VWD domain stabilization by autocatalytic Asp-Pro cleavage.

Domains known as von Willebrand factor type D (VWD) are found in extracellular and cell-surface proteins including von Willebrand factor, mucins, and various signaling molecules and receptors. Many VWD domains have a glycine-aspartate-proline-histidine (GDPH) amino-acid sequence motif, which is hydrolytically cleaved post-translationally between the aspartate (Asp) and proline (Pro). The Fc IgG binding protein (FCGBP), found in intestinal mucus secretions and other extracellular environments, contains 13 VWD domains, 11 of which have a GDPH cleavage site. In this study, we investigated the structural and biophysical consequences of Asp-Pro peptide cleavage in a representative FCGBP VWD domain. We found that endogenous Asp-Pro cleavage increases the resistance of the domain to exogenous proteolytic degradation. Tertiary structural interactions made by the newly generated chain termini, as revealed by a crystal structure of an FCGBP segment containing the VWD domain, may explain this observation. Notably, the Gly-Asp peptide bond, upstream of the cleavage site, assumed the cis configuration in the structure. In addition to these local features of the cleavage site, a global organizational difference was seen when comparing the FCGBP segment structure with the numerous other structures containing the same set of domains. Together, these data illuminate the outcome of GDPH cleavage and demonstrate the plasticity of proteins with VWD domains, which may contribute to their evolution for function in a dynamic extracellular environment.

Is "Water in Salt" Electrolytes the Ultimate Solution? Achieving High Stability of Organic Anodes in Diluted Electrolyte Solutions Via a Wise Anions Selection.

The introduction of the water-in-salt (WIS) electrolytes concept to prevent water splitting and widen the electrochemical stability window, has spurred extensive research efforts toward development of improved aqueous batteries. The successful implementation of these electrolyte solutions in many electrochemical systems shifts the focus from diluted to WIS electrolyte solutions. Considering the high costs and the tendency of these nearly saturated solutions to crystallize, this trend can be carefully re-evaluated. Herein we show that the stability of organic electrodes comprising the active material perylene-3,4,9,10-tetracarboxylic dianhydride (PTCDA), is strongly influenced by the solvation character of the anions rather than the concentration of the electrolyte solution. Even though the charging process of PTCDA involves solely insertion of cations (i.e., principal counter-ions), surprisingly, the dominant factor influencing its electrochemical performance, including long-term electrode stability, is the type of the co-ions (i.e., electrolytic anions). Using systematic electrochemical analysis combined with theoretical simulations, we show that the selection of kosmotropic anions results in fast fading of the PTCDA anodes, while a selection of chaotropic anions leads to excellent stability, even at electrolytes concentrations as low as 0.2 M. These findings provide a new conceptual approach for designing advanced electrolyte solutions for aqueous batteries.

Origin of Catalysis by Nitroalkane Oxidase.

The rate of proton abstraction of the carbon acid nitroethane by Asp402 is accelerated by a factor of 108 in the enzyme nitroalkane oxidase (NAO) relative to that by the organic base acetate ion in water. The Cα proton of nitroalkanes is known to exhibit an abnormal correlation between its acidity strength and the rate of deprotonation, with an unusually slow rate of deprotonation in water. This work examines the origin of NAO catalysis, revealing that the rate enhancement by the enzyme is due to transition-state stabilization, restoring the normal behavior of the linear free energy relationship of Bronsted acids. Interestingly, NAO employs the ubiquitous cofactor flavin adenosine diphosphate (FAD) to perform the subsequent oxidation. Does the FAD cofactor also affect the catalytic rate of the initial proton transfer process of the overall nitroalkane oxidation? Classical molecular dynamics and path-integral simulations using a reaction-specific combined quantum mechanics/molecular mechanics (QM/MM) approach were carried out to obtain the free energy reaction profiles, or the potentials of mean force, for the enzymatic reaction and for a model reaction in aqueous solution, as well as for the 2'-deoxy-FAD co-factor-modified NAO. Free energy perturbation calculations suggest that transition-state stabilization of the reactive fragment is the primary cause of the catalytic effect. It is found that the FAD cofactor plays a crucial role in increasing the Cα proton acidity, via specific hydrogen bonding and π-stacking interactions, although these factors have a smaller effect on the enhancement of the rate of deprotonation. Model QM calculations of the π-stacking complexes between the FAD isoalloxazine ring and the neutral and anionic nitroethane, respectively, reveal that the anionic π-stacking complex is more stable than the neutral one by 15.7 kcal/mol, and a net π-stacking energy of 17.3 kcal/mol is obtained. Hence, the isoalloxazine ring, in addition to serving as a very potent oxidizing agent via the formation of covalent intermediate structures, is able to exert a considerable amount of catalytic effect through noncovalent π-stacking interactions.

