Elasticity, friction, and pathway of γ-subunit rotation in FoF1-ATP synthase.
Ontology highlight
ABSTRACT: 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 k(B)T 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.
Project description:F(O)F(1)-ATP synthases are ubiquitous proton- or ion-powered membrane enzymes providing ATP for all kinds of cellular processes. The mechanochemistry of catalysis is driven by two rotary nanomotors coupled within the enzyme. Their different step sizes have been observed by single-molecule microscopy including videomicroscopy of fluctuating nanobeads attached to single enzymes and single-molecule Förster resonance energy transfer. Here we review recent developments of approaches to monitor the step size of subunit rotation and the transient elastic energy storage mechanism in single F(O)F(1)-ATP synthases.
Project description:The F(1)F(o)-type ATP synthase is the smallest motor enzyme known. Previous studies had established that the central gamma and epsilon subunits of the F(1) part rotate relative to a stator of alpha(3)beta(3) and delta subunits during catalysis. We now show that the ring of c subunits in the F(o) part moves along with the gamma and epsilon subunits. This was demonstrated by linking the three rotor subunits with disulfide bridges between cysteine residues introduced genetically at the interfaces between the gamma, epsilon, and c subunits. Essentially complete cross-linking of the gamma, epsilon, and c subunits was achieved by using CuCl(2) to induce oxidation. This fixing of the three subunits together had no significant effect on ATP hydrolysis, proton translocation, or ATP synthesis, and each of these functions retained inhibitor sensitivity. These results unequivocally place the c subunit oligomer in the rotor part of this molecular machine.
Project description:FoF1-ATP synthase (FoF1) is a motor enzyme that couples ATP synthesis/hydrolysis with a transmembrane proton translocation. F1, a water-soluble ATPase portion of FoF1, rotates by repeating ATP-waiting dwell, 80 degrees substep rotation, catalytic dwell, and 40 degrees -substep rotation. Compared with F1, rotation of FoF1 has yet been poorly understood, and, here, we analyzed ATP-driven rotations of FoF1. Rotation was probed with an 80-nm bead attached to the ring of c subunits in the immobilized FoF1 and recorded with a submillisecond fast camera. The rotation rates at various ATP concentrations obeyed the curve defined by a Km of approximately 30 microM and a Vmax of approximately 350 revolutions per second (at 37 degrees C). At low ATP, ATP-waiting dwell was seen and the kon-ATP was estimated to be 3.6 x 10(7) M(-1) x s(-1). At high ATP, fast, poorly defined stepwise motions were observed that probably reflect the catalytic dwells. When a slowly hydrolyzable substrate, adenosine 5'-[gamma-thio]triphosphate, was used, the catalytic dwells consisting of two events were seen more clearly at the angular position of approximately 80 degrees . The rotational behavior of FoF1 resembles that of F1. This finding indicates that "friction" in Fo motor is negligible during the ATP-driven rotation. Tributyltin chloride, a specific inhibitor of proton translocation, slowed the rotation rate by 96%. However, dwells at clearly defined angular positions were not observed under these conditions, indicating that inhibition by tributyltin chloride is complex.
Project description:RNA editing is the process of modifying RNA molecules by inserting, deleting, or substituting nucleotides. In flowering plants, RNA editing occurs predominantly in RNAs encoded by the organellar genomes of mitochondria and chloroplasts, and the main type of editing involves the substitution of cytidine with uridine at specific sites. Abnormal RNA editing in plants can affect gene expression, organelle function, plant growth, and reproduction. In this study, we report that ATPC1, the gamma subunit of ATP synthase in Arabidopsis chloroplasts, has an unexpected role in the regulation of editing at multiple sites of plastid RNAs. The loss of function of ATPC1 severely arrests chloroplast development, causing a pale-green phenotype and early seedling lethality. Disruption of ATPC1 increases the editing of matK-640, rps12-i-58, atpH-3'UTR-13210, and ycf2-as-91535 sites while decreasing the editing of rpl23-89, rpoA-200, rpoC1-488, and ndhD-2 sites. We further show that ATPC1 participates in RNA editing by interacting with known multiple-site chloroplast RNA editing factors, including MORFs, ORRM1, and OZ1. The transcriptome in the atpc1 mutant is profoundly affected, with a pattern of defective expression of chloroplast development-related genes. These results reveal that the ATP synthase γ subunit ATPC1 is involved in multiple-site RNA editing in Arabidopsis chloroplasts.
