F-type ATP synthases play a key role in oxidative and photophosphorylation processes generating adenosine triphosphate (ATP) for most biochemical reactions in living organisms. In contrast to the mitochondrial F_OF₁-ATP synthases, those of chloroplasts are known to be mostly monomers with approx. 15% fraction of oligomers interacting presumably non-specifically in a thylakoid membrane. To shed light on the nature of this difference we studied interactions of the chloroplast ATP synthases using small-angle X-ray scattering (SAXS) method. Here, we report evidence of I-shaped dimerization of solubilized F_OF₁-ATP synthases from spinach chloroplasts at different ionic strengths. The structural data were obtained by SAXS and demonstrated dimerization in response to ionic strength. The best model describing SAXS data was two ATP-synthases connected through F₁/F₁′ parts, presumably via their δ-subunits, forming “I” shape dimers. Such I-shaped dimers might possibly connect the neighboring lamellae in thylakoid stacks assuming that the F_OF₁ monomers comprising such dimers are embedded in parallel opposing stacked thylakoid membrane areas. If this type of dimerization exists in nature, it might be one of the pathways of inhibition of chloroplast F_OF₁-ATP synthase for preventing ATP hydrolysis in the dark, when ionic strength in plant chloroplasts is rising. Together with a redox switch inserted into a γ-subunit of chloroplast F_OF₁ and lateral oligomerization, an I-shaped dimerization might comprise a subtle regulatory process of ATP synthesis and stabilize the structure of thylakoid stacks in chloroplasts.

ATP synthases are large enzymes present in every living cell. They consist of a transmembrane and a soluble domain, each comprising multiple subunits. The transmembrane part contains an oligomeric rotor ring (c-ring), whose stoichiometry defines the ratio between the number of synthesized ATP molecules and the number of ions transported through the membrane. Currently, c-rings of F-Type ATP synthases consisting of 8 to 17 (except 16) subunits have been experimentally demonstrated. Here, we present an easy-to-use high-throughput computational approach based on AlphaFold that allows us to estimate the stoichiometry of all homooligomeric c-rings, whose sequences are present in genomic databases. We validate the approach on the available experimental data, obtaining the correlation as high as 0.94 for the reference data set, and use it to predict the existence of c-rings with stoichiometry varying from 8 to 27. We then conduct molecular dynamics simulations of two c-rings with stoichiometry above 17 to corroborate the machine learning-based predictions. Our work strongly suggests existence of rotor rings with previously undescribed high stoichiometry in natural organisms and highlights the utility of AlphaFold-based approaches for studying homooligomeric proteins.

F-type ATP synthases play a key role in oxidative and photophosphorylation processes producing adenosine triphosphate (ATP) for most biochemical reactions in living organisms. In contrast to the mitochondrial FOF1-ATP synthases those of chloroplasts are known to be mostly monomers with approx. 15% fraction of oligomers interacting presumably non-specifically in a thylakoid membrane. To shed light to the nature of this difference we studied interactions of the chloroplast ATP synthases using small-angle X-ray scattering (SAXS) method. Here, we report evidence of I-shaped dimerization of solubilized FOF1-ATP synthases from spinach chloroplasts at different salinity. The structural data were obtained by SAXS and showed dimerization in response to changes in ionic strength. The best model describing SAXS data was two ATP-synthases connected through F1/F1’ parts, presumably via their δ-subunits, forming dimers with the “I” shape. Such I-shaped dimers might possibly connect the neighboring lamellae in thylakoid stacks assuming that the FOF1 monomers comprising such dimers are embedded in different thylakoid membranes. If this type of dimerization exists in nature, it might be one of the pathways of inhibition of chloroplast FOF1-ATP synthase for preventing of ATP hydrolysis in dark, when salinity in plant chloroplasts is rising. Together with a redox switch inserted into a γ-subunit of chloroplast FOF1 and lateral oligomerization, an I-shaped dimerization might comprise a subtle regulatory process of ATP synthesis and stabilize the structure of thylakoid stacks in chloroplasts.

ATP synthase is a membrane protein complex that plays a crucial role in cellular bioenergetics. The formation of I-shaped dimers of spinach chloroplast ATP synthase in a model system (detergent micelles) with a high concentration of sodium chloride has been reported in the literature, but the presence and functional role of this mechanism in vivo remains unclear. We studied the impact of ionic strength on the thermostability and activity of spinach ATP synthase in liposomes. We measured ATP synthesis in the presence of NaCl, NaNO₃, and Na₂SO₄ at various concentrations and found that high ionic strength (~1 M) reduced ATP synthase activity by approximately 50%. Additionally, we determined the melting temperature of ATP synthase in the presence of NaCl and KCl, observing an increase from ~60.5 to ~62.0°C in solutions with high ionic strength. Our results demonstrate that salt ions affect ATP synthesis and thermostability in a non-specific manner. These findings provide support for the hypothesis that ATP synthase dimerization in vivo may serve as a regulatory mechanism for controlling its activity.