Cooperative Intrinsic Basicity and Hydrogen Bonding Render SmI2 More Azaphilic than Oxophilic.

It has been recently shown that SmI2 is more azaphilic than oxophilic. Density functional theory calculations reveal that coordination of 1-3 molecules of ethylenediamine is more exothermic by up to 10 kcal/mol than coordination of the corresponding number of ethylene glycol molecules. Taking into account also hydrogen bonds between ligands and tetrahydrofuran doubles this preference. The intrinsic affinity parallels the order of basicity. The cooperativity with the hydrogen bonding makes SmI2 more azaphilic than oxophilic.

Copper coordination states affect the flexibility of copper Metallochaperone Atox1: Insights from molecular dynamics simulations.

Copper is an essential element in nature but in excess, it is toxic to the living cell. The human metallochaperone Atox1 participates in copper homeostasis and is responsible for copper transmission. In a previous multiscale simulation study, we noticed a change in the coordination state of the Cu(I) ion, from 4 bound cysteine residues to 3, in agreement with earlier studies. Here, we perform and analyze classical molecular dynamic simulations of various coordination states: 2, 3, and 4. The main observation is an increase in protein flexibility as a result of a decrease in the coordination state. In addition, we identified several populated conformations that correlate well with double electron-electron resonance distance distributions or an X-ray structure of Cu(I)-bound Atox1. We suggest that the increased flexibility might benefit the process of ion transmission between interacting proteins. Further experiments can scrutinize this hypothesis and shed additional light on the mechanism of action of Atox1.

A Benchmark Study of Quantum Mechanics and Quantum Mechanics-Molecular Mechanics Methods for Carbocation Chemistry.

Carbocations play key roles in classical organic reactions and have also been implicated in several enzyme families. A hallmark of carbocation chemistry is multitudes of competing reaction pathways, and to be able to distinguish between pathways with quantum chemical calculations, it is necessary to approach chemical accuracy for relative energies between carbocations. Here, we present an extensive study of the performance of selected density functional theory (DFT) methods in describing the thermochemistry and kinetics of carbocations and their corresponding neutral alkenes both in the gas-phase and within a hybrid quantum mechanics-molecular mechanics (QM/MM) framework. The density functionals are benchmarked against accurate ab initio methods such as CBS-QB3 and DLPNO-CCSD(T). Based on the findings in the gas-phase calculations of carbocations and alkenes, the best functionals are chosen and tested further for non-covalent interactions in model systems using QM and QM/MM methods. We compute the interaction energies between a model carbocation/alkane and model π, dipole, and hydrophobic systems using DFT and QM(DFT)/MM and compare with DLPNO-CCSD(T). These latter model systems are representative of side chains of amino acids such as phenylalanine/tyrosine, tryptophan, asparagine/glutamine, serine/threonine, methionine, and other hydrophobic groups. The Lennard-Jones parameters of the QM atoms in QM(DFT)/MM calculations are modified to obtain an optimal fit with the QM energies. Finally, a selected carbocation reaction is studied in the gas phase and in implicit chloroform solvent using QM and in explicit chloroform solvent using QM/MM and umbrella sampling simulations. This study highlights the highest accuracy possible with selected density functionals and QM/MM methods but also some limitations in using QM/MM methods for carbocation systems.

Molecular Dynamics Simulations of the Apo and Holo States of the Copper Binding Protein CueR Reveal Principal Bending and Twisting Motions.

