Rotary properties of hybrid F1-ATPases consisting of subunits from different species

Engineering of IF1‐susceptive bacterial F1‐ATPase

“…We prepared four kinds of hybrid F 1 s composed of subunits from b MF 1 or TF 1 along our previous article, bb T, b T b , b TT, and TT b (Watanabe et al, 2023 ). The triplet characters represent the origins (‘ b ’ from b MF 1 or ‘T' from TF 1 ) of the three subunits, α, β, and γ, in this order; bb T represents the hybrid F 1 with α and β from b MF 1 and γ from TF 1 .…”

“…The proteins of b MF 1 (WT), TF 1 (WT), four hybrid F 1 s, and IF 1 were expressed and purified as described in the reference article (Watanabe et al, 2023 ). For TF 1 (β 5 ), TF 1 (α 1 β 5 γ 2 ), and b MF 1 (β 5 ), the artificially synthesized construct with mutations was introduced into the wild‐type TF 1 and b MF 1 plasmids, respectively.…”

“…This study aims to address this question by engineering TF 1 to confer the susceptibility to bovine mitochondrial IF 1 . To determine which subunit is responsible for the species specificity, we first tested the hybrid F 1 s, which are composed of each α, β, γ subunit from b MF 1 or TF 1 (Watanabe et al, 2023 ). Then, based on the sequence analysis, we constructed the quintuple and the octuple mutants of TF 1 , where the non‐conserved five or eight residues in contact with IF 1 were substituted to the corresponding residues of b MF 1 .…”

Inhibitory to non-inhibitory evolution of the ζ subunit of the F1FO-ATPase of Paracoccus denitrificans and α-proteobacteria as related to mitochondrial endosymbiosis

“…This is in concordance with the lack of inhibitory function of Sm-ζ on its own SmF 1 -ATPase and indicates that a larger energetic barrier exists in Sm-ζ to achieve the transition from the compact non-inhibitory conformation to its N-terminal α-helical extended inhibitory conformer. Somehow, the PdF 1 -ATPase, but not the SmF 1 -ATPase, is able to overcome this energetic barrier due to its tendency to work as a dominant PdF 1 -ATPase to induce functional conformations on the reconstituted ATP synthases’ heterologous subunits from other species, adapted to the PdF 1 -ATPase subunits ( Watanabe et al, 2023 ), thus taking the Sm-ζ to the N-terminal α-helical extended inhibitory conformer.…”

“…In this regard, we have recently shown that in hybrid F 1 -ATPases constructed with at least one PdF 1 -ATPase subunit (either Pd-α, Pd-β, or Pd-γ), these PdF 1 -ATPase subunits induce the functional conformations of F 1 subunits from other species (i.e., bovine or thermophilic bacterial subunits) to rotate according to the newly described 3 °× 120 °rotation unique of the PdF 1 -ATPase and not according to the multiple rotary stepping in the other bacterial or mitochondrial F 1 -ATPases (Zarco-Zavala et al, 2020). This indicates that the PdF 1 individual subunits have the potential to induce other heterologous F 1 subunits to adapt to their structure and catalytic rotary mechanism; thus, the PdF 1 subunits work as robust dominant subunits (Watanabe et al, 2023). It seems, therefore, suitable to suggest that the PdF 1 -ATPase, but not the SmF 1 , is similarly able to induce the structural transition from the compact 5α-helical non-inhibitory conformer of Sm-ζ (Figures 7, 8B), into its inhibitory N-terminal extended α-helical conformation (Figures 8D; Supplementary MD analyses have been previously helpful in analyzing the inhibitory and regulatory mechanisms of the ε subunit from nonα-proteobacteria (Krah and Takada, 2016;Krah et al, 2021;Krah et al, 2023) Frontiers in Molecular Biosciences frontiersin.org bound productively to the PdF 1 (Garcia-Trejo et al, 2016) or PdF 1 F O complexes (Morales-Rios et al, 2015).…”