RESUMO
A nonribosomal peptide-synthesizing molecular machine, RimK, adds l-glutamic acids to the C-terminus of ribosomal protein S6 (RpsF) in vivo and synthesizes poly-α-glutamates in vitro. However, the mechanism of the successive glutamate addition, which is fueled by ATP, remains unclear. Here, we investigate the successive peptide-synthesizing mechanism of RimK via the molecular dynamics (MD) simulation of glutamate binding. We first show that RimK adopts three stable structural states with respect to the ATP-binding loop and the triphosphate chain of the bound ATP. We then show that a glutamate in solution preferentially binds to a positively charged belt-like region of RimK and the bound glutamate exhibits Brownian motion along the belt. The binding-energy landscape shows that the open-to-closed transition of the ATP-binding loop and the bent-to-straight transition of the triphosphate chain of ATP can function as an electrostatic ratchet that guides the bound glutamate to the active site. We then show the binding site of the second glutamate, which allows us to infer the ligation mechanism. Consistent with MD results, the crystal structure of RimK we obtained in the presence of RpsF presents an electron density that is presumed to correspond to the C-terminus of RpsF. We finally propose a mechanism for the successive peptide synthesis by RimK and discuss its similarity to other molecular machines.
Assuntos
Ácido Glutâmico , Peptídeos , Ácido Glutâmico/metabolismo , Eletricidade Estática , Trifosfato de Adenosina/químicaRESUMO
Fo portion of ATP synthase is a proton-motive rotary motor. The Coulombic attraction between the conserved acidic residues in the c-ring and the arginine in the a-subunit (aR) was early proposed to drive the c-ring rotation relative to the a-subunit, and has been actually observed in our previous molecular dynamics simulation with full atomistic description of Fo embedded in the membrane. In this study, to quantify the driving force, we conducted the umbrella sampling (US) and obtained the free-energy landscape for the c-ring rotation. We first show that the free-energy gradient toward the ATP-synthesis direction appears in the deprotonated state of cE. Using the sampled snapshots that cover a wide range of the rotational angle, we further analyzed the rotational-angle dependence of the hydration and the protonation states and obtained the Coulomb-energy landscapes with a focus on the cE-aR interaction. The results indicate that both the Coulombic solvation energy of cE and the interaction energy between cE and aR contribute to the torque generation for the c-ring rotation.
Assuntos
Trifosfato de Adenosina , ATPases Translocadoras de Prótons , Rotação , Torque , Trifosfato de Adenosina/química , ATPases Translocadoras de Prótons/metabolismoRESUMO
The generalized Born (GB) model is an extension of the continuum dielectric theory of Born solvation energy and is a powerful method for accelerating the molecular dynamic (MD) simulations of charged biological molecules in water. While the effective dielectric constant of water that varies as a function of the separation distance between solute molecules is incorporated into the GB model, adjustment of the parameters is indispensable for accurate calculation of the Coulomb (electrostatic) energy. One of the key parameters is the lower limit of the spatial integral of the energy density of the electric field around a charged atom, known as the intrinsic radius ρ. Although ad hoc adjustment of ρ has been conducted to improve the Coulombic (ionic) bond stability, the physical mechanism by which ρ affects the Coulomb energy remains unclear. Via energetic analysis of three differently sized systems, here, we clarify that the Coulomb bond stability increases with increasing ρ and that the increased stability is caused by the interaction energy term, not by the self-energy (desolvation energy) term, as was supposed previously. Our results suggest that the use of larger values for the intrinsic radii of hydrogen and oxygen atoms, together with the use of a relatively small value for the spatial integration cutoff in the GB model, can better reproduce the Coulombic attraction between protein molecules.
