently of each other and of the method used to obtain the receptor structural representation.

Highly Dynamic Polynuclear Metal Cluster Revealed in a Single Metallothionein Molecule.

Human metallothionein (MT) is a small-size yet efficient metal-binding protein, playing an essential role in metal homeostasis and heavy metal detoxification. MT contains two domains, each forming a polynuclear metal cluster with an exquisite hexatomic ring structure. The apoprotein is intrinsically disordered, which may strongly influence the clusters and the metal-thiolate (M-S) bonds, leading to a highly dynamic structure. However, these features are challenging to identify due to the transient nature of these species. The individual signal from dynamic conformations with different states of the cluster and M-S bond will be averaged and blurred in classic ensemble measurement. To circumvent these problems, we combined a single-molecule approach and multiscale molecular simulations to investigate the rupture mechanism and chemical stability of the metal cluster by a single MT molecule, focusing on the Zn4S11 cluster in the α domain upon unfolding. Unusual multiple unfolding pathways and intermediates are observed for both domains, corresponding to different combinations of M-S bond rupture. None of the pathways is clearly preferred suggesting that unfolding proceeds from the distribution of protein conformational substates with similar M-S bond strengths. Simulations indicate that the metal cluster may rearrange, forming and breaking metal-thiolate bonds even when MT is folded independently of large protein backbone reconfiguration. Thus, a highly dynamic polynuclear metal cluster with multiple conformational states is revealed in MT, responsible for the binding promiscuity and diverse cellular functions of this metal-carrier protein.

Flexibility and inhibitor binding in cdc25 phosphatases.

Cdc25 phosphatases involved in cell cycle checkpoints are now active targets for the development of anti-cancer therapies. Rational drug design would certainly benefit from detailed structural information for Cdc25s. However, only apo- or sulfate-bound crystal structures of the Cdc25 catalytic domain have been described so far. Together with previously available crystalographic data, results from molecular dynamics simulations, bioinformatic analysis, and computer-generated conformational ensembles shown here indicate that the last 30-40 residues in the C-terminus of Cdc25B are partially unfolded or disordered in solution. The effect of C-terminal flexibility upon binding of two potent small molecule inhibitors to Cdc25B is then analyzed by using three structural models with variable levels of flexibility, including an equilibrium distributed ensemble of Cdc25B backbone conformations. The three Cdc25B structural models are used in combination with flexible docking, clustering, and calculation of binding free energies by the linear interaction energy approximation to construct and validate Cdc25B-inhibitor complexes. Two binding sites are identified on top and beside the Cdc25B active site. The diversity of interaction modes found increases with receptor flexibility. Backbone flexibility allows the formation of transient cavities or compact hydrophobic units on the surface of the stable, folded protein core that are unexposed or unavailable for ligand binding in rigid and densely packed crystal structures. The present results may help to speculate on the mechanisms of small molecule complexation to partially unfolded or locally disordered proteins.

Effects of lipid composition on membrane distribution and permeability of natural quinones.

Natural quinones are amphiphilic molecules that function as mobile charge carriers in biological energy transduction. Their distribution and permeation across membranes are important for binding to enzymatic complexes and for proton translocation. Here, we employ molecular dynamics simulations and free energy calculations with a carefully calibrated classical force-field to probe quinone distribution and permeation in a multi-component bilayer trying to mimic the composition of membranes involved in bioenergetic processes. Ubiquinone, ubiquinol, plastoquinone and menaquinone molecules with short and long isoprenoid tails are simulated. We find that penetration of water molecules bound to the polar quinone head increases considerably in the less ordered and porous bilayer formed by di-linoleoyl (18:2) phospholipids, resulting in a lower free energy barrier for quinone permeation and faster transversal diffusion. In equilibrium, quinone and quinol heads localize preferentially near lipid glycerol groups, but do not perform specific contacts with lipid polar heads. Quinone distribution is not altered significantly by the quinone head, tail and lipid composition in comparison to a single-component bilayer. This study highlights the role of lipid acyl chain unsaturation for permeation and transversal diffusion of polar molecules across biological membranes.

The catalytic acid in the dephosphorylation of the Cdk2-pTpY/CycA protein complex by Cdc25B phosphatase.

The development of anticancer therapeutics that target Cdc25 phosphatases is now an active area of research. A complete understanding of the Cdc25 catalytic mechanism would certainly allow a more rational inhibitor design. However, the identity of the catalytic acid used by Cdc25 has been debated and not established unambiguously. Results of molecular dynamics simulations with a calibrated hybrid potential for the first reaction step catalyzed by Cdc25B in complex with its natural substrate, the Cdk2-pTpY/CycA protein complex, are presented here. The calculated reaction free-energy profiles are in very good agreement with experimental measurements and are used to discern between different proposals for the general acid. In addition, the simulations give useful insight on interactions that can be explored for the design of inhibitors specific to Cdc25.

Mechanical Unfolding of Macromolecules Coupled to Bond Dissociation.

