Structures Of Intermediate States Research Articles

Kinetics and mechanism of the gas-phase reaction of the hydroxyl radical with meta-aminotoluene compound.

An ab initio investigation into the potential energy landscape of the meta-aminotoluene + •OHreaction has been conducted in this study. The calculated results reveal that the reaction channel leading to the product (NHC6H4CH3 + H2O) prevails under the 300-1700K temperature range, while the reaction path forming the product (NH2C6H4CH2 + H2O) dominates in the higher-temperature region (T ≥ 1800K). Within the specified temperature range, the product branching ratio for the former declines from 48 to 30%, while the latter shows an increase, reaching 29%. The overall second-order rate constants of the titled reaction obtained at the pressure 760Torr (N2) can be illustrated by the modified Arrhenius expression of ktotal = 1.46 × 10-13 T0.58 exp[(-0.759kcal.mol-1)/RT] cm3 molecule-1s-1 and ktotal = 1.86 × 10-22 T3.24 exp[(-5.086kcal.mol-1)/RT] cm3 molecule-1s-1, covering the temperature range of T = 300-600K and T > 600K, respectively. The total rate constant at the ambient conditions in this work, 1.43 × 10-11 cm3 molecule-1s-1, has been found to be roughly one order of magnitude lower than the available experimental data, ~ 1.2 × 10-10 cm3 molecule-1s-1, measured by Atkinson et al., Rinke et al., and Witte et al., or the theoretical value, 4.4 × 10-10 cm3 molecule-1s-1, and calculated by Abdel-Rahman and co-workers for the aniline + •OH reaction. The structures of reactants, transition states, intermediate states, and products of the meta-aminotoluene + •OHreaction are calculated with the aug-cc-pVTZ basis set and the methods DFT/B3LYP and CCSD(T). The rate constants and branching ratios in the 300-2000K temperature range are calculated with the statistical theoretical TST and RRKM master equation computations including tunneling corrections, with potential energy surface constructed by the CCSD(T)//B3LYP/aug-cc-pVTZ approach.

Antiviral drug recognition and elevator-type transport motions of CNT3.

Nucleoside analogs have broad clinical utility as antiviral drugs. Key to their systemic distribution and cellular entry are human nucleoside transporters. Here, we establish that the human concentrative nucleoside transporter 3 (CNT3) interacts with antiviral drugs used in the treatment of coronavirus infections. We report high-resolution single-particle cryo-electron microscopy structures of bovine CNT3 complexed with antiviral nucleosides N4-hydroxycytidine, PSI-6206, GS-441524 and ribavirin, all in inward-facing states. Notably, we found that the orally bioavailable antiviral molnupiravir arrests CNT3 in four distinct conformations, allowing us to capture cryo-electron microscopy structures of drug-loaded outward-facing and drug-loaded intermediate states. Our studies uncover the conformational trajectory of CNT3 during membrane transport of a nucleoside analog antiviral drug, yield new insights into the role of interactions between the transport and the scaffold domains in elevator-like domain movements during drug translocation, and provide insights into the design of nucleoside analog antiviral prodrugs with improved oral bioavailability.

Capturing the pre-power stroke structure of actomyosin V through time resolved cryo-electron microscopy

The actomyosin complex is an archetypical molecular machine, which converts the chemical energy of ATP into mechanical energy allowing myosins to carry out physiological functions including muscle contraction and vesicular transport. The structures adopted by myosins alone and in complex with actin are relatively well studied, but to date high-resolution studies have been limited to equilibrium conditions. As a result, structures of important intermediate states in the catalytic cycle remain elusive.