Assuntos
Proteínas , Rádio (Anatomia) , Eletricidade Estática , Termodinâmica , Proteínas/química , Água/químicaRESUMO
Hydroxymethylbilane synthase (HMBS), which is involved in the heme biosynthesis pathway, has a dipyrromethane cofactor and combines four porphobilinogen (PBG) molecules to form a linear tetrapyrrole, hydroxymethylbilane. Enzyme kinetic study of human HMBS using a PBG-derivative, 2-iodoporphobilinogen (2-I-PBG), exhibited noncompetitive inhibition with the inhibition constant being 5.4 ± 0.3â µM. To elucidate the reaction mechanism of HMBS in detail, crystal structure analysis of 2-I-PBG-bound holo-HMBS and its reaction intermediate possessing two PBG molecules (ES2), and inhibitor-free ES2 was performed at 2.40, 2.31, and 1.79â Å resolution, respectively. Their overall structures are similar to that of inhibitor-free holo-HMBS, and the differences are limited near the active site. In both 2-I-PBG-bound structures, 2-I-PBG is located near the terminus of the cofactor or the tetrapyrrole chain. The propionate group of 2-I-PBG interacts with the side chain of Arg173, and its acetate group is associated with the side chains of Arg26 and Ser28. Furthermore, the aminomethyl group and pyrrole nitrogen of 2-I-PBG form hydrogen bonds with the side chains of Gln34 and Asp99, respectively. These amino acid residues form a single substrate-binding site, where each of the four PBG molecules covalently binds to the cofactor (or oligopyrrole chain) consecutively, ultimately forming a hexapyrrole chain. Molecular dynamics simulation of the ES2 intermediate suggested that the thermal fluctuation of the lid and cofactor-binding loops causes substrate recruitment and oligopyrrole chain shift needed for consecutive condensation. Finally, the hexapyrrole chain is hydrolyzed self-catalytically to produce hydroxymethylbilane.
Assuntos
Hidroximetilbilano Sintase/química , Hidroximetilbilano Sintase/metabolismo , Porfobilinogênio/metabolismo , Uroporfirinogênios/metabolismo , Catálise , Domínio Catalítico , Cristalografia por Raios X , Humanos , Simulação de Dinâmica Molecular , Conformação Proteica , Domínios Proteicos , Especificidade por SubstratoRESUMO
KIF1A is a kinesin family protein that moves over a long distance along the microtubule (MT) to transport synaptic vesicle precursors in neurons. A single KIF1A molecule can move toward the plus-end of MT in the monomeric form, exhibiting the characteristics of biased Brownian motion. However, how the bias is generated in the Brownian motion of KIF1A has not yet been firmly established. To elucidate this, we conducted a set of molecular dynamics simulations and observed the binding of KIF1A to MT. We found that KIF1A exhibits biased Brownian motion along MT as it binds to MT. Furthermore, we show that the bias toward the plus-end is generated by the ratchet-like energy landscape for the KIF1A-MT interaction, in which the electrostatic interaction and the negatively-charged C-terminal tail (CTT) of tubulin play an essential role. The relevance to the post-translational modifications of CTT is also discussed.
Assuntos
Fenômenos Biofísicos , Cinesinas/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Domínios e Motivos de Interação entre Proteínas , Tubulina (Proteína)/química , Algoritmos , Transporte Axonal , Cinesinas/metabolismo , Microtúbulos/química , Microtúbulos/metabolismo , Modelos Teóricos , Neurônios/metabolismo , Ligação Proteica , Tubulina (Proteína)/metabolismoRESUMO
Ingestion of marine invertebrates often causes food allergy, where the major allergens have been reported to be derived from tropomyosin (TM). Intact or the digestive fragments of food allergens generally show resistance to digestion, which is usually attributable to the structural stability (or rigidity). The difference in the structural and dynamical characteristics between the epitope and the non-epitope regions in TM has not yet been well understood. In the present study, molecular dynamics simulation was performed at constant pHs for shrimp TM. By analyzing the main-chain dihedral angle fluctuations and local α-helix contents, we found that the epitope regions are more stable than the non-epitope counterparts, providing a possible physical reason for the resistance to digestion in the epitopes regions. The difference of the structural stability between the epitope and the non-epitope regions was largest at low pHs, even though pH dependence of the structural stability in itself was not significant in both regions. The lower content of the Ala cluster in the epitope region is considered to cause the higher stability of the epitope region.
Assuntos
Alérgenos/química , Epitopos/química , Penaeidae/química , Tropomiosina/química , Sequência de Aminoácidos , Animais , Concentração de Íons de Hidrogênio , Estrutura Secundária de Proteína , TemperaturaRESUMO
A high dielectric constant is one of the peculiar properties of liquid water, indicating that the electrostatic interaction between charged substances is largely reduced in water. We show by molecular dynamics simulation that the dielectric constant of water is decreased near the hydrophobic surface. We further show that the decrease in the dielectric constant is due to both the decreased water density and the reduced water dipole correlation in the direction perpendicular to the surface. We finally demonstrate that electrostatic interaction in water is actually strengthened near the hydrophobic surface.