Single-molecule force spectroscopy has become a powerful tool to investigate molecular mechanisms in biophysics and materials science. In particular, the new field of polymer mechanochemistry has emerged to study how tension may induce chemical reactions in a macromolecule. A rich example is the mechanical unfolding of the metalloprotein rubredoxin coupled to dissociation of iron-sulfur bonds that has recently been studied in detail by atomic force microscopy. Here, we present a simple molecular model composed of a classical all-atom force field description, implicit solvation, and steered molecular dynamics simulation to describe the mechanical properties and mechanism of forced unfolding coupled to covalent bond dissociation of macromolecules. We apply this model and test it extensively to simulate forced rubredoxin unfolding, and we dissect the sensitivity of the calculated mechanical properties with model parameters. The model provides a detailed molecular explanation of experimental observables such as force-extension profiles and contour length increments. Changing the points of force application along the macromolecule results in different unfolding mechanisms, characterized by disruption of hydrogen bonds and secondary protein structure, and determines the degree of solvent access to the reactive center. We expect that this molecular model will be broadly applicable to simulate (bio)polymer mechanochemistry.

Approximate Multiconfigurational Treatment of Spin-Coupled Metal Complexes.

The weak interaction between unpaired electrons in polynuclear transition-metal complexes is often described by exchange and spin polarization mechanisms. The resulting intrinsic multiconfigurational electronic structure for such complexes may be calculated with wave function-based methods (e.g., complete active space configuration interaction and complete active space self-consistent field), but computations become extremely demanding and even unfeasible for polynuclear complexes with a large number of open-shells. Here, several levels of selection of configurations and symmetry considerations that still capture the essential physics of exchange and spin polarization mechanisms are presented. The proposed approximations result in significantly smaller configuration interaction expansions and are equally valid for ab initio and semiempirical methods. Tests are performed in simple molecular systems and in small transition-metal complexes that cover a range of valence and charge states. In particular, superexchange contributions can be calculated to good accuracy using only single ionic excitations. Further reduction in the size of the configuration expansions is possible but restricts the description to low-lying spin ladders. The proposed configuration interaction schemes may be used to resolve space and spin symmetries in the calculation of electronic structures, exchange coupling constants, and other properties pertinent to polynuclear transition-metal complexes.

Specific parametrisation of a hybrid potential to simulate reactions in phosphatases.

Phosphatases are key biomolecules because they regulate many cellular processes. These enzymes have been studied for many years, but there are still doubts about the catalytic mechanism. Computer simulations can be used to shed light on these questions. Here we develop a new and specific parametrisation, and present extensive tests of a hybrid potential that can be used to reliably simulate reactions catalysed by phosphatases. High level ab initio data for phosphate ester thiolysis and alcoholysis is used in the training set. The parametrised quantum mechanical Hamiltonian reproduces ab initio energies with a root mean-squared deviation of 3 kcal mol(-1) for species along the pathway of various phosphate ester reactions. Preliminary results for simulation with the calibrated hybrid potential of catalysis by the phosphatase VHR indicate the calculated reaction barriers are in very good agreement with experiment.

Thiolysis and alcoholysis of phosphate tri- and monoesters with alkyl and aryl leaving groups. An ab initio study in the gas phase.

Phosphate esters are important compounds in living systems. Their biological reactions with alcohol and thiol nucleophiles are catalyzed by a large superfamily of phosphatase enzymes. However, very little is known about the intrinsic reactivity of these nucleophiles with phosphorus centers. We have performed ab initio calculations on the thiolysis and alcoholysis at phosphorus of trimethyl phosphate, dimethyl phenyl phosphate, methyl phosphate, and phenyl phosphate. Results in the gas phase are a reference for the study of the intrinsic reactivity of these compounds. Thiolysis of triesters was much slower and less favorable than the corresponding alcoholysis. Triesters reacted through an associative mechanism. Monoesters can react by both associative and dissociative mechanisms. The basicity of the attacking and leaving groups and the possibility of proton transfers can modulate the reaction mechanisms. Intermediates formed along associative reactions did not follow empirically proposed rules for ligand positioning. Our calculations also allow re-interpretation of some experimental results, and new experiments are proposed to trace reactions that are normally not observed, both in the gas phase and in solution.

Sobre as proteínas tirosina-fosfatases. Reatividade intrínseca de ésteres de fosfato e simulação computacional dos mecanismos de reação enzimática / About the protein tyrosine phosphatases. Intrinsic reactivity of phosphate esters and computational simulation of the mechanisms of enzymatic reaction

Proteínas tirosina-fosfatases (PTPs) catalisam a hidrólise de fosfotirosina de outras proteínas e, assim, regulam importantes processos bioquímicos. Dois representantes desta família são as fosfatases de dupla especificidade VHR e CDC25B. A primeira etapa de reação catalisada é um ataque nucleofílico da cadeia lateral de uma cisteína sobre o fósforo do substrato, com uma possível transferência de H+ de um ácido geral para o grupo de saída, formando uma PTP intermediária tiofosforilada e desfosforilando o substrato. Dúvidas ainda persistem sobre esta etapa, envolvendo os estados de protonação do substrato e do nucleófilo enzimático, a inatividade de certos mutantes e a identificação do cido geral nas CDC25s...