Catalytic robustness and torque generation of the F1-ATPase.
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ABSTRACT:
SUBMITTER: Noji H
PROVIDER: S-EPMC5380711 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Molecular machines fueled by NTP play pivotal roles in a wide range of cellular activities. One common feature among NTP-driven molecular machines is that NTP binding is a major force-generating step among the elementary reaction steps comprising NTP hydrolysis. To understand the mechanism in detail,in this study, we conducted a single-molecule rotation assay of the ATP-driven rotary motor protein F1-ATPase using uridine triphosphate (UTP) and a base-free nucleotide (ribose triphosphate) to investigate the impact of a pyrimidine base or base depletion on kinetics and force generation. Although the binding rates of UTP and ribose triphosphate were 10(3) and 10(6) times, respectively, slower than that of ATP, they supported rotation, generating torque comparable to that generated by ATP. Affinity change of F1 to UTP coupled with rotation was determined, and the results again were comparable to those for ATP, suggesting that F1 exerts torque upon the affinity change to UTP via rotation similar to ATP-driven rotation. Thus, the adenine-ring significantly enhances the binding rate, although it is not directly involved in force generation. Taking into account the findings from another study on F1 with mutated phosphate-binding residues, it was proposed that progressive bond formation between the phosphate region and catalytic residues is responsible for the rotation-coupled change in affinity.
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Project description:Based on recent structural and functional findings, we have constructed a mathematical model for the sodium-driven Fo motor of the F1Fo-ATPase from the anaerobic bacterium Propionigenium modestum. The model reveals the mechanochemical principles underlying the Fo motor's operation, and explains all of the existing experimental data on wild-type and mutant Fo motors. In particular, the model predicts a nonmonotonic dependence of the ATP hydrolysis activity on the sodium concentration, a prediction confirmed by new experiments. To explain experimental observations, the positively charged stator residue (R227) must assume different positions in the ATP synthesis and hydrolysis directions. This work also illustrates how to extract a motor mechanism from dynamical experimental observations in the absence of complete structural information.
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Project description:F1-ATPase is a powerful rotary molecular motor that can rotate an object several hundred times as large as the motor itself against the viscous friction of water. Forced reverse rotation has been shown to lead to ATP synthesis, implying that the mechanical work against the motor's high torque can be converted into the chemical energy of ATP. The minimal composition of the motor protein is α3β3γ subunits, where the central rotor subunit γ turns inside a stator cylinder made of alternately arranged α3β3 subunits using the energy derived from ATP hydrolysis. The rotor consists of an axle, a coiled coil of the amino- and carboxyl-terminal α-helices of γ, which deeply penetrates the stator cylinder, and a globular protrusion that juts out from the stator. Previous work has shown that, for a thermophilic F1, significant portions of the axle can be truncated and the motor still rotates a submicron sized bead duplex, indicating generation of up to half the wild-type (WT) torque. Here, we inquire if any specific interactions between the stator and the rest of the rotor are needed for the generation of a sizable torque. We truncated the protruding portion of the rotor and replaced part of the remaining axle residues such that every residue of the rotor has been deleted or replaced in this or previous truncation mutants. This protrusionless construct showed an unloaded rotary speed about a quarter of the WT, and generated one-third to one-half of the WT torque. No residue-specific interactions are needed for this much performance. F1 is so designed that the basic rotor-stator interactions for torque generation and control of catalysis rely solely upon the shape and size of the rotor at very low resolution. Additional tailored interactions augment the torque to allow ATP synthesis under physiological conditions.
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Project description:Spin-orbit coupling (SOC), the interaction between the electron spin and the orbital angular momentum, can unlock rich phenomena at interfaces, in particular interconverting spin and charge currents. Conventional heavy metals have been extensively explored due to their strong SOC of conduction electrons. However, spin-orbit effects in classes of materials such as epitaxial 5d-electron transition-metal complex oxides, which also host strong SOC, remain largely unreported. In addition to strong SOC, these complex oxides can also provide the additional tuning knob of epitaxy to control the electronic structure and the engineering of spin-to-charge conversion by crystalline symmetry. Here, we demonstrate room-temperature generation of spin-orbit torque on a ferromagnet with extremely high efficiency via the spin-Hall effect in epitaxial metastable perovskite SrIrO3 We first predict a large intrinsic spin-Hall conductivity in orthorhombic bulk SrIrO3 arising from the Berry curvature in the electronic band structure. By manipulating the intricate interplay between SOC and crystalline symmetry, we control the spin-Hall torque ratio by engineering the tilt of the corner-sharing oxygen octahedra in perovskite SrIrO3 through epitaxial strain. This allows the presence of an anisotropic spin-Hall effect due to a characteristic structural anisotropy in SrIrO3 with orthorhombic symmetry. Our experimental findings demonstrate the heteroepitaxial symmetry design approach to engineer spin-orbit effects. We therefore anticipate that these epitaxial 5d transition-metal oxide thin films can be an ideal building block for low-power spintronics.
