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The phototroph-specific β-hairpin structure of the γ subunit of F_oF₁-ATP synthase is important for efficient ATP synthesis of cyanobacteria.

ABSTRACT: The F_oF₁ synthase produces ATP from ADP and inorganic phosphate. The γ subunit of F_oF₁ ATP synthase in photosynthetic organisms, which is the rotor subunit of this enzyme, contains a characteristic β-hairpin structure. This structure is formed from an insertion sequence that has been conserved only in phototrophs. Using recombinant subcomplexes, we previously demonstrated that this region plays an essential role in the regulation of ATP hydrolysis activity, thereby functioning in controlling intracellular ATP levels in response to changes in the light environment. However, the role of this region in ATP synthesis has long remained an open question because its analysis requires the preparation of the whole F_oF₁ complex and a transmembrane proton-motive force. In this study, we successfully prepared proteoliposomes containing the entire F_oF₁ ATP synthase from a cyanobacterium, Synechocystis sp. PCC 6803, and measured ATP synthesis/hydrolysis and proton-translocating activities. The relatively simple genetic manipulation of Synechocystis enabled the biochemical investigation of the role of the β-hairpin structure of F_oF₁ ATP synthase and its activities. We further performed physiological analyses of Synechocystis mutant strains lacking the β-hairpin structure, which provided novel insights into the regulatory mechanisms of F_oF₁ ATP synthase in cyanobacteria via the phototroph-specific region of the γ subunit. Our results indicated that this structure critically contributes to ATP synthesis and suppresses ATP hydrolysis.

SUBMITTER: Kondo K

PROVIDER: S-EPMC8390522 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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:The F-ATP synthase, consisting of F1 and FO motors connected by a central rotor and the stators, is the enzyme responsible for synthesizing the majority of ATP in all organisms. The F1 (αβ)3 ring stator contains three catalytic sites. Single-molecule F1 rotation studies revealed that ATP hydrolysis at each catalytic site (0°) precedes a power-stroke that rotates subunit-γ 120° with angular velocities that vary with rotational position. Catalytic site conformations vary relative to subunit-γ position (βE, empty; βD, ADP bound; βT, ATP-bound). During a power stroke, βE binds ATP (0°-60°) and βD releases ADP (60°-120°). Årrhenius analysis of the power stroke revealed that elastic energy powers rotation via unwinding the γ-subunit coiled-coil. Energy from ATP binding at 34° closes βE upon subunit-γ to drive rotation to 120° and forcing the subunit-γ to exchange its tether from βE to βD, which changes catalytic site conformations. In F1FO, the membrane-bound FO complex contains a ring of c-subunits that is attached to subunit-γ. This c-ring rotates relative to the subunit-a stator in response to transmembrane proton flow driven by a pH gradient, which drives subunit-γ rotation in the opposite direction to force ATP synthesis in F1. Single-molecule studies of F1FO embedded in lipid bilayer nanodisks showed that the c-ring transiently stopped F1-ATPase-driven rotation every 36° (at each c-subunit in the c10-ring of E. coli F1FO) and was able to rotate 11° in the direction of ATP synthesis. Protonation and deprotonation of the conserved carboxyl group on each c-subunit is facilitated by separate groups of subunit-a residues, which were determined to have different pKa's. Mutations of any of any residue from either group changed both pKa values, which changed the occurrence of the 11° rotation proportionately. This supports a Grotthuss mechanism for proton translocation and indicates that proton translocation occurs during the 11° steps. This is consistent with a mechanism in which each 36° of rotation the c-ring during ATP synthesis involves a proton translocation-dependent 11° rotation of the c-ring, followed by a 25° rotation driven by electrostatic interaction of the negatively charged unprotonated carboxyl group to the positively charged essential arginine in subunit-a.

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The phototroph-specific β-hairpin structure of the γ subunit of F_oF₁-ATP synthase is important for efficient ATP synthesis of cyanobacteria.

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The phototroph-specific β-hairpin structure of the γ subunit of FoF1-ATP synthase is important for efficient ATP synthesis of cyanobacteria.

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OmicsDI is part of the ELIXIR infrastructure

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The phototroph-specific β-hairpin structure of the γ subunit of F_oF₁-ATP synthase is important for efficient ATP synthesis of cyanobacteria.