Project description:Mycobacteria regulate their energy (ATP) levels to sustain their survival even in stringent living conditions. Recent studies have shown that mycobacteria not only slow down their respiratory rate but also block ATP hydrolysis of the F-ATP synthase (α3:β3:γ:δ:ε:a:b:b':c9) to maintain ATP homeostasis in situations not amenable for growth. The mycobacteria-specific α C-terminus (α533-545) has unraveled to be the major regulative of latent ATP hydrolysis. Its deletion stimulates ATPase activity while reducing ATP synthesis. In one of the six rotational states of F-ATP synthase, α533-545 has been visualized to dock deep into subunit γ, thereby blocking rotation of γ within the engine. The functional role(s) of this C-terminus in the other rotational states are not clarified yet and are being still pursued in structural studies. Based on the interaction pattern of the docked α533-545 region with subunit γ, we attempted to study the druggability of the α533-545 motif. In this direction, our computational work has led to the development of an eight-featured α533-545 peptide pharmacophore, followed by database screening, molecular docking, and pose selection, resulting in eleven hit molecules. ATP synthesis inhibition assays using recombinant ATP synthase as well as mycobacterial inverted membrane vesicles show that one of the hits, AlMF1, inhibited the mycobacterial F-ATP synthase in a micromolar range. The successful targeting of the α533-545-γ interaction motif demonstrates the potential to develop inhibitors targeting the α site to interrupt rotary coupling with ATP synthesis.
Project description:F1 domain of ATP synthase is a rotary ATPase complex in which rotation of central γ-subunit proceeds in 120° steps against a surrounding α3β3 fueled by ATP hydrolysis. How the ATP hydrolysis reactions occurring in three catalytic αβ dimers are coupled to mechanical rotation is a key outstanding question. Here we describe catalytic intermediates of the F1 domain in FoF1 synthase from Bacillus PS3 sp. during ATP mediated rotation captured using cryo-EM. The structures reveal that three catalytic events and the first 80° rotation occur simultaneously in F1 domain when nucleotides are bound at all the three catalytic αβ dimers. The remaining 40° rotation of the complete 120° step is driven by completion of ATP hydrolysis at αDβD, and proceeds through three sub-steps (83°, 91°, 101°, and 120°) with three associated conformational intermediates. All sub-steps except for one between 91° and 101° associated with phosphate release, occur independently of the chemical cycle, suggesting that the 40° rotation is largely driven by release of intramolecular strain accumulated by the 80° rotation. Together with our previous results, these findings provide the molecular basis of ATP driven rotation of ATP synthases.
Project description:ATP synthase uses a unique rotational mechanism to convert chemical energy into mechanical energy and back into chemical energy. The helix-turn-helix structure in the C-terminal domain of the β subunit containing the conserved DELSEED motif, termed "DELSEED-loop," was suggested to be involved in coupling between catalysis and rotation. If this is indeed the role of the loop, it must have a critical length, the minimum length required to sustain its function. Here, the critical length of the DELSEED-loop was determined by functional analysis of mutants of Bacillus PS3 ATP synthase that had 7-14 amino acids within the loop deleted. A 10 residue deletion lost the ability to catalyze ATP synthesis, but was still an active ATPase. Deletion of 14 residues abolished any enzymatic activity. Modeling indicated that in both deletion mutants the DELSEED-loop was shortened by ∼10 Å; fluorescence resonance energy transfer experiments confirmed the modeling results. This appears to define the minimum length for DELSEED-loop required for coupling of catalysis and rotation. In addition, we could demonstrate that the loss of high-affinity binding to the catalytic site(s) that had been observed previously in two deletion mutants with 3-4 residues removed was not due to the loss of negative charged residues of the DELSEED motif in these mutants. An AALSAAA mutant in which all negative charges of the DELSEED motif were removed showed a normal pattern for MgATP binding to the catalytic sites, with a clearly present high-affinity site.
