Kei-ichi Okazaki

Dynamic energy landscape view of coupled binding and protein conformational change: Induced-fit versus population-shift mechanisms

Allostery, the coupling between ligand binding and protein conformational change, is the heart of biological network and it has often been explained by two representative models, the induced-fit and the population-shift models. Here, we clarified for what systems one model fits better than the other by performing molecular simulations of coupled binding and conformational change. Based on the dynamic energy landscape view, we developed an implicit ligand-binding model combined with the double-basin Hamiltonian that describes conformational change. From model simulations performed for a broad range of parameters, we uncovered that each of the two models has its own range of applicability, stronger and longer-ranged interaction between ligand and protein favors the induced-fit model, and weaker and shorter-ranged interaction leads to the population-shift model. We further postulate that the protein binding to small ligand tends to proceed via the population-shift model, whereas the protein docking to macromolecules such as DNA tends to fit the induced-fit model.

Multiple-basin energy landscapes for large-amplitude conformational motions of proteins: Structure-based molecular dynamics simulations

Biomolecules often undergo large-amplitude motions when they bind or release other molecules. Unlike macroscopic machines, these biomolecular machines can partially disassemble (unfold) and then reassemble (fold) during such transitions. Here we put forward a minimal structure-based model, the “multiple-basin model,” that can directly be used for molecular dynamics simulation of even very large biomolecular systems so long as the endpoints of the conformational change are known. We investigate the model by simulating large-scale motions of four proteins: glutamine-binding protein, S100A6, dihydrofolate reductase, and HIV-1 protease. The mechanisms of conformational transition depend on the protein basin topologies and change with temperature near the folding transition. The conformational transition rate varies linearly with driving force over a fairly large range. This linearity appears to be a consequence of partial unfolding during the conformational transition.

Phosphate release coupled to rotary motion of F1-ATPase

Significance F1-ATPase is the catalytic domain of FoF1-ATP synthase, the rotary molecular motor at the core of the energy transduction machinery in all of life. We use atomistic molecular dynamics simulations to study a key event in its catalytic cycle, the release of inorganic phosphate (Pi) produced by the hydrolysis of ATP. We determine the timing, kinetics, and molecular mechanism of Pi release and clarify its role in torque generation. We also obtain an atomically detailed structure of a crystallographically unresolved intermediate formed after the 40° substep. Our results help reconcile conflicting interpretations of earlier biochemical, crystallographic, and single-molecule studies; shed light on the functional requirements of efficient ATP synthesis; and establish connections to other motors such as myosin. F1-ATPase, the catalytic domain of ATP synthase, synthesizes most of the ATP in living organisms. Running in reverse powered by ATP hydrolysis, this hexameric ring-shaped molecular motor formed by three αβ-dimers creates torque on its central γ-subunit. This reverse operation enables detailed explorations of the mechanochemical coupling mechanisms in experiment and simulation. Here, we use molecular dynamics simulations to construct a first atomistic conformation of the intermediate state following the 40° substep of rotary motion, and to study the timing and molecular mechanism of inorganic phosphate (Pi) release coupled to the rotation. In response to torque-driven rotation of the γ-subunit in the hydrolysis direction, the nucleotide-free αβE interface forming the “empty” E site loosens and singly charged Pi readily escapes to the P loop. By contrast, the interface stays closed with doubly charged Pi. The γ-rotation tightens the ATP-bound αβTP interface, as required for hydrolysis. The calculated rate for the outward release of doubly charged Pi from the αβE interface 120° after ATP hydrolysis closely matches the ∼1-ms functional timescale. Conversely, Pi release from the ADP-bound αβDP interface postulated in earlier models would occur through a kinetically infeasible inward-directed pathway. Our simulations help reconcile conflicting interpretations of single-molecule experiments and crystallographic studies by clarifying the timing of Pi exit, its pathway and kinetics, associated changes in Pi protonation, and changes of the F1-ATPase structure in the 40° substep. Important elements of the molecular mechanism of Pi release emerging from our simulations appear to be conserved in myosin despite the different functional motions.