RESUMO
A protein molecule is a dielectric substance, so the binding of a ligand is expected to induce dielectric response in the protein molecule, considering that ligands are charged or polar in general. We previously reported that binding of adenosine triphosphate (ATP) to molecular motor myosin actually induces such a dielectric response in myosin due to the net negative charge of ATP. By this dielectric response, referred to as "dielectric allostery," spatially separated two regions in myosin, the ATP-binding region and the actin-binding region, are allosterically coupled. In this study, from the statistically stringent analyses of the extensive molecular dynamics simulation data obtained in the ATP-free and the ATP-bound states, we show that there exists the dielectric allostery that transmits the signal of ATP binding toward the distant lever-arm region. The ATP-binding-induced electrostatic potential change observed on the surface of the main domain induced a movement of the converter subdomain from which the lever arm extends. The dielectric response was found to be caused by an underlying large-scale concerted rearrangement of the electrostatic bond network, in which highly conserved charged/polar residues are involved. Our study suggests the importance of the dielectric property for molecular machines in exerting their function.
Assuntos
Trifosfato de Adenosina/metabolismo , Subfragmentos de Miosina/metabolismo , Miosina Tipo II/metabolismo , Pectinidae/metabolismo , Animais , Simulação de Dinâmica Molecular , Subfragmentos de Miosina/química , Miosina Tipo II/química , Pectinidae/química , Ligação Proteica , Domínios Proteicos , Eletricidade EstáticaRESUMO
The intrinsically disordered protein (IDP) has distinct properties both physically and biologically: it often becomes folded when binding to the target and is frequently involved in signal transduction. The physical property seems to be compatible with the biological property where fast association and dissociation between IDP and the target are required. While fast association has been well studied, fueled by the fly-casting mechanism, the dissociation kinetics has received less attention. We here study how the intrinsic disorder affects the dissociation kinetics, as well as the association kinetics, paying attention to the interaction strength at the binding site (i.e., the quality of the "fly lure"). Coarse-grained molecular dynamics simulation of the pKID-KIX system, a well-studied IDP system, shows that the association rate becomes larger as the disorder-inducing flexibility that was imparted to the model is increased, but the acceleration is marginal and turns into deceleration as the quality of the fly lure is worsened. In contrast, the dissociation rate is greatly enhanced as the disorder is increased, indicating that intrinsic disorder serves for rapid signal switching more effectively through dissociation than association. Proteins 2016; 84:1124-1133. © 2016 Wiley Periodicals, Inc.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/química , Proteínas Intrinsicamente Desordenadas/química , Simulação de Dinâmica Molecular , Sítios de Ligação , Cinética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , TermodinâmicaRESUMO
An important unresolved problem associated with actomyosin motors is the role of Brownian motion in the process of force generation. On the basis of structural observations of myosins and actins, the widely held lever-arm hypothesis has been proposed, in which proteins are assumed to show sequential structural changes among observed and hypothesized structures to exert mechanical force. An alternative hypothesis, the Brownian motion hypothesis, has been supported by single-molecule experiments and emphasizes more on the roles of fluctuating protein movement. In this study, we address the long-standing controversy between the lever-arm hypothesis and the Brownian motion hypothesis through in silico observations of an actomyosin system. We study a system composed of myosin II and actin filament by calculating free-energy landscapes of actin-myosin interactions using the molecular dynamics method and by simulating transitions among dynamically changing free-energy landscapes using the Monte Carlo method. The results obtained by this combined multi-scale calculation show that myosin with inorganic phosphate (Pi) and ADP weakly binds to actin and that after releasing Pi and ADP, myosin moves along the actin filament toward the strong-binding site by exhibiting the biased Brownian motion, a behavior consistent with the observed single-molecular behavior of myosin. Conformational flexibility of loops at the actin-interface of myosin and the N-terminus of actin subunit is necessary for the distinct bias in the Brownian motion. Both the 5.5-11 nm displacement due to the biased Brownian motion and the 3-5 nm displacement due to lever-arm swing contribute to the net displacement of myosin. The calculated results further suggest that the recovery stroke of the lever arm plays an important role in enhancing the displacement of myosin through multiple cycles of ATP hydrolysis, suggesting a unified movement mechanism for various members of the myosin family.
Assuntos
Actomiosina/química , Coloides , Método de Monte Carlo , Conformação Proteica , Eletricidade EstáticaRESUMO
The actomyosin molecular motor, the motor composed of myosin II and actin filament, is responsible for muscle contraction, converting chemical energy into mechanical work. Although recent single molecule and structural studies have shed new light on the energy-converting mechanism, the physical basis of the molecular-level mechanism remains unclear because of the experimental limitations. To provide a clue to resolve the controversy between the lever-arm mechanism and the Brownian ratchet-like mechanism, we here report an in silico single molecule experiment of an actomyosin motor. When we placed myosin on an actin filament and allowed myosin to move along the filament, we found that myosin exhibits a unidirectional Brownian motion along the filament. This unidirectionality was found to arise from the combination of a nonequilibrium condition realized by coupling to the ATP hydrolysis and a ratchet-like energy landscape inherent in the actin-myosin interaction along the filament, indicating that a Brownian ratchet-like mechanism contributes substantially to the energy conversion of this molecular motor.