Copper is essential for proper functioning of cells but is dangerous in unregulated concentrations. One of the members in the bacterial system responsible for facilitating copper homeostasis is the copper efflux regulator (CueR) protein. Upon copper binding, CueR induces transcription of additional copper homeostasis proteins via a cascade of events. There are some available crystal structures of CueR, in the holo (copper-bound), active (copper- and DNA-bound), and repressed (only DNA-bound) states, and these structures suggest that transcription initiation involves a distortion in the promoter DNA strand. In this work, we study the dynamic behavior of the protein, using molecular dynamics simulations, and compare with available electron paramagnetic resonance measurements for validation. We develop simple force-field parameters to describe the copper-binding motif, thus enabling the use of simplified, classical physics equations. This enabled us to access reasonable simulation times that illustrate global motions of the protein. Both in the holo and apo states of CueR, we observed large-scale helical bending motions that could be involved in the bending of a bound DNA molecule so that transcription activation can take place. Additionally, copper binding might afford increased rigidification of the active state via helix α6.

Enhancement of Structural, Electrochemical, and Thermal Properties of High-Energy Density Ni-Rich LiNi0.85Co0.1Mn0.05O2 Cathode Materials for Li-Ion Batteries by Niobium Doping.

Ni-rich layered oxide LiNi1 - x - yCoxMnyO2 (1 - x - y > 0.5) materials are favorable cathode materials in advanced Li-ion batteries for electromobility applications because of their high initial discharge capacity. However, they suffer from poor cycling stability because of the formation of cracks in their particles during operation. Here, we present improved structural stability, electrochemical performance, and thermal durability of LiNi0.85Co0.1Mn0.05O2(NCM85). The Nb-doped cathode material, Li(Ni0.85Co0.1Mn0.05)0.997Nb0.003O2, has enhanced cycling stability at different temperatures, outstanding capacity retention, improved performance at high discharge rates, and a better thermal stability compared to the undoped cathode material. The high electrochemical performance of the doped material is directly related to the structural stability of the cathode particles. We further propose that Nb-doping in NCM85 improves material stability because of partial reduction of the amount of Jahn-Teller active Ni3+ ions and formation of strong bonds between the dopant and the oxygen ions, based on density functional theory calculations. Structural studies of the cycled cathodes reveal that doping with niobium suppresses the formation of cracks during cycling, which are abundant in the undoped cycled material particles. The Nb-doped NCM85 cathode material also displayed superior thermal characteristics. The coherence between the improved electrochemical, structural, and thermal properties of the doped material is discussed and emphasized.

Temperature-Dependent Kinetic Isotope Effects in R67 Dihydrofolate Reductase from Path-Integral Simulations.

Calculation of temperature-dependent kinetic isotope effects (KIE) in enzymes presents a significant theoretical challenge. Additionally, it is not trivial to identify enzymes with available experimental accurate intrinsic KIEs in a range of temperatures. In the current work, we present a theoretical study of KIEs in the primitive R67 dihydrofolate reductase (DHFR) enzyme and compare with experimental work. The advantage of R67 DHFR is its significantly lower kinetic complexity compared to more evolved DHFR isoforms. We employ mass-perturbation-based path-integral simulations in conjunction with umbrella sampling and a hybrid quantum mechanics-molecular mechanics Hamiltonian. We obtain temperature-dependent KIEs in good agreement with experiments and ascribe the temperature-dependent KIEs primarily to zero-point energy effects. The active site in the primitive enzyme is found to be poorly preorganized, which allows excessive water access to the active site and results in loosely bound reacting ligands.

The Impression of a Nonexisting Catalytic Effect: The Role of CotB2 in Guiding the Complex Biosynthesis of Cyclooctat-9-en-7-ol.

