The “stator stalk” of F₁F_o-ATP synthase is essential for rotational catalysis as it connects the nonrotating portions of the enzyme. In Escherichia coli, the stator stalk consists of two (identical) b subunits and the δ subunit. In mycobacteria, one of the b subunits and the δ subunit are replaced by a b/δ fusion protein; the remaining b subunit is of the shorter b′ type. In the present study, it is shown that it is possible to generate a functional E. coli ATP synthase containing a b/δ fusion protein. This construct allowed the analysis of the roles of the individual b subunits. The full-length b subunit (which in this case is covalently linked to δ in the fusion protein) is responsible for connecting the stalk to the catalytic F₁ subcomplex. It is not required for interaction with the membrane-embedded F_o subcomplex, as its transmembrane helix can be removed. Attachment to F_o is the function of the other b subunit which in turn has only a minor (if any at all) role in binding to δ. Also in E. coli the second b subunit can be shortened to a b′ type.

P-glycoprotein (Pgp) pumps an array of hydrophobic compounds out of cells, and has major roles in drug pharmacokinetics and cancer multidrug resistance. Yet, polyspecific drug binding and ATP hydrolysis-driven drug export in Pgp are poorly understood. Fluorescence spectroscopy using tryptophans (Trp) inserted at strategic positions is an important tool to study ligand binding. In Pgp, this method will require removal of 11 endogenous Trps, including highly conserved Trps that may be important for function, protein-lipid interactions, and/or protein stability. Here, we developed a directed evolutionary approach to first replace all eight transmembrane Trps and select for transport-active mutants in Saccharomyces cerevisiae. Surprisingly, many Trp positions contained non-conservative substitutions that supportedin vivoactivity, and were preferred over aromatic amino acids. The most active construct, W(3Cyto), served for directed evolution of the three cytoplasmic Trps, where two positions revealed strong functional bias towards tyrosine. W(3Cyto) and Trp-less Pgp retained wild-type-like protein expression, localization and transport function, and purified proteins retained drug stimulation of ATP hydrolysis and drug binding affinities. The data indicate preferred Trp substitutions specific to the local context, often dictated by protein structural requirements and/or membrane lipid interactions, and these new insights will offer guidance for membrane protein engineering.

With the uncontrolled growth of multidrug-resistant bacteria, there is an urgent need to search for new therapeutic targets, to develop drugs with novel modes of bactericidal action. FoF1-ATP synthase plays a crucial role in bacterial bioenergetic processes, and it has emerged as an attractive antimicrobial target, validated by the pharmaceutical approval of an inhibitor to treat multidrug-resistant tuberculosis. In this work, we aimed to design, through two types of in silico strategies, new allosteric inhibitors of the ATP synthase, by targeting the catalytic β subunit, a centerpiece in communication between rotor subunits and catalytic sites, to drive the rotary mechanism. As a model system, we used the F1 sector of Escherichia coli, a bacterium included in the priority list of multidrug-resistant pathogens. Drug-like molecules and an IF1-derived peptide, designed through molecular dynamics simulations and sequence mining approaches, respectively, exhibited in vitro micromolar inhibitor potency against F1. An analysis of bacterial and Mammalia sequences of the key structural helix-turn-turn motif of the C-terminal domain of the β subunit revealed highly and moderately conserved positions that could be exploited for the development of new species-specific allosteric inhibitors. To our knowledge, these inhibitors are the first binders computationally designed against the catalytic subunit of FOF1-ATP synthase.

ATP synthase uses a rotary mechanism to couple transmembrane proton translocation to ATP synthesis and hydrolysis, which occur at the catalytic sites in the β subunits. In presence of Mg²⁺, the three catalytic sites of ATP synthase have vastly different affinities for nucleotides, and the position of the central γ subunit determines which site has high, medium, or low affinity. Affinity differences and their changes as rotation progresses underpin the ATP synthase catalytic mechanism. Here, we used a series of variants with up to 45- and 60-residue-long truncations of the N- and C-terminal helices of the γ subunit, respectively, to identify the segment(s) responsible for the affinity differences of the catalytic sites. We found that each helix carries an affinity-determining segment of ~ 10 residues. Our findings suggest that the affinity regulation by these segments is transmitted to the catalytic sites by the DELSEED-loop in the C-terminal domain of the β subunits. For the N-terminal truncation variants, presence of the affinity-determining segment and therefore emergence of a high-affinity binding site resulted in wild-type-like catalytic activity. At the C-terminus, additional residues outside of the affinity-determining segment were required for optimal enzymatic activity. Alanine substitutions revealed that the affinity changes of the catalytic sites required no specific interactions between amino acid side chains in the γ and α₃β₃ subunits but were caused by the presence of the helices themselves. Our findings help unravel the molecular basis for the affinity changes of the catalytic sites during ATP synthase rotation.