Structures and mechanisms of actin ATP hydrolysis.

The major cytoskeleton protein actin undergoes cyclic transitions between the monomeric G-form and the filamentous F-form, which drive organelle transport and cell motility. This mechanical work is driven by the ATPase activity at the catalytic site in the F-form. For deeper understanding of the actin cellular functions, the reaction mechanism must be elucidated. Here, we show that a single actin molecule is trapped in the F-form by fragmin domain-1 binding and present their crystal structures in the ATP analog-, ADP-Pi-, and ADP-bound forms, at 1.15-Å resolutions. The G-to-F conformational transition shifts the side chains of Gln137 and His161, which relocate four water molecules including W1 (attacking water) and W2 (helping water) to facilitate the hydrolysis. By applying quantum mechanics/molecular mechanics calculations to the structures, we have revealed a consistent and comprehensive reaction path of ATP hydrolysis by the F-form actin. The reaction path consists of four steps: 1) W1 and W2 rotations; 2) PG-O3B bond cleavage; 3) four concomitant events: W1-PO3- formation, OH- and proton cleavage, nucleophilic attack by the OH- against PG, and the abstracted proton transfer; and 4) proton relocation that stabilizes the ADP-Pi-bound F-form actin. The mechanism explains the slow rate of ATP hydrolysis by actin and the irreversibility of the hydrolysis reaction. While the catalytic strategy of actin ATP hydrolysis is essentially the same as those of motor proteins like myosin, the process after the hydrolysis is distinct and discussed in terms of Pi release, F-form destabilization, and global conformational changes.

Short-lived intermediate in N2O generation by P450 NO reductase captured by time-resolved IR spectroscopy and XFEL crystallography.

Nitric oxide (NO) reductase from the fungus Fusarium oxysporum is a P450-type enzyme (P450nor) that catalyzes the reduction of NO to nitrous oxide (N2O) in the global nitrogen cycle. In this enzymatic reaction, the heme-bound NO is activated by the direct hydride transfer from NADH to generate a short-lived intermediate ( I ), a key state to promote N-N bond formation and N-O bond cleavage. This study applied time-resolved (TR) techniques in conjunction with photolabile-caged NO to gain direct experimental results for the characterization of the coordination and electronic structures of I TR freeze-trap crystallography using an X-ray free electron laser (XFEL) reveals highly bent Fe-NO coordination in I , with an elongated Fe-NO bond length (Fe-NO = 1.91 Å, Fe-N-O = 138°) in the absence of NAD+ TR-infrared (IR) spectroscopy detects the formation of I with an N-O stretching frequency of 1,290 cm-1 upon hydride transfer from NADH to the Fe3+-NO enzyme via the dissociation of NAD+ from a transient state, with an N-O stretching of 1,330 cm-1 and a lifetime of ca. 16 ms. Quantum mechanics/molecular mechanics calculations, based on these crystallographic and IR spectroscopic results, demonstrate that the electronic structure of I is characterized by a singly protonated Fe3+-NHO•- radical. The current findings provide conclusive evidence for the N2O generation mechanism via a radical-radical coupling of the heme nitroxyl complex with the second NO molecule.

Development of serial X-ray fluorescence holography for radiation-sensitive protein crystals.

X-ray fluorescence holography (XFH) is a powerful atomic resolution technique capable of directly imaging the local atomic structure around atoms of a target element within a material. Although it is theoretically possible to use XFH to study the local structures of metal clusters in large protein crystals, the experiment has proven difficult to perform, especially on radiation-sensitive proteins. Here, the development of serial X-ray fluorescence holography to allow the direct recording of hologram patterns before the onset of radiation damage is reported. By combining a 2D hybrid detector and the serial data collection used in serial protein crystallography, the X-ray fluorescence hologram can be directly recorded in a fraction of the measurement time needed for conventional XFH measurements. This approach was demonstrated by obtaining the Mn Kα hologram pattern from the protein crystal Photosystem II without any X-ray-induced reduction of the Mn clusters. Furthermore, a method to interpret the fluorescence patterns as real-space projections of the atoms surrounding the Mn emitters has been developed, where the surrounding atoms produce large dark dips along the emitter-scatterer bond directions. This new technique paves the way for future experiments on protein crystals that aim to clarify the local atomic structures of their functional metal clusters, and for other related XFH experiments such as valence-selective XFH or time-resolved XFH.