Assuntos
Actinas/metabolismo , Modelos Biológicos , Proteínas Motores Moleculares/metabolismo , Movimento/fisiologia , Miosinas/metabolismo , Actinas/genética , Trifosfato de Adenosina/metabolismo , Animais , Galinhas , Mutagênese , Miosinas/genética , Processos EstocásticosRESUMO
The electrochemical potential difference of protons across the membrane is used to synthesize ATP through the proton-motive rotatory motion of the membrane-embedded region of ATP synthase called Fo. In this study, we illuminate the unsolved proton-motive rotary mechanism of Fo on the basis of atomistic simulation with full description of protein, lipid, and water molecules, and highlight the underlying Coulombic design. We first show that a water channel is spontaneously formed at the interfacial region between the rotor (c-ring) and the stator (a-subunit). The observed water channel is a full channel penetrating the membrane, but a Coulomb barrier by a strictly conserved arginine of the a-subunit dominates at the midpoint of the full channel, preventing proton leakage. Our molecular dynamics simulation further demonstrates that the Coulomb attraction between the arginine and the essential glutamic acid of the c-subunit drives the c-ring rotation. We finally illustrate that the charge-state changes of the glutamic acids, enabled by the electrochemical potential difference of proton and the thermal motion, can produce unidirectional rotation of the c-ring.
Assuntos
Simulação de Dinâmica Molecular , Prótons , Rotação , Trifosfato de Adenosina/metabolismo , Arginina , ATPases Translocadoras de Prótons/químicaRESUMO
Association of protein molecules constitutes the basis for the interaction network in a cell. Despite its fundamental importance, the thermodynamic aspect of protein-protein binding, particularly the issues relating to the entropy change upon binding, remains elusive. The binding of actin and myosin, which are vital proteins in motility, is a typical example, in which two different binding mechanisms have been argued: the binding affinity increases with increasing temperature and with decreasing salt-concentration, indicating the entropy-driven binding and the enthalpy-driven binding, respectively. How can these thermodynamically different binding mechanisms coexist? To address this question, which is of general importance in understanding protein-protein bindings, we conducted an in silico titration of the actin-myosin system by molecular dynamics simulation using a residue-level coarse-grained model, with particular focus on the role of the electrostatic interaction. We found a good agreement between in silico and in vitro experiments on the salt-concentration dependence and the temperature dependence of the binding affinity. We then figured out how the two binding mechanisms can coexist: the enthalpy (due to electrostatic interaction between actin and myosin) provides the basal binding affinity, and the entropy (due to the orientational disorder of water molecules) enhances it at higher temperatures. In addition, we analyzed the actin-myosin complex structures observed during the simulation and obtained a variety of weak-binding complex structures, among which were found an unusual binding mode suggested by an earlier experiment and precursor structures of the strong-binding complex proposed by electron microscopy. These results collectively indicate the potential capability of a residue-level coarse-grained model to simulate the association-dissociation dynamics (particularly for transient weak-bindings) exhibited by larger and more complicated systems, as in a cell.
Assuntos
Actinas/metabolismo , Simulação de Dinâmica Molecular , Miosinas/metabolismo , Eletricidade Estática , Temperatura , Actinas/química , Animais , Miosinas/química , Ligação Proteica , Conformação ProteicaRESUMO
Biophysics in Waseda University was started in 1965 as one of the three key research areas that constitute the Physics Department. In the biophysics group, one theoretical lab and two experimental labs are now working on the cutting-edge themes on biophysics, disseminating the ideas and knowledge of biophysics to undergraduate and graduate students from the viewpoint of physics.
RESUMO
Single protein molecules are regarded as contact networks of amino-acid residues. Relationships between the shortest path lengths and the numbers of residues within single molecules in the native structures are examined for various sized proteins. A universal scaling among proteins is obtained, which shows that the residue networks are fractal networks. This universal fractal network is characterized with three kinds of dimensions: the network topological dimension D{c} approximately 1.9 , the fractal dimension D{f} approximately 2.5 , and the spectral dimension D{s} approximately 1.3 . These values are in surprisingly good coincidence with those of the three-dimensional critical percolation cluster. Hence the residue contact networks in the protein native structures belong to the universality class of the three-dimensional percolation cluster. The criticality is relevant to the ambivalence in the protein native structures, the coexistence of stability and instability, both of which are necessary for protein functions.
