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Title: A New Type of Na+-Driven ATP Synthase Membrane Rotor with a Two-Carboxylate Ion-Coupling Motif}
Article:
Abstract:
The anaerobic bacterium Fusobacterium nucleatum uses glutamate decarboxylation to generate a transmembrane gradient of Na+. Here, we demonstrate that this ion-motive force is directly coupled to ATP synthesis, via an F1Fo-ATP synthase with a novel Na+ recognition motif, shared by other human pathogens. Molecular modeling and free-energy simulations of the rotary element of the enzyme, the c-ring, indicate Na+ specificity in physiological settings. Consistently, activity measurements showed Na+ stimulation of the enzyme, either membrane-embedded or isolated, and ATP synthesis was sensitive to the Na+ ionophore monensin. Furthermore, Na+ has a protective effect against inhibitors targeting the ion-binding sites, both in the complete ATP synthase and the isolated c-ring. Definitive evidence of Na+ coupling is provided by two identical crystal structures of the c11 ring, solved by X-ray crystallography at 2. 2 and 2. 6 Å resolution, at pH 5. 3 and 8. 7, respectively. Na+ ions occupy all binding sites, each coordinated by four amino acids and a water molecule. Intriguingly, two carboxylates instead of one mediate ion binding. Simulations and experiments demonstrate that this motif implies that a proton is concurrently bound to all sites, although Na+ alone drives the rotary mechanism. The structure thus reveals a new mode of ion coupling in ATP synthases and provides a basis for drug-design efforts against this opportunistic pathogen.
Introduction:
Synthesis of ATP, the most prominent energy source in biological cells, is largely mediated by the ATP synthase, an enzyme that resides in the membranes of bacteria, mitochondria, and chloroplasts. This enzyme catalyzes the phosphorylation of ADP by a rotary mechanism powered by a transmembrane electrochemical gradient, or ion-motive force, of either H+ or Na+( proton-motive force[PMF] or sodium-motive force[SMF], respectively). The ATP synthase consists of two sub-complexes: the water-soluble F1 sector[1],[2], which harbors the catalytic centers, and the membrane-embedded Fo complex, which mediates ion translocation across the membrane. These functionally distinct units are mechanically coupled by two additional elements, referred to as central and peripheral stalks[3],[4]. In the Fo sector, eight to 15 copies of subunit c are assembled into a closed ring[5], which rotates around its axis as ions permeate across the enzyme. The c-ring harbors a series of identical ion-binding sites, typically one per c-subunit, which selectively recognize the coupling ion[6]–[8]. Ion binding is facilitated by a conserved carboxylic amino acid, usually glutamate; however, it is the neighboring chemical groups in the protein side-chains and backbone, and sometimes a bound water molecule[9]–[11] that ultimately determine the specificity of the c-ring binding sites[8]. Na+ specific sites typically involve an intricate hydrogen-bonded network of polar groups, while H+-binding sites are simpler, and consist mainly of hydrophobic moieties. Either way, one complete rotation of the c-ring results in the translocation of one ion per binding site and the production of three ATP molecules[12],[13]; the stoichiometry of the c-ring thus defines the ion-to-ATP ratio of the enzyme, i. e., the minimum ion-motive force required for ATP synthesis[14]. In this study, we characterize the structure, ion specificity, and stoichiometry of the c-ring of the ATP synthase from F. nucleatum, a Gram-negative bacterium implicated in the etiology of periodontal diseases. F. nucleatum grows anaerobically, using amino acids as the preferred carbon source[15]. In particular, glutamate fermentation involves the glutaconyl-CoA decarboxylase, which uses the free energy of decarboxylation to generate a SMF across the cytoplasmic membrane[16],[17]. Comparison of the amino-acid sequence of the F. nucleatum c-subunit with those of other Na+-driven ATP synthases suggests that F. nucleatum utilizes the SMF directly to produce ATP( Figure S1), but this remains to be experimentally demonstrated. Sequence analysis also suggests that ion coordination in the F. nucleatum c-ring could involve not only one but possibly two carboxyl side-chains. This is an unusual and interesting feature, shared by other pathogenic bacteria, whose mechanistic implications are unclear. It is conceivable that the second carboxyl group could alter the assumed ion specificity of the c-ring, the ion-to-ATP ratio, or that it confers a novel coupling or regulatory mechanism to the enzyme[18]. It is also unknown what c-subunit stoichiometry is characteristic of F. nucleatum. Crystal structures have revealed a wide range of possible stoichiometries in H+-coupled c-rings( reviewed in[5]); whether Na+-dependent c-rings from F-type ATP synthases are similarly diverse is unclear, since to date only one such ring has been characterized structurally at high resolution[6]. To clarify these questions, we have isolated the F. nucleatum F1Fo-ATP synthase, and analyzed its ion-coupling mechanism using microbiological, biochemical, structural and computer-simulation methods, both classical and quantum-mechanical.