Molecular Mechanism of Force Generation by the Actin-Myosin Complex During Cardiac Muscle Contraction

Cardiac muscle contraction is performed by arrays of contractile proteins in the sarcomere. Serious heart diseases, such as cardiomyopathy, can often be results of mutations in myosin and actin, which slide along each other to generate force using the energy from ATP hydrolysis. All-atom molecular dynamics (MD) simulations have been growing its power in predicting protein structure-function relationships. However, such approaches are limited in the study of the myosin crossbridge cycle owing to the slow timescale as well as the lack of high-resolution structures of various intermediate states for the human cardiac myosin-actin complex. Here, combining comparative modeling and enhanced sampling MD simulations, we characterize how the myosin-actin interactions in human are influenced by the myosin functional states. By incorporating structural information from multiple templates, our comparative modeling provides conformational ensembles for different myosin-actin states, which enable us to efficiently sample the energy landscape using Gaussian accelerated MD simulations. Our results reveal novel interactions between key myosin loops and actin, which are consistent with recent cryo-EM density data. Furthermore, we find that the myosin cleft motion is impacted by the ATP binding site dynamics and that ADP release is coupled to the lever arm swing. Our approach demonstrates the potential to link genotype and molecular structures to the physical properties of the sarcomere.

Structures of catalytic cycle intermediates of the Pyrococcus furiosus methionine adenosyltransferase demonstrate negative cooperativity in the archaeal orthologues

Methionine adenosyltransferases catalyse the biosynthesis of S-adenosylmethionine, the primary methyl group donor in biochemical reactions, through the condensation of methionine and ATP. Here, we report the structural analysis of the Pyrococcus furiosus methionine adenosyltransferase (PfMAT) captured in the unliganded, substrate- and product-bound states. The conformational changes taking place during the enzymatic catalytic cycle are allosterically propagated by amino acid residues conserved in the archaeal orthologues to induce an asymmetric dimer structure. The distinct occupancy of the active sites within a PfMAT dimer is consistent with a half-site reactivity that is mediated by a product-induced negative cooperativity. The structures of intermediate states of PfMAT reported here suggest a distinct molecular mechanism for S-adenosylmethionine synthesis in Archaea, likely consequence of the evolutionary pressure to achieve protein stability under extreme conditions.

Water Plays a Cocatalytic Role in Epoxide Ring Opening Reaction in Aspartate Proteases: A QM/MM Study.

Aspartate proteases are potential targets for various diseases, and many of their inhibitors are FDA-approved drugs. However, these peptidomimetic and reversibly bound drugs become ineffective upon prolonged use. Attempts have been made to design and synthesize various nonpeptidic epoxide-based irreversible inhibitors to combat the drug-resistance enigma. Here, we study the mechanism of epoxide ring opening in two widely studied aspartate proteases, HIV-1 protease and pepsin. Our results from QM/MM molecular dynamics show that the epoxide ring opening in aspartate proteases follow a two-step mechanism with the formation of an oxyanion intermediate, stabilized by a set of water molecules in the protein active site. These water molecules by virtue of "low-barrier hydrogen bonds" with the epoxide ring reduce the intrinsic reaction barrier while remaining structurally unperturbed, thus playing a cocatalytic role. We validated our results by reproducing the experimentally observed protease/pepsin-epoxide covalent complexes as end products. The observed stability of our oxyanion intermediate in a four-water-coordinated state is also consistent with the reported stable state of the hydroxide ion in water as OH-(H2O)4. Our study could pave the way for the design of new class "HIV protease irreversible inhibitors" from the acquired knowledge of the structures of intermediate and transition states traced during the explored reaction mechanism.

A "Stepping Stone" Approach for Obtaining Quantum Free Energies of Hydration.

We present a method which uses DFT (quantum, QM) calculations to improve free energies of binding computed with classical force fields (classical, MM). To overcome the incomplete overlap of configurational spaces between MM and QM, we use a hybrid Monte Carlo approach to generate quickly correct ensembles of structures of intermediate states between a MM and a QM/MM description, hence taking into account a great fraction of the electronic polarization of the quantum system, while being able to use thermodynamic integration to compute the free energy of transition between the MM and QM/MM. Then, we perform a final transition from QM/MM to full QM using a one-step free energy perturbation approach. By using QM/MM as a stepping stone toward the full QM description, we find very small convergence errors (<1 kJ/mol) in the transition to full QM. We apply this method to compute hydration free energies, and we obtain consistent improvements over the MM values for all molecules we used in this study. This approach requires large-scale DFT calculations as the full QM systems involved the ligands and all waters in their simulation cells, so the linear-scaling DFT code ONETEP was used for these calculations.