Computational Exploration of Minimum Energy Reaction Pathway of N2O Formation from Intermediate I of P450nor Using an Active Center Model.

P450nor is a heme-containing enzyme that catalyzes the conversion of nitric oxide (NO) to nitrous oxide (N2O). Its catalytic mechanism has attracted attention in chemistry, biology, and environmental engineering. The catalytic cycle of P450nor is proposed to consist of three major steps. The reaction mechanism for the last step, N2O generation, remains unknown. In this study, the reaction pathway of the N2O generation from the intermediate I was explored with the B3LYP calculations using an active center model after the examination of the validity of the model. In the validation, we compared the heme distortions between P450nor and other oxidoreductases, suggesting a small effect of protein environment on the N2O generation reaction in P450nor. We then evaluated the electrostatic environment effect of P450nor on the hydride affinity to the active site with quantum mechanics/molecular mechanics (QM/MM) calculations, confirming that the affinity was unchanged with or without the protein environment. The active center model for P450nor showed that the N2O generation process in the enzymatic reaction undergoes a reasonable barrier height without protein environment. Consequently, our findings strongly suggest that the N2O generation reaction from the intermediate I depends sorely on the intrinsic reactivity of the heme cofactor bound on cysteine residue.

Unraveling Binding Mechanism and Stability of Urease Inhibitors: A QM/MM MD Study.

Soil bacteria can produce urease, which catalyzes the hydrolysis of urea to ammonia (NH3) and carbamate. A variety of urease inhibitors have been proposed to reduce NH3 volatilization by interfering with the urease activity. We report a quantum mechanics/molecular mechanics molecular dynamics (QM/MM MD) study on the mechanism employed for the inhibition of urease by three representative competitive inhibitors; namely, acetohydroxamic acid (AHA), hydroxyurea (HU), and N-(n-butyl)phosphorictriamide (NBPTO). The possible connections between the structural and thermodynamical properties and the experimentally observed inhibition efficiency were evaluated and characterized. We demonstrate that the binding affinity decreases in the order NBPTO >> AHA > HU in terms of the computed activation and reaction free energies. This trend also indicates that NBPTO shows the highest inhibitory activity and the lowest IC50 value of 2.1 nM, followed by AHA (42 µM) and HU (100 µM). It was also found that the X=O moiety (X = carbon or phosphorous) plays a crucial role in the inhibitor binding process. These findings not only elucidate why the potent urease inhibitors are effective but also have implications for the design of new inhibitors.

Global Analysis of Heme Proteins Elucidates the Correlation between Heme Distortion and the Heme-Binding Pocket.

Heme proteins play diverse and important biological roles, from electron transfer and chemical catalysis to oxygen transport and/or storage. Although the distortion of heme porphyrin correlates with the physical properties of heme, such as the redox potential and oxygen affinity, the relationship between heme distortion and the heme protein environment is unclear. Here, we tested the hypothesis that the protein environment of the heme-binding pocket determines heme distortion (conformation). We analyzed the correlations between the amino acid composition of the heme-binding pocket and the magnitude of heme distortion along 12 vibrational modes using machine learning. A correlation was detected in the three lowest vibrational modes. Analysis of heme distortions in nearly the same environments of the heme-binding pocket supported this notion. Our analyses indicate that the heme-binding pocket environment is a major factor impacting the distortion of heme porphyrin along the three lowest vibrational modes. In addition, statistical analysis of the distortion of heme porphyrin revealed that the peaks of distributions of the ruffling and breathing distortions are shifted from 0 (the equilibrium structure). Both the ruffling and breathing distortions are correlated with the redox potential of heme, so that heme molecules with these distortions have a lower redox potential than planar molecules. These findings explain the structure-function relationship of heme.