Discussion:
We describe the structure of a novel type of Na+-binding site in the c-ring of the F1Fo-ATP synthase from F. nucleatum and demonstrate that this enzyme is physiologically coupled to the SMF. The c-ring features two glutamate residues in each of its 11 ion-binding sites, instead of a single carboxylate side-chain as is typical. While such a motif has been observed in other Na+-binding proteins, and had been anticipated for c-rings in particular[9],[18], the implications in regard to the mechanism of ion transport and the specificity of the enzyme were unclear. A previous biochemical study of the PMF-driven ATP synthase from E. coli analyzed mutants with a two-carboxylate motif in the c-ring proton-binding sites, concluding that the engineered carboxylate( Asp24), which replaces a hydrophobic side-chain( Ala24), must be protonated for the enzyme to function, while it is the aspartate side-chain found in the wild-type enzyme( Asp61) that likely mediates H+ translocation[32]. The substitution of Ala24 by Asp or Glu, however, induces a pH dependence on the H+ transport activity in the physiological pH range, not observed for the wild-type enzyme. In a subsequent study of the two-carboxylate E. coli mutants, this dependence led to the proposal that the second carboxylate found in the binding sites of some c-rings might function as a regulatory high-pH sensor to inhibit the ion-translocation mechanism[18]. In light of our results, however, we interpret this pH dependence differently. In our view, it likely reflects a down-shift in the overall pKa of the proton-binding sites in the mutagenized E. coli c-ring, as a result of the constitutive protonation of the added carboxylic side-chain, which naturally diminishes the affinity for a second proton( i. e., the one transported across the membrane), relative to the wild-type. This interpretation would also be consistent with the diminished rate of H+ translocation mediated by these two-carboxylate mutants. In support of our hypothesis that the second carboxylate group is constitutively protonated, we find that under physiological conditions the F. nucleatum ATP synthase mediates the translocation of Na+ only, i. e., protons are not co-transported during either ATP hydrolysis or ATP synthesis, even though they are concurrently bound to the c-ring binding sites, as the crystal structures and calculations demonstrate. The structural basis for the ion specificity of the ATP synthase can be rationalized on the basis of the amino acid composition of the c-ring binding sites, and the concentrations of the two competing ions in the environment[8]. This study indicates that the F. nucleatum c-ring preferentially uses Na+ as the coupling ion, but only under conditions in which Na+ is in large excess over H+, e. g., in physiological settings. The c-ring is in fact selective for H+, like all other rings characterized thus far. In particular it appears to be ∼100-fold more H+ selective than the ring of the Na+-driven V-type ATPase from E. hirae, which reportedly is almost non-selective[21]. The finding that the additional glutamate side-chain in the c-ring binding sites is constitutively protonated implies that its energetic contribution to ion selectivity is largely equivalent to that of a glutamine or asparagine side-chain at the same position. We expect this equivalence to hold true also for H+-driven ATP synthases with a double-carboxylate motif, such as those in Streptococcus pneumoniae[33] and M. tuberculosis( Figure S1). These c-rings probably bind two H+ concurrently to each site( as in Figure 3A), only one of which is likely to be transported. This perspective would also be consistent with a recent study of the ATP synthase from the archaeon Methanosarcina acetivorans[19], whose c-ring binding sites were also predicted to feature two glutamate side-chains, one of which was proposed to be constitutively protonated. The M. acetivorans ATP synthase is reportedly coupled to both Na+ and H+ translocation under physiological conditions[19]. However, this is not due to the deprotonation of the atypical carboxyl side-chain in the c-ring( the equivalent to Glu32 in F. nucleatum), but rather to the increased H+ specificity of its binding sites, relative to Na+-driven c-ring rotors( Figure 3B). Physiologically, i. e., with Na+ in large excess over H+, the more pronounced H+ selectivity of the M. acetivorans c-ring makes