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The functional mechanism of the F1Fo ATP synthase, like many membrane transporters and pumps, entails a conformational cycle that is coupled to the movement of H+ or Na+ ions across its transmembrane domain, down an electrochemical gradient. This coupling is an efficient means of energy transduction and regulation, provided that ion binding to the membrane domain, known as Fo, is appropriately selective. In this study we set out to establish the structural and energetic basis for the ion-binding selectivity of the membrane-embedded Fo rotors of two representative ATP synthases. First, we use a biochemical approach to demonstrate the inherent binding selectivity of these rotors, that is, independently from the rest of the enzyme. We then use atomically detailed computer simulations of wild-type and mutagenized rotors to calculate and rationalize their selectivity, on the basis of the structure, dynamics and coordination chemistry of the binding sites. We conclude that H+ selectivity is most likely a robust property of all Fo rotors, arising from the prominent presence of a conserved carboxylic acid and its intrinsic chemical propensity for protonation, as well as from the structural plasticity of the binding sites. In H+-coupled rotors, the incorporation of hydrophobic side chains to the binding sites enhances this inherent H+ selectivity. Size restriction may also favor H+ over Na+, but increasing size alone does not confer Na+ selectivity. Rather, the degree to which Fo rotors may exhibit Na+ coupling relies on the presence of a sufficient number of suitable coordinating side chains and/or structural water molecules. These ligands accomplish a shift in the relative binding energetics, which under some physiological conditions may be sufficient to provide Na+ dependence.

The crystal structure of the c-ring from the proton-coupled F1Fo ATP synthase from Spirulina platensis is shown at 2.1-A resolution. The ring includes 15 membrane-embedded c subunits forming an hourglass-shaped assembly. The structure demonstrates that proton translocation across the membrane entails protonation of a conserved glutamate located near the membrane center in the c subunit outer helix. The proton is locked in this site by a precise hydrogen bond network reminiscent of that in Na+-dependent ATP synthases. However, the structure suggests that the different coordination chemistry of the bound proton and the smaller curvature of the outer helix drastically enhance the selectivity of the H+ site against other cations, including H3O+. We propose a model for proton translocation whereby the c subunits remain in this proton-locked state when facing the membrane lipid. Proton exchange would occur in a more hydrophilic and electrostatically distinct environment upon contact with the a subunit interface.

The interaction between the c(11)ring and the gammaepsilon complex, forming the rotor of the Ilyobacter tartaricus ATP synthase, was probed by surface plasmon resonance spectroscopy and in vitro reconstitution analysis. The results provide, for the first time, a direct and quantitative assessment of the stability of the rotor. The data indicated very tight binding between the c(11)ring and the gammaepsilon complex, with an apparent K(d) value of approximately 7.4nm. The rotor assembly was primarily dependent on the interaction of the cring with the gammasubunit, and binding of the cring to the free epsilon subunit was not observed. Mutagenesis of selected conserved amino acid residues of all three rotor components (cR45, cQ46, gammaE204, gammaF203 and epsilonH38) severely affected rotor assembly. The interaction kinetics between the gammaepsilon complex and c(11)ring mutants suggested that the assembly of the c(11)gammaepsiloncomplex was governed by interactions of low and high affinity. Low-affinity binding was observed between the polar loops of the cring subunits and the bottom part of the gamma subunit. High-affinity interactions, involving the two residues gammaE204 and epsilonH38, stabilized the holo-c(11)gammaepsilon complex. NMR experiments indicated the acquisition of conformational order in otherwise flexible C- and N-terminal regions of the gamma subunit on rotor assembly. The results of this study suggest that docking of the central stalk of the F(1)complex to the rotor ring of F(o) to form tight, but reversible, contacts provides an explanation for the relative ease of dissociation and reconstitution of F(1)F(o)complexes.

Despite the thermodynamic problem imposed on alkaliphilic bacteria of synthesizing adenosine triphosphate (ATP) against a large inverted pH gradient and consequently a low electrochemical proton potential, these bacteria still utilize a proton-coupled F(1)F(o)-ATP synthase to synthesize ATP. One potential solution to this apparent thermodynamic problem would be the operation of a larger oligomeric c ring, which would raise the ion to ATP ratio, thus facilitating the conversion of a low electrochemical potential into a significant phosphorylation potential. To address this hypothesis, we have purified the oligomeric c ring from the thermoalkaliphilic bacterium Bacillus sp. strain TA2.A1 and determined the number of c-subunits using a novel mass spectrometry method, termed 'laser-induced liquid bead ion desorption' (LILBID). This technique allows the mass determination of non-covalently assembled, detergent-solubilized membrane protein complexes, and hence enables an accurate determination of c ring stoichiometries. We show that the Bacillus sp. strain TA2.A1 ATP synthase harbours a tridecameric c ring. The operation of a c ring with 13 subunits renders the thermodynamic problem of ATP synthesis at alkaline pH less severe and may represent a strategy for ATP synthesis at low electrochemical potential.