Intermediate states in the binding process of folic acid to folate receptor α: insights by molecular dynamics and metadynamics

Folate receptor α (FRα) is a cell surface, glycophosphatidylinositol-anchored protein which has focussed attention as a therapeutic target and as a marker for the diagnosis of cancer. It has a high affinity for the dietary supplemented folic acid (FOL), carrying out endocytic transport across the cell membrane and delivering the folate at the acidic pH of the endosome. Starting from the recently reported X-ray structure at pH 7, 100 ns classical molecular dynamics simulations have been carried out on the FRα-FOL complex; moreover, the ligand dissociation process has been studied by metadynamics, a recently reported method for the analysis of free-energy surfaces (FES), providing clues on the intermediate states and their energy terms. Multiple dissociation runs were considered to enhance the configurational sampling; a final clustering of conformations within the averaged FES provides the representative structures of several intermediate states, within an overall barrier for ligand escape of about 75 kJ/mol. Escaping of FOL to solvent occurs while only minor changes affect the FRα conformation of the binding pocket. During dissociation, the FOL molecule translates and rotates around a turning point located in proximity of the receptor surface. FOL at this transition state assumes an "L" shaped conformation, with the pteridin ring oriented to optimize stacking within W102 and W140 residues, and the negatively charged glutamate tail, outside the receptor, interacting with the positively charged R103 and R106 residues, that contrary to the bound state, are solvent exposed. We show that metadynamics method can provide useful insights at the atomistic level on the effects of point-mutations affecting functionality, thus being a very promising tool for any study related to folate-targeted drug delivery or cancer therapies involving folate uptake.

Ultrafast Dynamical Study Using Time-resolved Laue Diffraction

Electron transfer reactions are fundamental processes in chemistry and also in biology [1-3]. Light harvesting complexes are functional centers in plants where sunlight is converted into chemical energy. In this, optical excitation in a chromophore unit leads to the transfer of electrons within the system. However, due to the complexity of the biological photo-reaction centre, recent spectroscopic efforts have concentrated on a smaller chemical model which share characteristic with their biological counter parts. A promising model is the so-called D-A (donor-acceptor) systems, which are chemically synthetic molecules with electron transfer capabilities. The electrical conductivity is a function of the optical state of the system. An optically switching diode is an interesting application of donor-acceptor molecules. We aimed to determine photo-induced structural changes in Pyrene-N,N-dimethylaniline (PyDMA). Static structures for many molecules are available at high resolution but the mechanism by which these molecules function and the structures of intermediate states often remain elusive. Knowledge of the geometry of molecular excited states at atomic resolution is crucial for a full understanding of photo-induced chemical processes. Time-resolved X-ray diffraction (TR-XRD) using polychromatic synchrotron radiation allows a detailed study of the time evolution of structural intermediates and short living states of chemical systems at wide range of time-scales, drawing a complete picture of the photo-induced charge transfer process. Investigation of photo-excitation processes in molecular single crystals, where the initial photo-excitation processes occur on extremely short time-scales (femto-/picosecond time domain) and have been in the focus of scientific investigations due to their possible applications, e.g. as optical switches.

Lights, X-Rays, Oxygen!

Photosystem II uses metal ions to oxidize water to form O2. Two recent papers employ the new technique of serial femtosecond crystallography utilizing X-ray free-electron lasers and nanocrystals to obtain initial structures of intermediate states of photosystem II catalysis at the site of oxygen production.

Structures of intermediate transport states of ZneA, a Zn(II)/proton antiporter

Efflux pumps belonging to the ubiquitous resistance-nodulation-cell division (RND) superfamily transport substrates out of cells by coupling proton conduction across the membrane to a conformationally driven pumping cycle. The heavy metal-resistant bacteria Cupriavidus metallidurans CH34 relies notably on as many as 12 heavy metal efflux pumps of the RND superfamily. Here we show that C. metallidurans CH34 ZneA is a proton driven efflux pump specific for Zn(II), and that transport of substrates through the transmembrane domain may be electrogenic. We report two X-ray crystal structures of ZneA in intermediate transport conformations, at 3.0 and 3.7 Å resolution. The trimeric ZneA structures capture protomer conformations that differ in the spatial arrangement and Zn(II) occupancies at a proximal and a distal substrate binding site. Structural comparison shows that transport of substrates through a tunnel that links the two binding sites, toward an exit portal, is mediated by the conformation of a short 14-aa loop. Taken together, the ZneA structures presented here provide mechanistic insights into the conformational changes required for substrate efflux by RND superfamily transporters.