Depolarizing Effects in Hydrogen Bond Energy in 310-Helices Revealed by Quantum Chemical Analysis.

Hydrogen-bond (H-bond) energies in 310-helices of short alanine peptides were systematically examined by precise DFT calculations with the negative fragmentation approach (NFA), a modified method based on the molecular tailoring approach. The contribution of each H-bond was evaluated in detail from the 310-helical conformation of total energies (whole helical model, WH3-10 model), and the results were compared with the property of H-bond in α-helix from our previous study. The H-bond energies of the WH3-10 model exhibited tendencies different from those exhibited by the α-helix in that they depended on the helical position of the relevant H-bond pair. H-bond pairs adjacent to the terminal H-bond pairs were observed to be strongly destabilized. The analysis of electronic structures indicated that structural characteristics cause the destabilization of the H-bond in 310-helices. We also found that the longer the helix length, the more stable the H-bond in the terminal pairs of the WH3-10 model, suggesting the action of H-bond cooperativity.

Effects of Active-Center Reduction of Plant-Type Ferredoxin on Its Structure and Dynamics: Computational Analysis Using Molecular Dynamics Simulations.

"Plant-type" ferredoxins (Fds) in the thylakoid membranes of plants, algae, and cyanobacteria possess a single [2Fe-2S] cluster in active sites and mediate light-induced electron transfer from Photosystem I reaction centers to various Fd-dependent enzymes. Structural knowledge of plant-type Fds is relatively limited to static structures, and the detailed behavior of oxidized and reduced Fds has not been fully elucidated. It is important that the investigations of the effects of active-center reduction on the structures and dynamics for elucidating electron-transfer mechanisms. In this study, model systems of oxidized and reduced Fds were constructed from the high-resolution crystal structure of Chlamydomonas reinhardtii Fd1, and three 200 ns molecular dynamics simulations were performed for each system. The force field parameters of the oxidized and reduced active centers were independently obtained using quantum chemical calculations. There were no substantial differences in the global conformations of the oxidized and reduced forms. In contrast, active-center reduction affected the hydrogen-bond network and compactness of the surrounding residues, leading to the increased flexibility of the side chain of Phe61, which is essential for the interaction between Fd and the target protein. These computational results will provide insight into the electron-transfer mechanisms in the Fds.

Spin-projected QM/MM Free Energy Simulations for Oxidation Reaction of Guanine in B-DNA by Singlet Oxygen.

Guanine is the most susceptible base to oxidation damage induced by reactive oxygen species including singlet oxygen (1 O2 , 1 Δg ). We clarify whether the first step of guanine oxidation in B-DNA proceeds via either a zwitterionic or a diradical intermediate. The free energy profiles are calculated by means of a combined quantum mechanical and molecular mechanical (QM/MM) method coupled with the adaptive biasing force (ABF) method. To describe the open-shell electronic structure of 1 O2 correctly, the broken-symmetry spin-unrestricted density functional theory (BS-UDFT) with an approximate spin projection (AP) correction is applied to the QM region. We find that the effect of spin contamination on the activation and reaction free energies is up to ∼8 kcal mol-1 , which is too large to be neglected. The QM(AP-ULC-BLYP)/MM-based free energy calculations also reveal that the reaction proceeds through a diradical transition state, followed by a conversion to a zwitterionic intermediate. Our computed activation energy of 5.2 kcal mol-1 matches experimentally observed range (0∼6 kcal mol-1 ).

Hydrogen bond donors and acceptors are generally depolarized in α-helices as revealed by a molecular tailoring approach.