Terpene synthases generate terpenes employing diversified carbocation chemistry, including highly specific ring formations, proton and hydride transfers, and methyl as well as methylene migrations, followed by reaction quenching. In this enzyme family, the main catalytic challenge is not rate enhancement, but rather structural and reactive control of intrinsically unstable carbocations in order to guide the resulting product distribution. Here we employ multiscale modeling within classical and quantum dynamics frameworks to investigate the reaction mechanism in the diterpene synthase CotB2, commencing with the substrate geranyl geranyl diphosphate and terminating with the carbocation precursor to the final product cyclooctat-9-en-7-ol. The 11-step in-enzyme carbocation cascade is compared with the same reaction in the absence of the enzyme. Remarkably, the free energy profiles in gas phase and in CotB2 are surprisingly similar. This similarity contrasts the multitude of strong π-cation, dipole-cation, and ion-pair interactions between all intermediates in the reaction cascade and the enzyme, suggesting a remarkable balance of interactions in CotB2. We ascribe this balance to the similar magnitude of the interactions between the carbocations along the reaction coordinate and the enzyme environment. The effect of CotB2 mutations is studied using multiscale mechanistic docking, machine learning, and X-ray crystallography, pointing the way for future terpene synthase design.

Enzymatic control of product distribution in terpene synthases: insights from multiscale simulations.

In this opinion, we review some recent work on terpene biosynthesis using multiscale simulation approaches, with special focus on contributions from our group. Terpene synthases generate terpenes employing rich carbocation chemistry, including highly specific ring formations, proton, hydride, methyl, and methylene migrations, followed by reaction quenching. In these enzymes, the main catalytic challenge is not rate enhancement, but rather control of intrinsically reactive carbocations and the resulting product distribution. Herein, we review multiscale simulations of selected mono-, sesqui-, and diterpene synthases. We point to the many tools adopted by terpene synthases to achieve correct substrate fold, carbocation formation, carbocation reaction environment, and reaction quenching. A better understanding of the toolbox employed by terpene synthases is expected to aid in the search for new enzymatic and biomimetic synthetic routes to natural and unnatural terpenes.

Cu(I) Controls Conformational States in Human Atox1 Metallochaperone: An EPR and Multiscale Simulation Study.

Atox1 is a human copper metallochaperone that is responsible for transferring copper ions from the main human copper transporter, hCtr1, to ATP7A/B in the Golgi apparatus. Atox1 interacts with the Ctr1 C-terminal domain as a dimer, although it transfers the copper ions to ATP7A/B in a monomeric form. The copper binding site in the Atox1 dimer involves Cys12 and Cys15, while Lys60 was also suggested to play a role in the copper binding. We recently showed that Atox1 can adopt various conformational states, depending on the interacting protein. In the current study, we apply EPR experiments together with hybrid quantum mechanics-molecular mechanics molecular dynamics simulations using a recently developed semiempirical density functional theory approach, to better understand the effect of Atox1's conformational states on copper coordination. We propose that the flexibility of Atox1 occurs owing to protonation of one or more of the cysteine residues, and that Cys15 is an important residue for Atox1 dimerization, while Cys12 is a critical residue for Cu(I) binding. We also show that Lys60 electrostatically stabilizes the Cu(I)-Atox1 dimer.

Role of Microsolvation and Quantum Effects in the Accurate Prediction of Kinetic Isotope Effects: The Case of Hydrogen Atom Abstraction in Ethanol by Atomic Hydrogen in Aqueous Solution.

Hydrogen abstraction from ethanol by atomic hydrogen in aqueous solution is studied using two theoretical approaches: the multipath variational transition state theory (MP-VTST) and a path-integral formalism in combination with free-energy perturbation and umbrella sampling (PI-FEP/UM). The performance of the models is compared to experimental values of H kinetic isotope effects (KIE). Solvation models used in this study ranged from purely implicit, via mixed-microsolvation treated quantum mechanically via the density functional theory (DFT) to fully explicit representation of the solvent, which was incorporated using a combined quantum mechanical-molecular mechanical (QM/MM) potential. The effects of the transition state conformation and the position of microsolvating water molecules interacting with the solute on the KIE are discussed. The KIEs are in good agreement with experiment when MP-VTST is used together with a model that includes microsolvation of the polar part of ethanol by five or six water molecules, emphasizing the importance of explicit solvation in KIE calculations. Both, MP-VTST and PI-FEP/UM enable detailed characterization of nuclear quantum effects accompanying the hydrogen atom transfer reaction in aqueous solution.

Current understanding and biotechnological application of the bacterial diterpene synthase CotB2.