Isomerization Mechanismof Xylene Catalyzed by H-ZSM-5 Molecular Sieve

The isomerization mechanism of xylene over H-ZSM-5 molecular sieve has been examined using the density functional theory(DFT) and our own-N-layered integrated molecular orbital + molecular mechanics(ONIOM) methods.The structures of intermediate species and transition states are described.The adsorption of reactant and desorption of product significantly affect the tendency of xylene to isomerize.Calculated activation energies suggest that isomerization occurs during the formation of meta-xylene within the extended pore structure of H-ZSM-5 molecular sieve.However,the produced meta-xylene is retained within the pore because of a high desorption energy,and further isomerization to form para-xylene is kinetically favorable.The acid sites within the pores of the molecular sieve allow selective formation of para-xylene.On the external surface of H-ZSM-5 molecular sieve,which lacks the steric constraints of the extended pore structure,xylene isomerizes to form meta-xylene,which can readily desorb from the active site.Such non-selective isomerization decreases the selectivity for para-xylene.Thus,external surface modification of H-ZSM-5 molecular sieve should suppress the non-selective isomerization of xylene,thereby increasing the selectivity for para-xylene by restricting isomerization to inside the pores of the molecular sieve.Calculated relative reaction rate constants for xylene isomerization also indicate that xylene isomerization occurring on the external surface of H-ZSM-5 with meta-xylene as the product has the highest reaction rate.The selectivity for para-xylene is decreased as the reaction temperature is increased.

Structures of dNTP Intermediate States during DNA Polymerase Active Site Assembly

DNA polymerase and substrate conformational changes are essential for high-fidelity DNA synthesis. Structures of DNA polymerase (pol) β in complex with DNA show the enzyme in an "open" conformation. Subsequent to binding the nucleotide, the polymerase "closes" around the nascent base pair with two metals positioned for chemistry. However, structures of substrate/active site intermediates prior to closure are lacking. By destabilizing the closed complex, we determined unique ternary complex structures of pol β with correct and incorrect incoming nucleotides bound to the open conformation. These structures reveal that Watson-Crick hydrogen bonding is assessed upon initial complex formation. Importantly, nucleotide-bound states representing intermediate metal coordination states occur with active site assembly. The correct, but not incorrect, nucleotide maintains Watson-Crick hydrogen bonds during interconversion of these states. These structures indicate that the triphosphate of the incoming nucleotide undergoes rearrangement prior to closure, providing an opportunity to deter misinsertion and increase fidelity.

Conserved activation pathways in G-protein-coupled receptors

GPCRs (G-protein-coupled receptors) are seven-transmembrane helix proteins that transduce exogenous and endogenous signals to modulate the activity of downstream effectors inside the cell. Despite the relevance of these proteins in human physiology and pharmaceutical research, we only recently started to understand the structural basis of their activation mechanism. In the period 2008-2011, nine active-like structures of GPCRs were solved. Among them, we have determined the structure of light-activated rhodopsin with all the features of the active metarhodopsin-II, which represents so far the most native-like model of an active GPCR. This structure, together with the structures of other inactive, intermediate and active states of rhodopsin constitutes a unique structural framework on which to understand the conserved aspects of the activation mechanism of GPCRs. This mechanism can be summarized as follows: retinal isomerization triggers a series of local structural changes in the binding site that are amplified into three intramolecular activation pathways through TM (transmembrane helix) 5/TM3, TM6 and TM7/TM2. Sequence analysis strongly suggests that these pathways are conserved in other GPCRs. Differential activation of these pathways by ligands could be translated into the stabilization of different active states of the receptor with specific signalling properties.

