The Inhibitory Mechanism of the ζ Subunit of the F1FO-ATPase Nanomotor of Paracoccus denitrificans and Related α-Proteobacteria

Abstract

The subunit is a novel inhibitor of the F 1 F O -ATPase of Paracoccus denitrificans and related ␣-proteobacteria. It is different from the bacterial (⑀) and mitochondrial (IF 1 ) inhibitors. The N terminus of blocks rotation of the ␥ subunit of the F 1 -ATPase of P. denitrificans (Zarco-Zavala, M., Morales-Ríos, E., Mendoza-Hernández, G., Ramírez-Silva, L., Pérez-Hernández, G., and García-Trejo, J. J. (2014) FASEB J. 24, 599 -608) by a hitherto unknown quaternary structure that was first modeled here by structural homology and protein docking. The F 1 -ATPase and F 1 -models of P. denitrificans were supported by crosslinking, limited proteolysis, mass spectrometry, and functional data. The final models show that enters into F 1 -ATPase at the open catalytic ␣ E /␤ E interface, and two partial ␥ rotations lock the N terminus of in an "inhibition-general core region," blocking further ␥ rotation, while the globular domain anchors it to the closed ␣ DP /␤ DP interface. Heterologous inhibition of the F 1 -ATPase of P. denitrificans by the mitochondrial IF 1 supported both the modeled binding site at the ␣ DP /␤ DP /␥ interface and the endosymbiotic ␣-proteobacterial origin of mitochondria. In summary, the subunit blocks the intrinsic rotation of the nanomotor by inserting its N-terminal inhibitory domain at the same rotor/stator interface where the mitochondrial IF 1 or the bacterial ⑀ binds. The proposed pawl mechanism is coupled to the rotation of the central ␥ subunit working as a ratchet but with structural differences that make it a unique control mechanism of the nanomotor to favor the ATP synthase activity over the ATPase turnover in the ␣-proteobacteria.

Deleting the IF 1 -like ζ subunit from Paracoccus denitrificans ATP synthase is not sufficient to activate ATP hydrolysis

“…The biochemical work of García-Trejo and co-workers [ 29 , 30 , 36 ], together with the structure of P. denitrificans ATP synthase that revealed the ζ subunit bound in a manner analogous to the eukaryotic inhibitor protein IF 1 [ 13 ], indicated that the ζ subunit is responsible for preventing ATP hydrolysis and enforcing unidirectional catalysis in the P. denitrificans enzyme. However, deleting the ζ subunit caused only very moderate increases in ATP hydrolysis (less than twofold), and the rates remain very low in comparison with the rates observed from the mammalian and E. coli enzymes ( table 2 ).…”

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

“…Still, the sequence of its inhibitory region is weakly related to that of the mitochondrial inhibitor IF 1 (32). Biochemical and crystallographic evidence supports the notion that the ζ-inhibitory mechanism resembles that of IF 1 , in which the ζ-intrinsically disordered inhibitory domain adopts a helical structure after occupying its inhibitory position in the α D β D -catalytic interface of its cognate F 1 , equivalent to IF 1 (26,33).…”

A systematic assessment of mycobacterial F1‐ATPase subunit ε’s role in latent ATPase hydrolysis

“…Latency of ATP hydrolysis activity of the mycobacterial F‐ATP synthase has also been observed for the thermoalkaliphilic Ct F 1 ‐ATPase [39,40] and the α‐proteobacterium Paracoccus denitrificans F‐ATP synthase [41,42]. Based on the Ct F 1 ‐ATPase crystallographic structure, the hydrolytic bound product of ADP and Pi within the β E ‐catalytic site was described to be likely the basis of inhibition of ATP hydrolysis of this enzyme [39].…”

“…Based on the Ct F 1 ‐ATPase crystallographic structure, the hydrolytic bound product of ADP and Pi within the β E ‐catalytic site was described to be likely the basis of inhibition of ATP hydrolysis of this enzyme [39]. In comparison, besides subunit ε the P. denitrificans F‐ATP synthase contains an additional subunit called ζ, which is different in structure from the bacterial ε and mitochondrial inhibitor protein, IF 1 [41]. ζ harbours the N‐terminal inhibitory domain and a globular anchoring domain.…”