CotB2 catalyzes the first committed step in cyclooctatin biosynthesis of the soil bacterium Streptomyces melanosporofaciens. To date, CotB2 represents the best studied bacterial diterpene synthase. Its reaction mechanism has been addressed by isoptope labeling, targeted mutagenesis and theoretical computations in the gas phase, as well as full enzyme molecular dynamic simulations. By X-ray crystallography different snapshots of CotB2 from the open, inactive, to the closed, active conformation have been obtained in great detail, allowing us to draw detailed conclusions regarding the catalytic mechanism at the molecular level. Moreover, numerous alternative geranylgeranyl diphosphate cyclization products obtained by CotB2 mutagenesis have exciting applications for the sustainable production of high value bioactive substances.

Crystal structure of LepI, a multifunctional SAM-dependent enzyme which catalyzes pericyclic reactions in leporin biosynthesis.

LepI is a novel multifunctional enzyme that catalyzes stereoselective dehydration, Diels-Alder reaction, and retro-Claisen rearrangement. Here we report the crystal structure of LepI in complex with its co-factor S-adenosyl methionine (SAM). LepI forms a tetramer via the N-terminal helical domain and binds to a SAM molecule in the C-terminal catalytic domain. The binding modes of various LepI substrates are investigated by docking simulations, which suggest that the substrates are bound via both hydrophobic and hydrophilic forces, as well as cation-π interactions with the positively charged SAM. The reaction starts with a dehydration step in which H133 possibly deprotonates the pyridone hydroxyl group of the substrate, while D296 might protonate an alkyl-chain hydroxyl group. Subsequent pericyclization may be facilitated by the correct fold of the substrate's alkyl chain and a thermodynamic driving force towards σ-bonds at the expense of π-bonds. These results provide structural insights into LepI catalysis and are important in understanding the mechanism of enzymatic pericyclization.

Endogenous Dynamic Nuclear Polarization for Natural Abundance 17O and Lithium NMR in the Bulk of Inorganic Solids.

In recent years magic angle spinning-dynamic nuclear polarization (MAS-DNP) has developed as an excellent approach for boosting the sensitivity of solid-state NMR (ssNMR) spectroscopy, thereby enabling the characterization of challenging systems in biology and chemistry. Most commonly, MAS-DNP is based on the use of nitroxide biradicals as polarizing agents. In materials science, since the use of nitroxides often limits the signal enhancement to the materials' surface and subsurface layers, there is need for hyperpolarization approaches which will provide sensitivity in the bulk of micron sized particles. Recently, an alternative in the form of paramagnetic metal ions has emerged. Here we demonstrate the remarkable efficacy of Mn(II) dopants, used as endogenous polarization agents for MAS-DNP, in enabling the detection of 17O at a natural abundance of only 0.038%. Distinct oxygen sites are identified in the bulk of micron-sized crystals, including battery anode materials Li4Ti5O12 (LTO) and Li2ZnTi3O8, as well as the phosphor materials NaCaPO4 and MgAl2O4, all doped with Mn(II) ions. Density functional theory calculations are used to assign the resonances to specific oxygen environments in these phases. Depending on the Mn(II) dopant concentration, we obtain significant signal enhancement factors, 142 and 24, for 6Li and 7Li nuclei in LTO, respectively. We furthermore follow the changes in the 6,7Li LTO resonances and determine their enhancement factors as a function of Mn(II) concentration. The results presented show that MAS-DNP from paramagnetic metal ion dopants provides an efficient approach for probing informative nuclei such as 17O, despite their low gyromagnetic ratio and negligible abundance, without isotope enrichment.

Evolutionary Effects on Bound Substrate p Ka in Dihydrofolate Reductase.

In the present study, we address the effect of active site structure and dynamics of different dihydrofolate reductase (DHFR) isoforms on the p Ka of the bound substrate 7,8-dihydrofolate, in an attempt to understand possible evolutionary trends. We apply a hybrid QM/MM free energy perturbation method to estimate the p Ka of the N5 position of the bound substrate. We observe a gradual increase in N5 basicity as we move from primitive to more evolved DHFR isoforms. Structural analysis of these isoforms reveals a gradual sequestering of water molecules from the active site in the more evolved enzymes, thereby modulating the local dielectric environment near the substrate. Furthermore, the present study reveals a clear correlation between active site hydration and the N5 p Ka of the substrate. We emphasize the role of the M20 loop in controlling the active site hydration level, via a preorganized active site with a more hydrophobic environment and reduced loop flexibility as evolution progresses from bacterial to the human enzyme.