Structural Comparison of F1-ATPase: Interplay among Enzyme Structures, Catalysis, and Rotations

F(1)-ATPase, a rotary motor powered by adenosine triphosphate hydrolysis, has been extensively studied by various methods. Here, we performed a systematic comparison of 29 X-ray crystal structures of F(1)-complexes, finding fine interplay among enzyme structures, catalysis, and rotations. First, analyzing the 87 structures of enzymatic αβ-subunits, we confirmed that the two modes, the hinge motion of β-subunit and the loose/tight motion of the αβ-interface, dominate the variations. The structural ensemble was nearly contiguous bridging three clusters, αβ(TP), αβ(DP), and αβ(E). Second, the catalytic site analysis suggested the correlation between the phosphate binding and the tightening of the αβ-interface. Third, addressing correlations of enzymatic structures with the orientations of the central stalk γ, we found that the γ rotation highly correlates with loosening of αβ(E)-interface and β(DP) hinge motions. Finally, calculating the helix 6 angle of β, we identified the recently observed partially closed conformation being consistent with β(HC).

Temperature-Enhanced Association of Proteins Due to Electrostatic Interaction: A Coarse-Grained Simulation of Actin–Myosin Binding

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.

Paddling mechanism for the substrate translocation by AAA+ motor revealed by multiscale molecular simulations

Hexameric ring-shaped AAA+ molecular motors have a key function of active translocation of a macromolecular chain through the central pore. By performing multiscale molecular dynamics (MD) simulations, we revealed that HslU, a AAA+ motor in a bacterial homologue of eukaryotic proteasome, translocates its substrate polypeptide via paddling mechanism during ATP-driven cyclic conformational changes. First, fully atomistic MD simulations showed that the HslU pore grips the threaded signal peptide by the highly conserved Tyr-91 and Val-92 firmly in the closed form and loosely in the open form of the HslU. The grip depended on the substrate sequence. These features were fed into a coarse-grained MD, and conformational transitions of HslU upon ATP cycles were simulated. The simulations exhibited stochastic unidirectional translocation of a polypeptide. This unidirectional translocation is attributed to paddling motions of Tyr-91s between the open and the closed forms: downward motions of Tyr-91s with gripping the substrate and upward motions with slipping on it. The paddling motions were caused by the difference between the characteristic time scales of the pore-radius change and the up-down displacements of Tyr-91s. Computational experiments on mutations at the pore and the substrate were in accord with several experiments.

Elasticity, friction, and pathway of γ-subunit rotation in FoF1-ATP synthase

Significance FoF1-ATP synthase produces the ATP essential for cellular functions from bacteria to humans. Rotation of its central γ-subunit couples proton translocation in the membrane-embedded Fo motor to ATP synthesis in the catalytic F1 motor. To explain its high efficiency, determine its top speed, and characterize its mechanism, we construct a viscoelastic model of the F1 rotary motor from molecular dynamics simulation trajectories. We find that the γ-subunit is just flexible enough to compensate for the incommensurate eightfold and threefold rotational symmetries of mammalian Fo and F1 motors, respectively. The resulting energetic constraints dictate a unique pathway for the coupled rotations of the Fo and F1 rotary motors, and explain the fine stepping seen in single-molecule experiments. We combine molecular simulations and mechanical modeling to explore the mechanism of energy conversion in the coupled rotary motors of FoF1-ATP synthase. A torsional viscoelastic model with frictional dissipation quantitatively reproduces the dynamics and energetics seen in atomistic molecular dynamics simulations of torque-driven γ-subunit rotation in the F1-ATPase rotary motor. The torsional elastic coefficients determined from the simulations agree with results from independent single-molecule experiments probing different segments of the γ-subunit, which resolves a long-lasting controversy. At steady rotational speeds of ∼1 kHz corresponding to experimental turnover, the calculated frictional dissipation of less than kBT per rotation is consistent with the high thermodynamic efficiency of the fully reversible motor. Without load, the maximum rotational speed during transitions between dwells is reached at ∼1 MHz. Energetic constraints dictate a unique pathway for the coupled rotations of the Fo and F1 rotary motors in ATP synthase, and explain the need for the finer stepping of the F1 motor in the mammalian system, as seen in recent experiments. Compensating for incommensurate eightfold and threefold rotational symmetries in Fo and F1, respectively, a significant fraction of the external mechanical work is transiently stored as elastic energy in the γ-subunit. The general framework developed here should be applicable to other molecular machines.

F1-ATPase conformational cycle from simultaneous single-molecule FRET and rotation measurements.

