Control of rotation of the FF-ATP synthase nanomotor by an inhibitory α-helix from unfolded ε or intrinsically disordered ζ and IF proteins.

The ATP synthase is a ubiquitous nanomotor that fuels life by the synthesis of the chemical energy of ATP. In order to synthesize ATP, this enzyme is capable of rotating its central rotor in a reversible manner. In the clockwise (CW) direction, it functions as ATP synthase, while in counter clockwise (CCW) sense it functions as an proton pumping ATPase. In bacteria and mitochondria, there are two known canonical natural inhibitor proteins, namely the ε and IF subunits. These proteins regulate the CCW FF-ATPase activity by blocking γ subunit rotation at the α/β/γ subunit interface in the F domain. Recently, we discovered a unique natural F-ATPase inhibitor in Paracoccus denitrificans and related α-proteobacteria denoted the ζ subunit. Here, we compare the functional and structural mechanisms of ε, IF, and ζ, and using the current data in the field, it is evident that all three regulatory proteins interact with the α/β/γ interface of the F-ATPase. In order to exert inhibition, IF and ζ contain an intrinsically disordered N-terminal protein region (IDPr) that folds into an α-helix when inserted in the α/β/γ interface. In this context, we revised here the mechanism and role of the ζ subunit as a unidirectional F-ATPase inhibitor blocking exclusively the CCW FF-ATPase rotation, without affecting the CW-FF-ATP synthase turnover. In summary, the ζ subunit has a mode of action similar to mitochondrial IF, but in α-proteobacteria. The structural and functional implications of these intrinsically disordered ζ and IF inhibitors are discussed to shed light on the control mechanisms of the ATP synthase nanomotor from an evolutionary perspective.