Towards a comprehensive understanding of the structural dynamics of a bacterial diterpene synthase during catalysis.

Terpenes constitute the largest and structurally most diverse natural product family. Most terpenoids exhibit a stereochemically complex macrocyclic core, which is generated by C-C bond forming of aliphatic oligo-prenyl precursors. This reaction is catalysed by terpene synthases (TPSs), which are capable of chaperoning highly reactive carbocation intermediates through an enzyme-specific reaction. Due to the instability of carbocation intermediates, the proteins' structural dynamics and enzyme:substrate interactions during TPS catalysis remain elusive. Here, we present the structure of the diterpene synthase CotB2, in complex with an in crystallo cyclised abrupt reaction product and a substrate-derived diphosphate. We captured additional snapshots of the reaction to gain an overview of CotB2's catalytic mechanism. To enhance insights into catalysis, structural information is augmented with multiscale molecular dynamic simulations. Our data represent fundamental TPS structure dynamics during catalysis, which ultimately enable rational engineering towards tailored terpene macrocycles that are inaccessible by conventional chemical synthesis.

Effect of Asp122 Mutation on the Hydride Transfer in E. coli DHFR Demonstrates the Goldilocks of Enzyme Flexibility.

Dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate (THF) in the presence of NADPH. The key hydride transfer step in the reaction is facilitated by a combination of enzyme active site preorganization and correlated protein motions in the Michaelis-Menten (E:NADPH:DHF) complex. The present theoretical study employs mutagenesis to examine the relation between structural and functional properties of the enzyme. We mutate Asp122 in Escherichia coli DHFR, which is a conserved amino acid in the DHFR family. The consequent effect of the mutation on enzyme catalysis is examined from an energetic, structural and short-time dynamic perspective. Our investigations suggest that the structural and short-time dynamic perturbations caused by Asp122X mutations (X = Asn, Ser, Ala) are along the reaction coordinate and lower the rate of hydride transfer. Importantly, analysis of the correlated and principle component motions in the enzyme suggest that the mutation alters the coupled motions that are present in the wild-type enzyme. In the case of D122N and D122S, the mutations inhibit coupled motion, whereas in the case of D122A, the mutation enhances coupled motion, although all mutations result in similar rate reduction. These results emphasize a Goldilocks principle of enzyme flexibility, that is, enzymes should neither be too rigid nor too flexible.

Slow-Starter Enzymes: Role of Active-Site Architecture in the Catalytic Control of the Biosynthesis of Taxadiene by Taxadiene Synthase.

Taxadiene synthase (TXS) catalyzes the formation of natural product taxa-4(5),11(12)-diene (henceforth taxadiene). Taxadiene is the precursor in the formation of Taxol, which is an important natural anticancer agent. In the current study, we present a detailed mechanistic view of the biosynthesis of taxadiene by TXS using a hybrid quantum mechanics-molecular mechanics potential in conjunction with free energy simulation methods. The obtained free-energy landscape displays initial endergonic steps followed by a stepwise downhill profile, which is an emerging free-energy fingerprint for type I terpene synthases. We identify an active-site Trp residue (W753) as a key feature of the TXS active-site architecture and propose that this residue stabilized intermediate cations via π-cation interactions. To validate our proposed active TXS model, we examine a previously reported W753H mutation, which leads to the exclusive formation of side product cembrene A. The simulations of the W753H mutant show that, in the mutant structure, the His side chain is in the perfect position to deprotonate the cembrenyl cation en route to cembrene formation and that this abortive deprotonation is an energetically facile process. On the basis of the current model, we propose that an analogous mutation of Y841 to His could possibly lead to verticillane. The current simulations stress the importance of the precise positioning of key active-site residues in stabilizing intermediate carbocations. In view of the great pharmaceutical importance of taxadiene, a detailed understanding of the TXS mechanism can provide important clues toward a synthetic strategy for Taxol manufacturing.