the likelihood of a state with two bound H+ comparable to that with one Na+ and one H+, in contrast to F. nucleatum, for which the latter is the most probable. Intriguingly, all Na+-coupled c-rings from F-type ATP synthases characterized so far consist of 11 α-helical hairpins, including those of I. tartaricus, P. modestum, A. woodii, C. paradoxum[24],[34]–[36], and now F. nucleatum. The only apparent commonality of these bacteria is that they grow under anaerobic conditions and in environments where Na+ is abundant( e. g., marine sediment). All these organisms thus use the SMF to sustain ATP production, but a phylogenetic relationship among them is not apparent. A more evident similarity, besides the near-identical amino acid composition of the Na+-binding sites, is a glycine-rich motif in the N-terminal helix of the c-subunit( Figure S1), which has been shown to have a strong influence on the stoichiometry of the c-ring[14],[37],[38]. It would be therefore tempting to hypothesize that the 11-hairpin architecture might reflect a universal feature of all Na+-driven c-rings in F-type ATP synthases. Nevertheless, studies of A- and V-type rotary ATPases indicate that Na+ specificity is compatible with other c-ring stoichiometries[20],[39],[40]. Consistently, single-point mutations of the glycine-motif in the c11 ring from I. tartaricus result in increased c-subunit stoichiometries, but do not necessarily impair Na+ coupling[14]. Further experimental data will be required to clarify this question conclusively. In recent years, the ATP synthase has come into focus as a novel drug target[41]. Of particular interest is the finding that the M. tuberculosis c-ring can be inhibited by diarylquinolines, a new class of antibiotic compounds against multi-drug resistant strains[42],[43]. Even in non-replicating or persister cells of M. tuberculosis, the PMF is obligatory for ATP homeostasis[44] and hence it is of medical interest to develop strategies to disrupt energy generation via the ATP synthase. This notion also holds true for F. nucleatum, which is a pervasive bacterium in dental plaque biofilms, and which is associated with periodontitis, one of the most common human infections[45]. The fact that the c-ring of the F. nucleatum ATP synthase is Na+-coupled and that its structure is significantly different from mitochondrial c-rings[13],[28] opens an opportunity for new selective drugs against this opportunistic human pathogen.
Essential cellular processes such as biosynthesis , transport , and motility are sustained by the energy released in the hydrolysis of ATP , the universal energy carrier in living cells . Most ATP in the cell is produced by a membrane-bound enzyme , the ATP synthase , through a rotary mechanism that is coupled to the translocation of ions across the membrane . The majority of ATP synthases are energized by transmembrane electrochemical gradients of protons ( proton-motive force ) , but a number of organisms , including some important human pathogens , use gradients of sodium ions instead ( sodium-motive force ) . The ion specificity of ATP synthases is determined by a membrane-embedded sub-complex , the c-ring , which is the smallest known biological rotor . The functional mechanism of the rotor ring and its variations among different organisms are of wide interest , because of this enzyme's impact on metabolism and disease , and because of its potential for nanotechnology applications . Here , we characterize a previously unrecognized type of Na+-driven ATP synthase from the opportunistic human pathogen Fusobacterium nucleatum , which is implicated in periodontal diseases . We analyzed this ATP synthase and its rotor ring through a multi-disciplinary approach , combining cell-growth and biochemical assays , X-ray crystallography and computer-simulation methods . Two crystal structures of the membrane rotor were solved , at low and high pH , revealing an atypical ion-recognition motif mediated by two carboxylate side-chains . This motif is shared by other human pathogens , such as Mycobacterium tuberculosis or Streptococcus pneumonia , whose ATP synthases are targets of novel antibiotic drugs . The implications of this ion-recognition mode on the mechanism of the ATP synthase and the cellular bioenergetics of F . nucleatum were thus examined . Our results provide the basis for future pharmacological efforts against this important pathogen .