Unidirectional regulation of the F1FO-ATP synthase nanomotor by the ζ pawl-ratchet inhibitor protein of Paracoccus denitrificans and related α-proteobacteria

Abstract

The ATP synthase is a reversible nanomotor that gyrates its central rotor clockwise (CW) to synthesize ATP and in counter clockwise (CCW) direction to hydrolyse it. In bacteria and mitochondria, two natural inhibitor proteins, namely the ε and IF subunits, prevent the wasteful CCW FF-ATPase activity by blocking γ rotation at the α/β/γ interface of the F portion. In Paracoccus denitrificans and related α-proteobacteria, we discovered a different natural F-ATPase inhibitor named ζ. Here we revise the functional and structural data showing that this novel ζ subunit, although being different to ε and IF, it also binds to the α/β/γ interface of the F of P. denitrificans. ζ shifts its N-terminal inhibitory domain from an intrinsically disordered protein region (IDPr) to an α-helix when inserted in the α/β/γ interface. We showed for the first time the key role of a natural ATP synthase inhibitor by the distinctive phenotype of a Δζ knockout mutant in P. denitrificans. ζ blocks exclusively the CCW FF-ATPase rotation without affecting the CW-FF-ATP synthase turnover, confirming that ζ is important for respiratory bacterial growth by working as a unidirectional pawl-ratchet PdFF-ATPase inhibitor, thus preventing the wasteful consumption of cellular ATP. In summary, ζ is a useful model that mimics mitochondrial IF but in α-proteobacteria. The structural, functional, and endosymbiotic evolutionary implications of this ζ inhibitor are discussed to shed light on the natural control mechanisms of the three natural inhibitor proteins (ε, ζ, and IF) of this unique ATP synthase nanomotor, essential for life.

Evolution of the Inhibitory and Non-Inhibitory ε, ζ, and IF1 Subunits of the F1FO-ATPase as Related to the Endosymbiotic Origin of Mitochondria

“…Perhaps the closest to an intermediate conformation in this “up” or “down” transition is the first structure of the isolated structure of the γ/ε complex of the E. coli ε subunit resolved via crystallography [ 40 ]. Therefore, a pawl/ratchet mechanism, as we also proposed for the inhibitory subunit ζ in Paracocus denitrificans [ 41 , 42 , 43 , 44 ] (see below), seems more likely to take place as the originally proposed ε ratchet mechanism than a gear/shift one.…”

The 3 × 120° rotary mechanism of Paracoccus denitrificans F 1 -ATPase is different from that of the bacterial and mitochondrial F 1 -ATPases

“…However, the other knockout caused only a moderate increase in PdF 1 F o ATP hydrolysis, which is insufficient to activate the membrane ATPase (74). We have previously hypothesized which differences in the strains could explain these apparent discrepancies (75).…”

Complex effects of macrolide venturicidins on bacterial F-ATPases likely contribute to their action as antibiotic adjuvants

“…7 , step 5 ) and/or the presence of endogenous activating oxyanions. The prominence of ε– and MgADP–inhibited states can vary between bacterial species 39 but has not been studied for many pathogens; in some species, inhibition by the εCTD may be supplemented or superseded by a unique subunit 48 or by a unique segment of another F 1 subunit 49 .…”