I-Shaped Dimers of a Plant Chloroplast FOF1-ATP Synthase in Response to Changes in Ionic Strength

Abstract

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.

Keywords: FOF1-ATP synthase; chloroplasts; dimers; membrane proteins; small-angle scattering.

Figure 1

Purification and characterization of cF_OF₁: (a) Overall view of ATP synthase from spinach chloroplasts. F_O part consists of abb′c₁₄ subunits, F₁ part consists of α₃β₃γεδ subunits and bb′ subunits form a peripheral stalk. The structure of ATP synthase from spinach chloroplasts (PDB ID: 6FKF [15]) was used for representation; (b) Characterization of the peak fractions of cF_OF₁ after anion exchange chromatography (AEX) by SAXS, experimental I(Q) 1D-profile showed as hollow grey stars, a model of cF_OF₁ (PDB ID: 6FKF) with a detergent belt was used for approximation of experimental data with χ² = 1.15 (blue line); (c) Characterization of the AEX peak fractions of cF_OF₁ by SDS PAGE, colored with Coomassie; (d) Anion exchange chromatography (AEX) of cF_OF₁ from spinach chloroplasts, highlighted fractions were merged and taken for structural studies, the region shown with a red box is described in details in Panel (e); (e) AEX peak fractions of cF_OF₁. Panels (a,c–e) were adapted from [12] with modifications.

Figure 2

SAXS characterization of cF_OF₁: (a) Experimental I(Q) 1D-profiles for AEX-purified and dialyzed against the same buffer with different NaCl concentrations samples of cF_OF₁, experimental data are shown as dots, regularized fits are shown as black lines. For clarity, SAXS data for 150, 250, 300, 350, and 450 mM NaCl were multiplied by 10, 10², 10³, 10⁴, and 10⁵, respectively; (b) Normalized pair-distance distribution function P(r) for cF_OF₁ at different NaCl concentrations; (c) Maximum size of the object (D_max) for samples of cF_OF₁ at different NaCl concentrations; (d) Radius of gyration (Rg) for samples of cF_OF₁ at different NaCl concentrations obtained from P(r); (e) Porod volume (Vp) for samples of cF_OF₁ at different NaCl concentrations. Gray lines show the values of D_max, Rg, Vp for an AEX-purified sample of cF_OF₁ without dialysis. The values used for plots in panels (c–e) are given in Table S1.

Figure 3

Dimerization of cF_OF₁ from Spinacia oleracea at 450 mM NaCl shown by SAXS: (a) Experimental I(Q) 1D-profile for cF_OF₁ at 450 mM NaCl (orange squares) and an approximation (blue line, χ² = 2.36) by using a model of a mixture of cF_OF₁ monomers and dimers formed by F_O-F_O contacts (see Macromolecular docking section in Materials and Methods); (b) Experimental I(Q) 1D-profile for cF_OF₁ at 450 mM NaCl (the same as in panel (a), orange squares) and an approximation (blue line, χ² = 1.20) by using a model of a mixture of cF_OF₁ monomers and dimers formed by F₁-F₁ contacts (see Macromolecular docking section in Materials and Methods); (c) Relative residues of the fit shown in panel (a); (d) Relative residues of the fit shown in panel (b). The models of monomeric and dimeric cF_OF₁, shown in panels (a,b), contain detergent belts obtained from the experimental SAXS data and built by the program MEMPROT. Volume fractions of monomers and dimers of cF_OF₁, used in the models for approximation, are shown.

Figure 4

Possible biological interpretation of observed cF_OF₁ I-shaped dimer: (a) Segmented subvolume of a thylakoid stack with connected stroma lamellae, cF_OF₁ are shown as yellow 12-nm spheres, cF_OF₁ is randomly distributed over stromal lamellae; (b) Surface rendering of a lateral cF_OF₁ dimer in isolated pea thylakoid membranes; (c) A model of an I-shaped dimer with F₁/F₁ interaction interface, presumably via δ-subunit, the distance between membrane F_O parts of different cF_OF₁ monomers is about 30 nm; (d) Electron Tomography of Vitreous Spinach Chloroplast Sections, stacked grana and unstacked stroma thylakoid membranes are shown, the distance between neighboring stroma lamellae approximately fits the size of the I-shaped cF_OF₁ dimer, the stroma thylakoids, which are continuous with a grana thylakoid is shown with green arrowheads, the stroma thylakoids merged with two adjacent grana thylakoids—blue arrowheads, the region shown with a red box is described in details in Panel (e); (e) Surface representation of connections between grana (green) and stroma (purple) thylakoids, the distance between neighboring stroma lamellae is about 30 nm, the region shown with a green box is described in details in Panel (f); (f) Schematic representation of an I-shaped cF_OF₁ dimer in between of two neighboring stroma lamellae; (g) A part of a tomographic slice showing neighboring stroma lamellae, the distance between them approximately fits the size of an I-shaped ATP-synthase dimer, Pink lines are 30 nm size and demonstrate the possible fit of an I-shaped cF_OF₁ dimer between the neighbor lamellae. Presumably, ATP synthases shown with red arrows might be these I-shaped dimers. Yellow arrowheads show separate cF_OF₁. Panels (a,b,d,e,g) were adapted from [3] with modifications.

Figure 5

Structure and disorder in the cF_OF₁ δ-subunit: (a) 3D structure of the cF_OF₁ δ-subunit generated by AlphaFold2; (b) Per-residue intrinsic disorder predisposition of the cF_OF₁ δ-subunit from the spinach chloroplasts. The intrinsic disorder profile was generated using the outputs of the Rapid Intrinsic Disorder Analysis Online (RIDAO) platform [38] that aggregates the results from a number of well-known disorder predictors, such as PONDR^® VLXT, PONDR^® VL3, PONDR^® VLS2, PONDR^® FIT, and IUPred2 (Short) and IUPred2 (Long) and also produces the mean disorder profile (MDP) and corresponding error distribution (pink shadow). The outputs of the evaluation of the per-residue disorder propensity by these tools are represented as real numbers between 1 (ideal prediction of disorder) and 0 (ideal prediction of order). A threshold of 0.5 is used to identify disordered residues and regions in query proteins. Residues with the disorder scores (DS) DS < 0.15 are considered as ordered, residues with 0.15 ≤ DS < 0.25 are taken as flexible, whereas residues with 0.25 ≤ DS < 0.5 are considered as moderately disordered.