Tracking Conformational Changes during Amyloidogenesis in Real-Time at Atomic-Resolution by NMR

Understanding the structural transitions that amyloid proteins undergo during amyloidogenesis would greatly enhance our understanding of this process. However, our knowledge has been currently largely limited to global conformational changes, with high-resolution structural information only available for the monomeric proteins and for a few structures of the final amyloid product. In particular, high-resolution structures of intermediate states have been notoriously absent with few exceptions. We show here that high-resolution structures of intermediates can be obtained by using SOFAST-HMQC, CPMG, NOESY, magic-angle-spinning, and other experiments in real-time to track the aggregation pathway at atomic-level detail and at a time-resolution of minutes. As examples, we show the aggregation pathways of Aβ and IAPP, two initially unstructured peptides implicated in Alzheimer's and type II Diabetes, respectively.While previous studies commonly show the Aβ1-40 is largely unstructured in solution before the formation of β-sheet oligomers, we show that Aβ1-40 gradually adopts a compact, partially folded helical structure over a period of several days. In this structure, the central hydrophobic region of the peptide forms a 310 helix from H13 to D23 and the N- and C-termini collapse against the helix due to the clustering of hydrophobic residues (pdb:2LFM). The formation of the helical intermediate is concentration dependent and can be partially reversed by dilution of the peptide. Helical intermediates have been predicted to be crucial on-pathway intermediates in amyloid fibrillogenesis, and the structure presented here presents a new target for structure-based intervention, shown here by the interaction of the helical intermediate with polyphenols. By contrast, the more amyloidogenic IAPP peptide shows only a gradual transition to the fiber form after an initial pH dependent formation of a micelle-like aggregate, with distinct β-sheet small oligomers forming only a small fraction of the observable population.

Thermal Decomposition of 1,5-Dinitrobiuret (DNB): Direct Dynamics Trajectory Simulations and Statistical Modeling

A large set of quasi-classical, direct dynamics trajectory simulations were performed for decomposition of 1,5-dinitrobiuret (DNB) over a temperature range from 4000 to 6000 K, aimed at providing insight into DNB decomposition mechanisms. The trajectories revealed various decomposition paths and reproduced the products (including HNCO, N(2)O, NO(2), NO, and water) observed in DNB pyrolysis experiments. Using trajectory results as a guide, structures of intermediate complexes and transition states that might be important for decomposition were determined using density functional theory calculations. Rice-Ramsperger-Kassel-Marcus (RRKM) theory was then utilized to examine behaviors of the energized reactant and intermediates and to determine unimolecular rates for crossing various transition states. According to RRKM predictions, the dominant initial decomposition path of energized DNB corresponds to elimination of HNNO(2)H via a concerted mechanism where the molecular decomposition is accompanied with intramolecular H-atom transfer from the central nitrogen to the terminal nitro oxygen. Other important paths correspond to elimination of NO(2) and H(2)NNO(2). NO(2) elimination is a simple N-N bond scission process. Formation and elimination of nitramide is, however, dynamically complicated, requiring twisting a -NHNO(2) group out of the molecular plane, followed by an intramolecular reaction to form nitramide before its elimination. These two paths become significant at temperatures above 1500 K, accounting for >17% of DNB decomposition at 2000 K. This work demonstrates that quasi-classical trajectory simulations, in conjunction with electronic structure and RRKM calculations, are able to extract mechanisms, kinetics, dynamics and product branching ratios for the decomposition of complex energetic molecules and to predict how they vary with decomposition temperature.