We isolated the c rings of F-ATP synthases from eight cyanobacterial strains belonging to four different taxonomic classes ( Chroococcales , Nostocales , Oscillatoriales , and Gloeobacteria ). These c rings showed different mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), probably reflecting their molecular masses. This supposition was validated with the previously characterized c 11 , c 14 , and c 15 rings, which migrated on SDS-PAGE in proportion to their molecular masses. Hence, the masses of the cyanobacterial c rings can conveniently be deduced from their electrophoretic mobilities and, together with the masses of the c monomers, allow the calculation of the c ring stoichiometries. The method is a simple and fast way to determine stoichiometries of SDS-stable c rings and hence a convenient means to unambiguously determine the ion-to-ATP ratio, a parameter reflecting the bioenergetic efficacy of F-ATP synthases. AFM imaging was used to prove the accuracy of the method and confirmed that the c ring of Synechococcus elongatus SAG 89.79 is a tridecameric oligomer. Despite the high conservation of the c-subunit sequences from cyanobacterial strains from various environmental groups, the stoichiometries of their c rings varied between c 13 and c 15 . This systematic study of the c-ring stoichiometries suggests that variability of c-ring sizes might represent an adaptation of the individual cyanobacterial species to their particular environmental and physiological conditions. Furthermore, the two new examples of c 15 rings underline once more that an F 1 /F o symmetry mismatch is not an obligatory feature of all F-ATP synthases.

Numerous membrane transporters and enzymes couple their mechanisms to the permeation of Na(+) or H(+), thereby harnessing the energy stored in the form of transmembrane electrochemical potential gradients to sustain their activities. The molecular and environmental factors that control and modulate the ion specificity of most of these systems are, however, poorly understood. Here, we use isothermal titration calorimetry to determine the Na(+)/H(+) selectivity of the ion-driven membrane rotor of an F-type ATP synthase. Consistent with earlier theoretical predictions, we find that this rotor is significantly H(+) selective, although not sufficiently to be functionally coupled to H(+), owing to the large excess of Na(+) in physiological settings. The functional Na(+) specificity of this ATP synthase thus results from two opposing factors, namely its inherent chemical selectivity and the relative availability of the coupling ion. Further theoretical studies of this membrane rotor, and of two others with a much stronger and a slightly weaker H(+) selectivity, indicate that, although the inherent selectivity of their ion-binding sites is largely set by the balance of polar and hydrophobic groups flanking a conserved carboxylic side chain, subtle variations in their structure and conformational dynamics, for a similar chemical makeup, can also have a significant contribution. We propose that the principle of ion selectivity outlined here may provide a rationale for the differentiation of Na(+)- and H(+)-coupled systems in other families of membrane transporters and enzymes.

ATP synthases are rotating nanomachines that couple ATP synthesis or hydrolysis to the transmembrane flow of protons or sodium ions down or against their electrochemical gradient. The key coupling element is a membrane-embedded subcomplex, the c-ring. We have recently proposed a principle that explains the ion selectivity of the c-ring, and validated this through functional studies of several ATP synthases. Specifically, we have proposed that a conserved Glu/Asp confers a universal H+ selectivity to all c-ring binding sites, and that additional amino-acids, which vary among species, have evolved to modulate this selectivity. In particular, we have shown that polar groups can suppress the H+ selectivity of the c-ring by a factor of 1-103. Thus, the enzyme becomes coupled to Na+, due to the large excess of Na+ over H+ under physiological conditions. Here, we further assess this theory by directly measuring the selectivity of a representative c-ring through Isothermal Titration Calorimetry. Specifically, we characterized the c-ring from the ATP synthase of Ilyobacter tartaricus. From titrations at different pH values, we established that Kd(Na+) ∼ 0.3 mM while Kd(H+) ∼ 0.3 μM, confirming the notion that this prototypical Na+-coupled c-ring is in fact H+ selective, although to a much smaller degree than those actually H+-driven. Comparing our results with those obtained for Enterococcus hirae demonstrates that, as predicted by our theory, the I. tartaricus c-ring is 100 times less Na+ selective. The weaker affinity for Na+ of the I.tartaricus ring is also coherent with the 10-fold difference in Km(Na+) values between these enzymes, at high pH. Taken together, these experiments demonstrate that the c-ring is the main determinant of the physiological ion specificity of rotary ATPases, and provide a conclusive validation of our theory.

The oligomeric c ring of the F-ATP synthase from the alkaliphilic cyanobacterium Spirulina platensis was isolated and characterized. Mass spectroscopy analysis indicated a mass of 8,210 Da, reflecting that of a c monomer. The mass increased by 206 Da after treatment with the c-subunit-specific inhibitor dicyclohexylcarbodiimide (DCCD), which indicated modification of the ion-binding carboxylate by DCCD. Atomic force microscopy topographs of c rings from S. platensis showed 15 symmetrically assembled subunits. The c15-mer reported here is the largest c ring that is isolated and does not show the classical c-ring mismatch to the three-fold symmetry of the F1 domain.

ATP synthase membrane rotors consist of a ring of c-subunits whose stoichiometry is constant for a given species but variable across different ones. We investigated the importance of c/c-subunit contacts by site-directed mutagenesis of a conserved stretch of glycines (GxGxGxGxG) in a bacterial c 11 ring. Structural and biochemical studies show a direct, specific influence on the c-subunit stoichiometry, revealing c , c 12 , c 13 , c 14 , and c >14 rings. Molecular dynamics simulations rationalize this effect in terms of the energetics and geometry of the c-subunit interfaces. Quantitative data from a spectroscopic interaction study demonstrate that the complex assembly is independent of the c-ring size. Real-time ATP synthesis experiments in proteoliposomes show the mutant enzyme, harboring the larger c 12 instead of c 11 , is functional at lower ion motive force. The high degree of compliance in the architecture of the ATP synthase rotor offers a rationale for the natural diversity of c-ring stoichiometries, which likely reflect adaptations to specific bioenergetic demands. These results provide the basis for bioengineering ATP synthases with customized ion-to-ATP ratios, by sequence modifications.