Hydrogen-bond (H-bond) interaction energies in α-helices of short alanine peptides were systematically examined by precise density functional theory calculations, followed by a molecular tailoring approach. The contribution of each H-bond interaction in α-helices was estimated in detail from the entire conformation energies, and the results were compared with those in the minimal H-bond models, in which only H-bond donors and acceptors exist with the capping methyl groups. The former interaction energies were always significantly weaker than the latter energies, when the same geometries of the H-bond donors and acceptors were applied. The chemical origin of this phenomenon was investigated by analyzing the differences among the electronic structures of the local peptide backbones of the α-helices and those of the minimal H-bond models. Consequently, we found that the reduced H-bond energy originated from the depolarizations of both the H-bond donor and acceptor groups, due to the repulsive interactions with the neighboring polar peptide groups in the α-helix backbone. The classical force fields provide similar H-bond energies to those in the minimal H-bond models, which ignore the current depolarization effect, and thus they overestimate the actual H-bond energies in α-helices. © 2019 The Authors. Journal of Computational Chemistry published by Wiley Periodicals, Inc.

Density functional study of porphyrin distortion effects on redox potential of heme.

Heme is involved in various biochemical roles in hemoproteins. In the present study, the effect of heme distortion on the redox potential was systematically investigated with density functional calculations. We focused on the ruffled and saddled distortions of heme, which correspond to the two lowest-frequency normal modes. Our computations demonstrated that the ruffled distortion tended to reduce the redox potential of heme and that the transition of the electronic configuration occurred from (dxz , dyz )3 (dxy )2 to (dxz , dyz )4 (dxy )1 . In contrast, the saddled distortion had a tendency toward an increase in the redox potential, and no transition of the electronic configuration occurred. In experiments, these tendencies were found in the relationship between with the heme distortions and the redox potentials in cytochrome c3 . © 2017 Wiley Periodicals, Inc.

Quantitative Assessment of rPM6 for Fluorine- and Chlorine-Containing Metal Complexes: Comparison with Experimental, First-Principles, and Other Semiempirical Results.

We report a reparameterization of PM6 parameters for fluorine and chlorine using our training set containing transition metal complexes. Spin unrestricted calculations with the resulting rPM6 (UrPM6) were examined quantitatively using two test sets: (i) the description of magnetic interactions in 25 dinuclear metal complexes and (ii) the prediction of barrier heights and reaction energies for epoxidation and fluorination reactions catalyzed by high-valent manganese-oxo species. The conventional UPM6 and UPM7 methods were also evaluated for comparison on the basis of either experimental or computational (the UB3LYP/SVP level) outcomes. The merits of UrPM6 are highlighted by both the test sets. As regards magnetic exchange coupling constants, the UrPM6 method had the smallest mean absolute errors from the experimental data (19 cm-1), followed by UPM7 (119 cm-1) and UPM6 (373 cm-1). For the epoxidation and fluorination reactions, all of the transition state searches were successful using UrPM6, while the success rates obtained by UPM6 and UPM7 were only 50%. The UrPM6-optimized stationary points also agreed well with the reference UB3LYP-optimized geometries. The accuracy for estimating reaction energies was also greatly remedied.

Direct Interaction between the Voltage Sensors Produces Cooperative Sustained Deactivation in Voltage-gated H+ Channel Dimers.

The voltage-gated H(+) channel (Hv) is a voltage sensor domain-like protein consisting of four transmembrane segments (S1-S4). The native Hv structure is a homodimer, with the two channel subunits functioning cooperatively. Here we show that the two voltage sensor S4 helices within the dimer directly cooperate via a π-stacking interaction between Trp residues at the middle of each segment. Scanning mutagenesis showed that Trp situated around the original position provides the slow gating kinetics characteristic of the dimer's cooperativity. Analyses of the Trp mutation on the dimeric and monomeric channel backgrounds and analyses with tandem channel constructs suggested that the two Trp residues within the dimer are functionally coupled during Hv deactivation but are less so during activation. Molecular dynamics simulation also showed direct π-stacking of the two Trp residues. These results provide new insight into the cooperative function of voltage-gated channels, where adjacent voltage sensor helices make direct physical contact and work as a single unit according to the gating process.