Significance The major source of ATP in life is ATP synthase, and its catalytic part is known to be the F1 rotary motor. F1’s structure and function have been characterized in spectacular detail by crystallography and single-molecule techniques, respectively. However, despite more than two decades of intense research, the correspondence of the observed functional states and crystal structures is uncertain. To match structures and states, we perform single-molecule fluorescence-based distance measurements and simultaneous rotary angle measurements on F1-ATPase, and then exploit the wealth of structural data in their analysis. The resulting comprehensive view of the F1’s ATPase cycle reveals the functional principles in the coupling of chemical reactions, stator conformations, and rotary angles for efficient ATP synthesis. Despite extensive studies, the structural basis for the mechanochemical coupling in the rotary molecular motor F1-ATPase (F1) is still incomplete. We performed single-molecule FRET measurements to monitor conformational changes in the stator ring-α3β3, while simultaneously monitoring rotations of the central shaft-γ. In the ATP waiting dwell, two of three β-subunits simultaneously adopt low FRET nonclosed forms. By contrast, in the catalytic intermediate dwell, two β-subunits are simultaneously in a high FRET closed form. These differences allow us to assign crystal structures directly to both major dwell states, thus resolving a long-standing issue and establishing a firm connection between F1 structure and the rotation angle of the motor. Remarkably, a structure of F1 in an ε-inhibited state is consistent with the unique FRET signature of the ATP waiting dwell, while most crystal structures capture the structure in the catalytic dwell. Principal component analysis of the available crystal structures further clarifies the five-step conformational transitions of the αβ-dimer in the ATPase cycle, highlighting the two dominant modes: the opening/closing motions of β and the loosening/tightening motions at the αβ-interface. These results provide a new view of tripartite coupling among chemical reactions, stator conformations, and rotary angles in F1-ATPase.

Processive chitinase is Brownian monorail operated by fast catalysis after peeling rail from crystalline chitin

Processive chitinase is a linear molecular motor which moves on the surface of crystalline chitin driven by processive hydrolysis of single chitin chain. Here, we analyse the mechanism underlying unidirectional movement of Serratia marcescens chitinase A (SmChiA) using high-precision single-molecule imaging, X-ray crystallography, and all-atom molecular dynamics simulation. SmChiA shows fast unidirectional movement of ~50 nm s−1 with 1 nm forward and backward steps, consistent with the length of reaction product chitobiose. Analysis of the kinetic isotope effect reveals fast substrate-assisted catalysis with time constant of ~3 ms. Decrystallization of the single chitin chain from crystal surface is the rate-limiting step of movement with time constant of ~17 ms, achieved by binding free energy at the product-binding site of SmChiA. Our results demonstrate that SmChiA operates as a burnt-bridge Brownian ratchet wherein the Brownian motion along the single chitin chain is rectified forward by substrate-assisted catalysis.Processive chitinase is a linear molecular motor which moves on the surface of crystalline chitin. Here authors use single-molecule imaging, X-ray crystallography and simulations on chitinase A (SmChiA) and show that Brownian motion along the single chitin chain is rectified forward by substrate-assisted catalysis.

Exploring an Intermediate State of F1-ATPase by Atomistic Molecular Dynamics Simulation

F1-ATPase, the catalytic domain of FoF1-ATP synthase, is a rotary molecular motor that reversibly interconverts ATP hydrolysis free energy and mechanical work associated with the rotation of the central stalk. In the structure, α- and β-subunits alternatingly arrange to form a hexameric ring, with the rod-like γ -subunit located at its center. Single-molecule experiments showed that the γ -subunit rotates in 120° steps, and that this step is further divided into 80° and 40° substeps. We thus expect two metastable conformations of F1, one before the 80° substep (binding dwell state) and the other before the 40° substep (catalytic dwell state). X-ray structures of F1-ATPase in the catalytic dwell state have provided tremendous functional insight. However, to complete our understanding of the mechanochemical coupling of the F1 motor, it is important to explore also the binding dwell state, and the transitions between the substates. In this work, we use molecular dynamics simulations to study the structure, motions, and ligand dissociation energetics of F1-ATPase as a function of the γ -subunit rotation angle. The γ -subunit is rotated with the help of a newly developed torque simulation method. In our all-atom/explicit-solvent molecular dynamics simulations we use enhanced sampling techniques to study phosphate (Pi) release in the transition from the catalytic to the binding dwell state, and to characterize the conformational responses of the α β-subunits. From the simulations, we obtain a detailed picture of the conformational changes in F1-ATPase induced by γ -subunit rotation, their energetics, and of their relevance to function.