Bacterial Inclusion Bodies of Alzheimer's Disease β‐Amyloid Peptides Can Be Employed To Study Native‐Like Aggregation Intermediate States

The structures of oligomeric intermediate states in the aggregation process of Alzheimer's disease β-amyloid peptides have been the subject of debate for many years. Bacterial inclusion bodies contain large amounts of small heat shock proteins (sHSPs), which are highly homologous to those found in the plaques of the brains of Alzheimer's disease patients. sHSPs break down amyloid fibril structure in vitro and induce oligomeric assemblies. Prokaryotic protein overexpression thus mimics the conditions encountered in the cell under stress and allows the structures of Aβ aggregation intermediate states to be investigated under native-like conditions, which is not otherwise technically possible. We show that IB40/IB42 fulfil all the requirements to be classified as amyloids: they seed fibril growth, are Congo red positive and show characteristic β-sheet-rich CD spectra. However, IB40 and IB42 are much less stable than fibrils formed in vitro and contain significant amounts of non-β-sheet regions, as seen from FTIR studies. Quantitative analyses of solution-state NMR H/D exchange rates show that the hydrophobic cores involving residues V18-F19-F20 adopt β-sheet conformations, whereas the C termini adopt α-helical coiled-coil structures. In the past, an α-helical intermediate-state structure has been postulated, but could not be verified experimentally. In agreement with the current literature, in which Aβ oligomers are described as the most toxic state of the peptides, we find that IB42 contains SDS-resistant oligomers that are more neurotoxic than Aβ42 fibrils. E. coli inclusion bodies formed by the Alzheimer's disease β-amyloid peptides Aβ40 and Aβ42 thus behave structurally like amyloid aggregation intermediate states and open the possibility of studying amyloids in a native-like, cellular environment.

Structural Snapshots of Escherichia coli Histidinol Phosphate Phosphatase along the Reaction Pathway

HisB from Escherichia coli is a bifunctional enzyme catalyzing the sixth and eighth steps of l-histidine biosynthesis. The N-terminal domain (HisB-N) possesses histidinol phosphate phosphatase activity, and its crystal structure shows a single domain with fold similarity to the haloacid dehalogenase (HAD) enzyme family. HisB-N forms dimers in the crystal and in solution. The structure shows the presence of a structural Zn(2+) ion stabilizing the conformation of an extended loop. Two metal binding sites were also identified in the active site. Their presence was further confirmed by isothermal titration calorimetry. HisB-N is active in the presence of Mg(2+), Mn(2+), Co(2+), or Zn(2+), but Ca(2+) has an inhibitory effect. We have determined structures of several intermediate states corresponding to snapshots along the reaction pathway, including that of the phosphoaspartate intermediate. A catalytic mechanism, different from that described for other HAD enzymes, is proposed requiring the presence of the second metal ion not found in the active sites of previously characterized HAD enzymes, to complete the second half-reaction. The proposed mechanism is reminiscent of two-Mg(2+) ion catalysis utilized by DNA and RNA polymerases and many nucleases. The structure also provides an explanation for the inhibitory effect of Ca(2+).

Crystal Structure of the Bromide-Bound D85S Mutant of Bacteriorhodopsin: Principles of Ion Pumping

We report the crystal structure of a bromide-bound form of the D85S mutant of bacteriorhodopsin, bR(D85S), a protein that uses light energy rather than ATP to pump halide ions across the cell membrane. Comparison of the structure of the halide-bound and halide-free states reveals that both displacements of individual side-chain positions and concerted helical movements occur on the extracellular side of the protein. Analysis of these structural changes reveals how this ion pump first facilitates ion uptake deep within the cell membrane and then prevents the backward escape of ions later in the pumping cycle. Together with the information provided by structures of intermediate states in the bacteriorhodopsin photocycle, this study also suggests the overall design principles that are necessary for ion pumping.

A DFT study of polymerization mechanisms of indole

Polymerization of unsubstituted indoles is studied by accurate density functional theory calculations. Relative stability of all possible dimers of indole is computed in order to understand the thermodynamics of polymerization. It is observed that 2-position is the most likely site to enhance polymerization. A selected set of trimers and tetramers which use a 2-position for linkages are generated to understand the further growth of polyindole. A study of local minima arising from different distributions of the torsional angles reveals that there are two equally probable conformations and the one with the torsional angle changing signs alternatively is slightly favored. The cyclic structures are also investigated and it is shown that it is possible to generate stable three- and four-membered cyclic structures. Finally, the structures of radical cations and intermediate states are fully optimized and the energetics of these metastable species are used to explain the competing mechanisms of radical–radical and radical–neutral pathways.