Project description:Living organisms rely on the FoF1 ATP synthase to maintain the non-equilibrium chemical gradient of ATP to ADP and phosphate that provides the primary energy source for cellular processes. How the Fo motor uses a transmembrane electrochemical ion gradient to create clockwise torque that overcomes F1 ATPase-driven counterclockwise torque at high ATP is a major unresolved question. Using single FoF1 molecules embedded in lipid bilayer nanodiscs, we now report the observation of Fo-dependent rotation of the c10 ring in the ATP synthase (clockwise) direction against the counterclockwise force of ATPase-driven rotation that occurs upon formation of a leash with Fo stator subunit a. Mutational studies indicate that the leash is important for ATP synthase activity and support a mechanism in which residues aGlu-196 and cArg-50 participate in the cytoplasmic proton half-channel to promote leash formation.
Project description:Here, we provide evidence that high ATP production by the mitochondrial ATP-synthase is a new therapeutic target for anticancer therapy, especially for preventing tumor progression. More specifically, we isolated a subpopulation of ATP-high cancer cells which are phenotypically aggressive and demonstrate increases in proliferation, stemness, anchorage-independence, cell migration, invasion and multi-drug resistance, as well as high antioxidant capacity. Clinically, these findings have important implications for understanding treatment failure and cancer cell dormancy. Using bioinformatic analysis of patient samples, we defined a mitochondrial-related gene signature for metastasis, which features the gamma-subunit of the mitochondrial ATP-synthase (ATP5F1C). The relationship between ATP5F1C protein expression and metastasis was indeed confirmed by immunohistochemistry. Next, we used MDA-MB-231 cells as a model system to functionally validate these findings. Importantly, ATP-high MDA-MB-231 cells showed a nearly fivefold increase in metastatic capacity in vivo. Consistent with these observations, ATP-high cells overexpressed (i) components of mitochondrial complexes I-V, including ATP5F1C, and (ii) markers associated with circulating tumor cells (CTCs) and metastasis, such as EpCAM and VCAM1. Knockdown of ATP5F1C expression significantly reduced ATP-production, anchorage-independent growth, and cell migration, as predicted. Similarly, therapeutic administration of the FDA-approved drug, Bedaquiline, downregulated ATP5F1C expression in vitro and prevented spontaneous metastasis in vivo. In contrast, Bedaquiline had no effect on the growth of non-tumorigenic mammary epithelial cells (MCF10A) or primary tumors in vivo. Taken together, our results suggest that mitochondrial ATP depletion is a new therapeutic strategy for metastasis prophylaxis, to avoid treatment failure. In summary, we conclude that mitochondrial ATP5F1C is a promising new biomarker and molecular target for future drug development, for the prevention of metastatic disease progression.
Project description:Proton-powered c-ring rotation in mitochondrial ATP synthase is crucial to convert the transmembrane protonmotive force into torque to drive the synthesis of adenosine triphosphate (ATP). Capitalizing on recent cryo-EM structures, we aim at a structural and energetic understanding of how functional directional rotation is achieved. We performed multi-microsecond atomistic simulations to determine the free energy profiles along the c-ring rotation angle before and after the arrival of a new proton. Our results reveal that rotation proceeds by dynamic sliding of the ring over the a-subunit surface, during which interactions with conserved polar residues stabilize distinct intermediates. Ordered water chains line up for a Grotthuss-type proton transfer in one of these intermediates. After proton transfer, a high barrier prevents backward rotation and an overall drop in free energy favors forward rotation, ensuring the directionality of c-ring rotation required for the thermodynamically disfavored ATP synthesis. The essential arginine of the a-subunit stabilizes the rotated configuration through a salt bridge with the c-ring. Overall, we describe a complete mechanism for the rotation step of the ATP synthase rotor, thereby illuminating a process critical to all life at atomic resolution.