Fluidity of structure and swiveling of helices in the subunit c ring of Escherichia coli ATP synthase as revealed by cysteine-cysteine cross-linking.
ABSTRACT Subunit c in the membrane-traversing F(0) sector of Escherichia coli ATP synthase is known to fold with two transmembrane helices and form an oligomeric ring of 10 or more subunits in the membrane. Models for the E. coli ring structure have been proposed based upon NMR solution structures and intersubunit cross-linking of Cys residues in the membrane. The E. coli models differ from the recent x-ray diffraction structure of the isolated Ilyobacter tartaricus c-ring. Furthermore, key cross-linking results supporting the E. coli model prove to be incompatible with the I. tartaricus structure. To test the applicability of the I. tartaricus model to the E. coli c-ring, we compared the cross-linking of a pair of doubly Cys substituted c-subunits, each of which was compatible with one model but not the other. The key finding of this study is that both A21C/M65C and A21C/I66C doubly substituted c-subunits form high yield oligomeric structures, c(2), c(3)... c(10), via intersubunit disulfide bond formation. The results indicate that helical swiveling, with resultant interconversion of the two conformers predicted by the E. coli and I. tartaricus models, must be occurring over the time course of the cross-linking experiment. In the additional experiments reported here, we tried to ascertain the preferred conformation in the membrane to help define the most likely structural model. We conclude that both structures must be able to form in the membrane, but that the helical swiveling that promotes their interconversion may not be necessary during rotary function.
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ABSTRACT: ATP, the universal energy currency of cells, is produced by F-type ATP synthases, which are ancient, membrane-bound nanomachines. F-type ATP synthases use the energy of a transmembrane electrochemical gradient to generate ATP by rotary catalysis. Protons moving across the membrane drive a rotor ring composed of 8-15 c-subunits. A central stalk transmits the rotation of the c-ring to the catalytic F1 head, where a series of conformational changes results in ATP synthesis. A key unresolved question in this fundamental process is how protons pass through the membrane to drive ATP production. Mitochondrial ATP synthases form V-shaped homodimers in cristae membranes. Here we report the structure of a native and active mitochondrial ATP synthase dimer, determined by single-particle electron cryomicroscopy at 6.2 Å resolution. Our structure shows four long, horizontal membrane-intrinsic α-helices in the a-subunit, arranged in two hairpins at an angle of approximately 70° relative to the c-ring helices. It has been proposed that a strictly conserved membrane-embedded arginine in the a-subunit couples proton translocation to c-ring rotation. A fit of the conserved carboxy-terminal a-subunit sequence places the conserved arginine next to a proton-binding c-subunit glutamate. The map shows a slanting solvent-accessible channel that extends from the mitochondrial matrix to the conserved arginine. Another hydrophilic cavity on the lumenal membrane surface defines a direct route for the protons to an essential histidine-glutamate pair. Our results provide unique new insights into the structure and function of rotary ATP synthases and explain how ATP production is coupled to proton translocation.Nature 02/2015; · 42.35 Impact Factor
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ABSTRACT: V-ATPase is an ATP-driven rotary motor that vectorially transports ions. Together with F-ATPase, a homologous protein, several models on the ion transport have been proposed, but their molecular mechanisms are yet unknown. V-ATPase from Enterococcus hirae forms a large supramolecular protein complex (total molecular weight: ~ 700,000) and physiologically transports Na+ and Li+ across a hydrophobic lipid bilayer. Stabilization of these cations in the binding site has been discussed on the basis of X-ray crystal structures of a membrane-embedded domain, the K-ring (Na+ and Li+ bound forms). Sodium or lithium ion binding-induced difference FTIR spectra of the intact E. hirae V-ATPase have been measured in aqueous solution at physiological temperature. The results suggest that sodium or lithium ion binding induces the deprotonation of Glu139, a hydrogen-bonding change in the tyrosine residue and rigid α-helical structures. Identical difference FTIR spectra between the entire V-ATPase complex and K-ring strongly suggest that protein interaction with the I subunit does not cause large structural changes in the K-ring. This result supports the previously proposed Na+ transport mechanism by V-ATPase stating that a flip-flop movement of a carboxylate group of Glu139 without large conformational changes in the K-ring accelerates the replacement of a Na+ ion in the binding site. This article is part of a Special Issue entitled: Vibrational Spectroscopies in Molecular Bioenergetics.Biochimica et Biophysica Acta (BBA) - Bioenergetics. 01/2014;
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ABSTRACT: Rotary catalysis in F1F0 ATP synthase is powered by proton translocation through the membrane-embedded F0 sector. Proton binding and release occur in the middle of the membrane at Asp61 on the second transmembrane helix (TMH) of subunit c, which folds in a hairpin-like structure with two TMHs. Previously, the aqueous accessibility of Cys substitutions in the transmembrane regions of subunit c was probed by testing the inhibitory effects of Ag+ or Cd2+ on function, and revealed extensive aqueous access in the region around Asp61 and on the half of TMH2 extending to the cytoplasm. In the current study, we surveyed the Ag+ and Cd2+ sensitivity of Cys substitutions in the loop of the helical hairpin and used a variety of assays to categorize the mechanisms by which Ag+ or Cd2+ chelation with the Cys thiolates caused inhibition. We identified two distinct metal sensitive regions in the cytoplasmic loop where function was inhibited by different mechanisms. Metal binding to Cys substitutions in the N-terminal half of the loop resulted in an uncoupling of F1 from F0, with release of F1 from the membrane. In contrast, substitutions in the C-terminal half of the loop retained membrane bound F1 after metal treatment. In several of these cases, inhibition was shown to be due to blockage of passive H+ translocation through F0, as assayed with F0 reconstituted into liposomes. The results suggest that the C-terminal domain of the cytoplasmic loop may function in gating H+ translocation to the cytoplasm.Journal of Biological Chemistry 12/2013; · 4.60 Impact Factor