Aqueous access pathways in subunit a of rotary ATP synthase extend to both sides of the membrane

ATP is the universal energy currency of living cells, and the majority of it is synthesized by the F 1 F 0 ATP synthase. Inhibitors of this enzyme are therefore potentially detrimental for all life forms. Tributyltin chloride (TBT-Cl) inhibits ATP hydrolysis by the Na ؉ -translocating ATP synthase of Ilyobacter tartaricus or the H ؉ -translocating counterpart of Escherichia coli with apparent K i of 200 nM. To target the site of this inhibition, we synthesized a tritium-labeled derivative of TBT-Cl in which one of the butyl groups was replaced by a photoactivatable aryldiazirine residue. Upon illumination, subunit a of the ATP synthase becomes specifically modified, and this labeling is suppressed in the presence of the original inhibitor. In case of the Na ؉ ATP synthase, labeling is also suppressed in the presence of Na ؉ ions, suggesting an interference in Na ؉ or TBT-Cl binding to subunit a. This interference is corroborated by the protection of ATP hydrolysis from TBT-Cl inhibition by 105 mM Na ؉ . TBT-Cl strongly inhibits Na ؉ exchange by the reconstituted I. tartaricus ATP synthase. Taken together these results indicate that the subunit a ion channel is the target site for ATPase inhibition by toxic organotin compounds. An inhibitor interacting specifically with this site has not been reported previously.

Section: Photoaffinity Labeling Of Atp Synthase and C-oligomer With Dmentioning

confidence: 95%

The ion channel of F-ATP synthase is the target of toxic organotin compounds

Ballmoos

Brunner

Dimroth

2004

“…The membrane block has a width of about 30 Å and the central Asp residues of the c-ring were placed almost at the middle of the membrane. Experimental evidences have suggested the presence of proton channels on the P and N side of the a/c interface that has access to the hydrophilic environment (28,29). In order to explore this effect in our CG model, we removed part of the membrane environment from the vicinity of the Asp-Arg pair in the interfacial regions.…”

Section: Resultsmentioning

confidence: 99%

Realistic simulations of the coupling between the protomotive force and the mechanical rotation of the F ₀ -ATPase

Mukherjee

Warshel

2012

The molecular origin of the action of the F 0 proton gradient-driven rotor presents a major puzzle despite significant structural advances. Although important conceptual models have provided guidelines of how such systems should work, it has been challenging to generate a structure-based molecular model using physical principles that will consistently lead to the unidirectional protondriven rotational motion during ATP synthesis. This work uses a coarse-grained (CG) model to simulate the energetics of the F 0 -ATPase system in the combined space defined by the rotational coordinate and the proton transport (PTR) from the periplasmic side (P) to the cytoplasmic side (N). The model establishes the molecular origin of the rotation, showing that this effect is due to asymmetry in the energetics of the proton path rather than only the asymmetry of the interaction of the Asp on the c-ring helices and Arg on the subunit-a. The simulation provides a clear conceptual background for further exploration of the electrostatic basis of proton-driven mechanochemical systems.T he F 0 F 1 -ATPase is a ubiquitous nanomotor in all living cells that generate the ATP molecules essential for maintaining large gamut of cellular functions (1). This system is comprised of two rotary motors; the mechanochemical F 1 -ATPase that synthesizes or hydrolyzes ATP, utilizing the mechanical torque generated from the rotation of the stalk, and the membranebound F 0 -ATPase that drives the mechanical rotation utilizing the ion-motive force established across the membrane. The detailed nature of the conversion and the utility of energy by the F 0 F 1 -ATPase has long been one of the fundamental questions in biology. Significant progress has been achieved in understanding the mechanochemical coupling in the F 1 -ATPase, utilizing information from several high-resolution crystal structures and a wealth of biochemical and single-molecule spectroscopic data (2-6). Recently, the molecular nature of the coupling between chemical and conformational coordinates in the F 1 -ATPase has been elucidated using coarse-grained (CG) theoretical modeling approaches (7). In contrast, the molecular basis of the conversion of the pH gradient across the membrane producing directional rotation of the F 0 -ATPase is less understood, despite significant conceptual progress (3). The problems are partially due to the limited structural information about the complete membranebound F 0 -ATPase complex and also reflects the inherent difficulty in modeling coupled long-range biological processes, such as proton transfer and mechanical rotation.The F 0 motor is consisted of a rotor part, known as the c-ring, connected with the stator subunit-a and dimer subunits-b. The c-ring is a tightly packed ring-like structure composed of several α-helical hairpins whose number varies in different species (3,8). Most of the central part of the c-ring is embedded in the membrane, except for the cytoplasmic loops and the periplasmic termini. Each c-ring helices consists of a highly conserved ...

“…A second set of Ag + -sensitive substitutions in subunit a mapped to the opposite face and periplasmic side of aTMH4 (18,19), and Ag + -sensitive substitutions were also found in TMHs 2, 3, and 5, where they extend from the center of the membrane to the periplasmic surface (19,20). The Ag + -sensitive substitutions on the periplasmic side of TMHs 2-5 cluster at the interior of the four-helix bundle predicted by cross-linking and could interact to form a continuous aqueous pathway extending from the periplasmic surface to the central region of the lipid bilayer (11,13,19,20). We have proposed that the movement of H + from the periplasmic half-channel and binding to the single ionized Asp61 in the c-ring is mediated by a swiveling of TMHs at the a-c subunit interface (16,(21)(22)(23)(24).…”

mentioning

confidence: 98%

“…The aqueous accessibility of Cys residues introduced into the five TMHs of subunit a has been probed on the basis of their reactivity with and inhibitory effects of Ag + and other thiolate-reactive agents (18)(19)(20). Two regions of aqueous access were found with distinctly different properties.…”

mentioning

confidence: 99%

Interacting cytoplasmic loops of subunits a and c of Escherichia coli F ₁ F ₀ ATP synthase gate H ⁺ transport to the cytoplasm

Steed

Kraft

Fillingame

2014

Self Cite

Significance H + transport through the membrane-traversing F 1 F 0 ATP synthase mechanically drives bond formation between ATP and P i . For the enzyme in the inner membrane of Escherichia coli , we show here that the H + transport pathway extends beyond the lipid bilayer into cytoplasmic loops connecting transmembrane helices in both subunits a and c of the transmembrane F 0 sector. On the basis of chemical cross-linking, we postulate that the multiple loop regions functioning in H + transport pack into a single domain, which then interacts with the cytoplasmic surface of the H + channel within F 0 . The interaction of the loop regions with the H + channel is proposed to specifically gate H + release to the cytoplasmic side of the membrane during ATP synthesis.