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Project description:F(1)-ATPase is an ATP-driven rotary molecular motor in which the central gamma-subunit rotates inside a stator cylinder made of alpha(3)beta(3) subunits. To elucidate the role of rotor-stator interactions in torque generation, we truncated the gamma-subunit at its carboxyl terminus, which forms an alpha helix that penetrates deeply into the stator cylinder. We used an alpha(3)beta(3)gamma subcomplex of F(1)-ATPase derived from thermophilic Bacillus PS3 and expressed it in Escherichia coli. We could obtain purified subcomplexes in which 14, 17, or 21 amino-acid residues were deleted. The rotary characteristics of the truncated mutants, monitored by attaching a duplex of 0.49-microm beads to the gamma-subunit, did not differ greatly from those of the wild-type over the ATP concentrations of 20 nM-2 mM, the most conspicuous effect being approximately 50% reduction in torque and approximately 70% reduction in the rate of ATP binding upon deletion of 21 residues. The ATP hydrolysis activity estimated in bulk samples was more seriously affected. The 21-deletion mutant, in particular, was >10-fold less active, but this is likely due to instability of this subcomplex. For torque generation, though not for rapid catalysis, most of the rotor-stator contacts on the deeper half of the penetrating portion of the gamma-subunit are dispensable.
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Project description:F1Fo ATP synthase interchanges phosphate transfer energy and proton motive force via a rotary catalysis mechanism. Isolated F1-ATPase catalytic cores can hydrolyze ATP, passing through six intermediate conformational states to generate rotation of their central γ-subunit. Although previous structural studies have contributed greatly to understanding rotary catalysis in the F1-ATPase, the structure of an important conformational state (the binding-dwell) has remained elusive. Here, we exploit temperature and time-resolved cryo-electron microscopy to determine the structure of the binding- and catalytic-dwell states of Bacillus PS3 F1-ATPase. Each state shows three catalytic β-subunits in different conformations, establishing the complete set of six states taken up during the catalytic cycle and providing molecular details for both the ATP binding and hydrolysis strokes. We also identify a potential phosphate-release tunnel that indicates how ADP and phosphate binding are coordinated during synthesis. Overall these findings provide a structural basis for the entire F1-ATPase catalytic cycle.
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Project description:Unraveling the molecular nature of the conversion of chemical energy (ATP hydrolysis in the α/β-subunits) to mechanical energy and torque (rotation of the γ-subunit) in F1-ATPase is very challenging. A major part of the challenge involves understanding the rotary-chemical coupling by a nonphenomenological structure-energy description, while accounting for the observed torque generated on the γ-subunit and its change due to mutation of this unit. Here we extend our previous study that used a coarse-grained model of the F1-ATPase to generate a structure-based free energy landscape of the rotary-chemical process. Our quantitative analysis of the landscape reproduced the observed torque for the wild-type enzyme. In doing so, we found that there are several possibilities of torque generation from landscapes with various shapes and demonstrated that a downhill slope along the chemical coordinate could still result in negligible torque, due to ineffective coupling of the chemistry to the γ-subunit rotation. We then explored the relationship between the functionality and the underlying sequence through systematic examination of the effect of various parts of the γ-subunit on free energy surfaces of F1-ATPase. Furthermore, by constructing several types of γ-deletion systems and calculating the corresponding torque generation, we gained previously unknown insights into the molecular nature of the F1-ATPase rotary motor. Significantly, our results are in excellent agreement with recent experimental findings and indicate that the rotary-chemical coupling is primarily established through electrostatic effects, although specific contacts through γ-ionizable residue side chains are not essential for establishing the basic features of the coupling.
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Project description:F1-ATPase (F1) is an ATP-driven rotary motor in which the three catalytic β subunits in the stator ring sequentially induce the unidirectional rotation of the rotary γ subunit. Many lines of evidence have revealed open-to-closed conformational transitions in the β subunit that swing the C-terminal domain inward. This conformational transition causes a C-terminal protruding loop with conserved sequence DELSEED to push the γ subunit. Previous work, where all residues of DELSEED were substituted with glycine to disrupt the specific interaction with γ and introduce conformational flexibility, showed that F1 still rotated, but that the torque was halved, indicating a remarkable impact on torque transmission. In this study, we conducted a stall-and-release experiment on F1 with a glycine-substituted DELSEED loop to investigate the impact of the glycine substitution on torque transmission upon ATP binding and ATP hydrolysis. The mutant F1 showed a significantly reduced angle-dependent change in ATP affinity, whereas there was no change in the equilibrium for ATP hydrolysis. These findings indicate that the DELSEED loop is predominantly responsible for torque transmission upon ATP binding but not for that upon ATP hydrolysis.
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