Assuntos
Aminoácidos/química , Modelos Químicos , Modelos Moleculares , Proteínas/química , Sequência de Aminoácidos , Simulação por Computador , Dados de Sequência Molecular , Conformação ProteicaRESUMO
Cytochrome P450 reductase (CPR) is the key protein that regulates the electron transfer from NADPH to various heme-containing monooxygenases. CPR has two flavin-containing domains: one with flavin adenine dinucleotide (FAD), called FAD domain, and the other with flavin mononucleotide (FMN), called FMN domain. It is considered that the electron transfer occurs via FAD and FMN (NADPH â FAD â FMN â monooxygenase) and is regulated by an interdomain open-close motion. It is generally thought that the structural state is coupled with the redox state, which, however, has not yet been firmly established. In this report, we studied the coupling of the redox and the structural states by full-scale molecular dynamics (MD) simulation of CPR (total 86.4 µs). Our MD result showed that while CPR predominantly adopts the closed state both in the oxidized and reduced states, it exhibits a tendency to open in the reduced state. We also found a correlation between the FAD-FMN distance and the predicted FMN-monooxygenase distance, which is embedded in the equilibrium thermal fluctuation of CPR. Based on these results, a physical mechanism for the electron transfer by CPR is discussed.
Assuntos
Simulação de Dinâmica Molecular , NADPH-Ferri-Hemoproteína Redutase/química , Oxirredução , Sítios de Ligação , Mononucleotídeo de Flavina/química , Mononucleotídeo de Flavina/metabolismo , Flavina-Adenina Dinucleotídeo/química , Flavina-Adenina Dinucleotídeo/metabolismo , Simulação de Acoplamento Molecular , NADP/química , NADP/metabolismo , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Ligação Proteica , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
In nonequilibrium steady states (NESS) far from equilibrium, it is known that the Einstein relation is violated. Then, the ratio of the diffusion coefficient to the mobility is called an effective temperature, and the physical relevance of this effective temperature has been studied in several works. Although the physical relevance is not yet completely clear, it has been found that the role of an effective temperature in NESS is indeed analogous to that of the temperature in equilibrium systems in a number of respects. In this paper, we find further evidence establishing this analogy. We employ a nonequilibrium Langevin system as a thermostat for a Hamiltonian system and find that the kinetic temperature of this Hamiltonian system is equal to the effective temperature of the thermostat.
RESUMO
Actin polymerization depends on the salt concentration, exhibiting a reentrant behavior: the polymerization is promoted by increasing KCl concentration up to 100 mM, and then depressed by further increase above 100 mM. We here investigated the physical mechanism of this reentrant behavior by calculating the polymerization energy, defined by the electrostatic energy change upon binding of an actin subunit to a filament, using an implicit solvent model based on the Poisson-Boltzmann (PB) equation. We found that the polymerization energy as a function of the salt concentration shows a non-monotonic reentrant-like behavior, with the minimum at about 100 mM (1:1 salt). By separately examining the salt concentration effect on the global electrostatic repulsion between the like-charged subunits and that on the local electrostatic attraction between the inter-subunit ionic-bond-forming residues in the filament, we clarified that the reentrant behavior is caused by the change in the balance between the two opposing electrostatic interactions. Our study showed that the non-specific nature of counterions, as described in the mean-field theory, plays an important role in the actin polymerization. We also discussed the endothermic nature of the actin polymerization and mentioned the effect of ATP hydrolysis on the G-F transformation, indicating that the electrostatic interaction is widely and intricately involved in the actin dynamics.
Assuntos
Actinas/química , Modelos Químicos , Multimerização Proteica , Eletricidade Estática , HumanosRESUMO
The generalize Born (GB) model is frequently used in MD simulations of biomolecular systems in aqueous solution. The GB model is usually based on the so-called Coulomb field approximation (CFA) for the energy density integration. In this study, we report that the GB model with CFA overdestabilizes the long-range electrostatic attraction between oppositely charged molecules (ionic bond forming two-helix system and kinesin-tubulin system) when the energy density integration cutoff, rmax, which is used to calculate the Born energy, is set to a large value. We show that employing large rmax, which is usually expected to make simulation results more accurate, worsens the accuracy so that the attraction is changed into repulsion. It is demonstrated that the overdestabilization is caused by the overestimation of the desolvation penalty upon binding that originates from CFA. We point out that the overdestabilization can be corrected by employing a relatively small cutoff (rmax = 10-15 Å), affirming that the GB models, even with CFA, can be used as a powerful tool to theoretically study the protein-protein interaction, particularly on its dynamical aspect, such as binding and unbinding.