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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.

The biological roles of the three natural FF-ATPase inhibitors, ε, ζ, and IF, on cell physiology remain controversial. The ζ subunit is a useful model for deletion studies since it mimics mitochondrial IF, but in the FF-ATPase of Paracoccus denitrificans (PdFF), it is a monogenic and supernumerary subunit. Here, we constructed a P. denitrificans 1222 derivative (PdΔζ) with a deleted ζ gene to determine its role in cell growth and bioenergetics. The results show that the lack of ζ in vivo strongly restricts respiratory P. denitrificans growth, and this is restored by complementation in trans with an exogenous ζ gene. Removal of ζ increased the coupled PdFF-ATPase activity without affecting the PdFF-ATP synthase turnover, and the latter was not affected at all by ζ reconstitution in vitro. Therefore, ζ works as a unidirectional pawl-ratchet inhibitor of the PdFF-ATPase nanomotor favoring the ATP synthase turnover to improve respiratory cell growth and bioenergetics.

A series of 37 benzolactam derivatives were synthesized, and their respective affinities for the dopamine D(2) and D(3) receptors evaluated. The relationships between structures and binding affinities were investigated using both ligand-based (3D-QSAR) and receptor-based methods. The results revealed the importance of diverse structural features in explaining the differences in the observed affinities, such as the location of the benzolactam carbonyl oxygen, or the overall length of the compounds. The optimal values for such ligand properties are slightly different for the D(2) and D(3) receptors, even though the binding sites present a very high degree of homology. We explain these differences by the presence of a hydrogen bond network in the D(2) receptor which is absent in the D(3) receptor and limits the dimensions of the binding pocket, causing residues in helix 7 to become less accessible. The implications of these results for the design of more potent and selective benzolactam derivatives are presented and discussed.

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