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A new type of Na(+)-driven ATP synthase membrane rotor with a two-carboxylate ion-coupling motif.

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 F?F?-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 c?? 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.

PROVIDER: S-EPMC3692424 | BioStudies |

REPOSITORIES: biostudies

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Project description:The proton-driven ATP synthase (FOF1) is comprised of two rotary, stepping motors (FO and F1) coupled by an elastic power transmission. The elastic compliance resides in the rotor module that includes the membrane-embedded FO c-ring. Proton transport by FO is firmly coupled to the rotation of the c-ring relative to other FO subunits (ab2). It drives ATP synthesis. We used a computational method to investigate the contribution of the c-ring to the total elastic compliance. We performed principal component analysis of conformational ensembles built using distance constraints from the bovine mitochondrial c-ring x-ray structure. Angular rotary twist, the dominant ring motion, was estimated to show that the c-ring accounted in part for the measured compliance. Ring rotation was entrained to rotation of the external helix within each hairpin-shaped c-subunit in the ring. Ensembles of monomer and dimers extracted from complete c-rings showed that the coupling between collective ring and the individual subunit motions was independent of the size of the c-ring, which varies between organisms. Molecular determinants were identified by covariance analysis of residue coevolution and structural-alphabet-based local dynamics correlations. The residue coevolution gave a readout of subunit architecture. The dynamic couplings revealed that the hinge for both ring and subunit helix rotations was constructed from the proton-binding site and the adjacent glycine motif (IB-GGGG) in the midmembrane plane. IB-GGGG motifs were linked by long-range couplings across the ring, while intrasubunit couplings connected the motif to the conserved cytoplasmic loop and adjacent segments. The correlation with principal collective motions shows that the couplings underlie both ring rotary and bending motions. Noncontact couplings between IB-GGGG motifs matched the coevolution signal as well as contact couplings. The residue coevolution reflects the physiological importance of the dynamics that may link proton transfer to ring compliance.

Project description:Two subunits within the transmembrane domain of the ATP synthase-the c-ring and subunit a-energize the production of 90% of cellular ATP by transducing an electrochemical gradient of H+ or Na+ into rotational motion. The nature of this turbine-like energy conversion mechanism has been elusive for decades, owing to the lack of definitive structural information on subunit a or its c-ring interface. In a recent breakthrough, several structures of this complex were resolved by cryo-electron microscopy (cryo-EM), but the modest resolution of the data has led to divergent interpretations. Moreover, the unexpected architecture of the complex has cast doubts on a wealth of earlier biochemical analyses conducted to probe this structure. Here, we use quantitative molecular-modeling methods to derive a structure of the a-c complex that is not only objectively consistent with the cryo-EM data, but also with correlated mutation analyses of both subunits and with prior cross-linking and cysteine accessibility measurements. This systematic, integrative approach reveals unambiguously the topology of subunit a and its relationship with the c-ring. Mapping of known Cd2+ block sites and conserved protonatable residues onto the structure delineates two noncontiguous pathways across the complex, connecting two adjacent proton-binding sites in the c-ring to the space on either side of the membrane. The location of these binding sites and of a strictly conserved arginine on subunit a, which serves to prevent protons from hopping between them, explains the directionality of the rotary mechanism and its strict coupling to the proton-motive force. Additionally, mapping of mutations conferring resistance to oligomycin unexpectedly reveals that this prototypical inhibitor may bind to two distinct sites at the a-c interface, explaining its ability to block the mechanism of the enzyme irrespective of the direction of rotation of the c-ring. In summary, this study is a stepping stone toward establishing the mechanism of the ATP synthase at the atomic level.

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A new type of Na(+)-driven ATP synthase membrane rotor with a two-carboxylate ion-coupling motif.

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