Molecular picture of the mechanochemical coupling in ATP synthase | Faculty of Chemistry at the Gdańsk University of Technology

ATP synthase (also known as F₀F₁-ATPase) is—to use a phrase due to Boyer—”a splendid molecular machine” that plays a central role in cellular bioenergetics, but due to its complex structure and multiple inhibition mechanisms has also recently emerged as an attractive target for the development of novel therapeutics. The primary biological role of ATP synthase in most cells is to use electrochemical proton gradients across energy-transducing membranes in mitochondria, chloroplasts and bacteria to synthesize ATP from ADP and inorganic phosphate. It consists of two opposing rotary motor proteins, the membrane-embedded, H⁺-translocating F₀ and hydrophilic, ATP-driven F₁, mechanically coupled by a common rotor, composed of the ring of 815 c subunits (c-ring) and the elongated coiled-coil γ subunit (γ-shaft). Despite numerous efforts no complete microscopic picture of the mechanochemical coupling in ATP synthase has been proposed that would account for the observed rotationa pattern of the γ-shaft and for the near-perfect thermodynamic efficiency of the motor.

The goal of the current project is to advance our understanding of the mechanism of action of ATP synthase, with a special emphasis on the possibility of selective inhibition of the microbial forms of the enzyme with small molecules. In particular, we intend to use state-of-the-art molecular simulation techniques to examine the energy conversion mechanism in the catalytic portion of ATP synthase and to identify differences in this mechanism that could allow for selective inhibition. Additionally, we would like to explore the possibility of extending the antimicrobial spectrum of existing drugs targeting the ion-translocating portion of ATP synthase and to elucidate the mechanism by which these drugs interfere with proton translocation. The proposed research will provide an important insight into the structural determinants of the mechanochemical coupling in a remarkable biological engine and will also contribute to developing innovative antimicrobial strategies.

In particular, the project focuses on the two primary research objectives that will be addressed using a wide range of multiscale modeling methods. First, we will attempt to explain the energy transduction mechanism in F₁-ATPase and identify possible differences in this mechanism between the homologous protein structures that could allow for selective inhibition. In order to achieve this goal, we intend to construct the complete free energy profile governing the rotary catalysis in F₁ that would allow us to correlate the sequence of substrate binding events known from single-molecule experiments with the conformational transitions within the the catalytic subunit and the angular position of the γ-shaft. Thus obtained model ofthe rotary catalysis, unifying the available structural and biochemical data, is expected to resolve long-standing controversies regarding the energetics and mechanism of mechanochemical coupling in F₁, as well as to provide detailed information about the conformations of all metastable states adopted during the catalytic cycle. The latter is especially important for structure-guided design of F₁ inhibitors, as it has proven to be notoriously difficult to obtain atomic resolution structures of these conformations by X-ray crystallography. Second, to assist in developing new F₀-directed agents and make the process more cost-efficient, we will explore the possibility of using the existing inhibitors of F₀ to selectively target other classes of human pathogens and elucidating the mechanism by which c-ring-binding inhibitors interfere with proton translocation. To this end, we will clarify the structural basis for specific recognition of the c-ring by bedaquiline, the F₀-directed antitubercular drug, using a recently determined X-ray structure of the mycobacterial c-ring complexed with the drug. Subsequently, using the obtained information, we will attempt to optimize the bedaquiline scaffold (in a structure-guided fragment-based docking procedure) into selective binders to the three selected microbial c-rings whose high-resolution structures are available in the literature: Gram-negative and Gram-positive bacteria and yeast. To advance a molecular-level understanding of the F₀ inhibition mechanism, we will construct the first atomic model of the entire membrane-embedded F₀ motor using the recently reported high-resolution cryo-EM maps of ATPase synthase as restraints for the molecular dynamics-based Bayesian refinement method. Extensive classical and ab initio molecular dynamics simulations of this model are expected to provide insight into functional dynamics of F₀, including the thermodynamics and kinetics of proton binding by a crucial acidic residue, and thus will facilitate design of new inhibitors interacting with the recognized drug-binding pocket of F₀ as well as help in identifying new inhibition sites.