Common folding processes of mini-proteins: Partial formations of secondary structures initiate the immediate protein folding.

The folding processes of mini-proteins (FSD-EY/FBPWW28 domain) were computationally investigated by an enhanced conformational sampling method. Through the analyses of trajectories, these mini-proteins had multiple folding pathways different from a simple two-state folding, and the multiple folding processes were initiated by partial formations of secondary structures. These findings can be used to understand the folding of large proteins, that is, which secondary structures are partially folded in the early process, and how the remaining parts are sequentially folded. It is found that FSD-EY (α/ß topology) folds by a simple diffusion-collision mechanism, while the folding process of the FBPWW28 domain (all-ß topology) requires a modification of the diffusion-collision theory to adequately treat the coil-sheet transition for the ß sheet formation. © 2017 Wiley Periodicals, Inc.

Inverse Ubbelohde effect in the short hydrogen bond of photosystem II: Relation between H/D isotope effect and symmetry in potential energy profile.

The short hydrogen bond between tyrosine Yz and D1-His190 of photosystem II (PSII) was investigated using multicomponent quantum mechanics, where the quantum fluctuation of a hydrogen nucleus was incorporated into electronic structure calculation. Our computation demonstrated that the deuteration for hydrogen in the short hydrogen bond of PSII led to the reduction of the O…N distance. It indicated an inverse Ubbelohde effect typically recognized in strong and symmetric hydrogen-bonding clusters such as FHF(-) and H3O2-. We confirmed that the relation between the geometric isotope effect and the symmetry of the potential energy profile of FHF(-) was reasonably agreed with that of PSII. According to this agreement, the short hydrogen bond in PSII can be regarded as a short strong hydrogen bond. © 2016 Wiley Periodicals, Inc.

Development of massive multilevel molecular dynamics simulation program, Platypus (PLATform for dYnamic Protein Unified Simulation), for the elucidation of protein functions.

A massively parallel program for quantum mechanical-molecular mechanical (QM/MM) molecular dynamics simulation, called Platypus (PLATform for dYnamic Protein Unified Simulation), was developed to elucidate protein functions. The speedup and the parallelization ratio of Platypus in the QM and QM/MM calculations were assessed for a bacteriochlorophyll dimer in the photosynthetic reaction center (DIMER) on the K computer, a massively parallel computer achieving 10 PetaFLOPs with 705,024 cores. Platypus exhibited the increase in speedup up to 20,000 core processors at the HF/cc-pVDZ and B3LYP/cc-pVDZ, and up to 10,000 core processors by the CASCI(16,16)/6-31G** calculations. We also performed excited QM/MM-MD simulations on the chromophore of Sirius (SIRIUS) in water. Sirius is a pH-insensitive and photo-stable ultramarine fluorescent protein. Platypus accelerated on-the-fly excited-state QM/MM-MD simulations for SIRIUS in water, using over 4000 core processors. In addition, it also succeeded in 50-ps (200,000-step) on-the-fly excited-state QM/MM-MD simulations for the SIRIUS in water.

Reparameterization of PM6 Applied to Organic Diradical Molecules.

We have performed a reparameterization of PM6 (called rPM6) to compute open-shell species, specifically organic diradical molecules, within a framework of the spin-unrestricted semiempirical molecular orbital (SE-UMO) method. The parameters for the basic elements (hydrogen, carbon, nitrogen, and oxygen) have been optimized simultaneously using the training set consisting of 740 reference data. On the basis of the GMTKN30 database, the mean absolute error of rPM6 is decreased from 16.1 to 14.1 kcal/mol, which reassures its accuracy for ground-state properties. Applications of the spin-unrestricted rPM6 (UrPM6) method to small diradicals and relatively large polycyclic aromatic hydrocarbons have provided substantial improvement over the standard SE-UMO methods like UAM1, UPM3, and the original UPM6. The UrPM6 calculation is much less susceptible to spin contamination and, therefore, reproduces geometric parameters and adiabatic singlet-triplet energy gaps obtained by UDFT (UB3LYP and/or UBHandHLYP) at much lower computational cost.

Mg2+-dependent interactions of ATP with the cystathionine-ß-synthase (CBS) domains of a magnesium transporter.

Ancient conserved domain protein/cyclin M (CNNM) family proteins are evolutionarily conserved Mg(2+) transporters. However, their biochemical mechanism of action remains unknown. Here, we show the functional importance of the commonly conserved cystathionine-ß-synthase (CBS) domains and reveal their unique binding ability to ATP. Deletion mutants of CNNM2 and CNNM4, lacking the CBS domains, are unable to promote Mg(2+) efflux. Furthermore, the substitution of one amino acid residue in the CBS domains of CNNM2, which is associated with human hereditary hypomagnesemia, abrogates Mg(2+) efflux. Binding analyses reveal that the CBS domains of CNNM2 bind directly to ATP and not AMP in a manner dependent on the presence of Mg(2+), which is inhibited in a similar pattern by the disease-associated amino acid substitution. The requirement of Mg(2+) for these interactions is a unique feature among CBS domains, which can be explained by the presence of highly electronegative surface potentials around the ATP binding site on CNNM2. These results demonstrate that the CBS domains play essential roles in Mg(2+) efflux, probably through interactions with ATP. Interactions with ATP, which mostly forms complexes with Mg(2+) in cells, may account for the rapid Mg(2+) transport by CNNM family proteins.

Enhanced conformational sampling method for proteins based on the TaBoo SeArch algorithm: application to the folding of a mini-protein, chignolin.

The conformational samplings are indispensible for obtaining reliable canonical ensembles, which provide statistical averages of physical quantities such as free energies. However, the samplings of vast conformational space of biomacromolecules by conventional molecular dynamics (MD) simulations might be insufficient, due to their inadequate accessible time-scales for investigating biological functions. Therefore, the development of methodologies for enhancing the conformational sampling of biomacromolecules still remains as a challenging issue in computational biology. To tackle this problem, we newly propose an efficient conformational search method, which is referred as TaBoo SeArch (TBSA) algorithm. In TBSA, an inverse energy histogram is used to select seeds for the conformational resampling so that states with high frequencies are inhibited, while states with low frequencies are efficiently sampled to explore the unvisited conformational space. As a demonstration, TBSA was applied to the folding of a mini-protein, chignolin, and automatically sampled the native structure (Cα root mean square deviation < 1.0 Å) with nanosecond order computational costs started from a completely extended structure, although a long-time 1-µs normal MD simulation failed to sample the native structure. Furthermore, a multiscale free energy landscape method based on the conformational sampling of TBSA were quantitatively evaluated through free energy calculations with both implicit and explicit solvent models, which enable us to find several metastable states on the folding landscape.

Protein folding pathways extracted by OFLOOD: Outlier FLOODing method.

The Outlier FLOODing method (OFLOOD) is proposed as an efficient conformational sampling method to extract biologically rare events such as protein folding. In OFLOOD, sparse distributions (outliers in the conformational space) were regarded as relevant states for the transitions. Then, the transitions were enhanced through conformational resampling from the outliers. This evidence indicates that the conformational resampling of the sparse distributions might increase chances for promoting the transitions from the outliers to other meta-stable states, which resembles a conformational flooding from the outliers to the neighboring clusters. OFLOOD consists of (i) detections of outliers from conformational distributions and (ii) conformational resampling from the outliers by molecular dynamics (MD) simulations. Cycles of (i) and (ii) are simply repeated. As demonstrations, OFLOOD was applied to folding of Chignolin and HP35. In both cases, OFLOOD automatically extracted folding pathways from unfolded structures with ns-order computational costs, although µs-order canonical MD failed to extract them.