Interactions involved in grasping and locking of the inhibitory peptide IF1 by mitochondrial ATP synthase.

When mitochondria become deenergized, futile ATP hydrolysis is prevented by reversible binding of an endogenous inhibitory peptide called IF1 to ATP synthase. Between initial IF1 binding and IF1 locking the enzyme experiences large conformational changes. While structural studies give access to analysis of the dead-end inhibited state, transient states have thus far not been described. Here, we studied both initial and final states by reporting, for the first time, the consequences of mutations of Saccharomyces cerevisiae ATP synthase on its inhibition by IF1. Kinetic studies allowed the identification of amino acids or motifs of the enzyme that are involved in recognition and/or locking of IF1 α-helical midpart. This led to an outline of IF1 binding process. In the recognition step, protruding parts of α and especially ß subunits grasp IF1, most likely by a few residues of its α-helical midpart. Locking IF1 within the αß interface involves additional residues of both subunits. Interactions of the α and ß subunits with the foot of the γ subunit might contribute to locking and stabilizing of the dead-end state.

How the N-terminal extremity of Saccharomyces cerevisiae IF1 interacts with ATP synthase: a kinetic approach.

The N-terminal part of the inhibitory peptide IF1 interacts with the central γ subunit of mitochondrial isolated extrinsic part of ATP synthase in the inhibited complex (J.R. Gledhill, M.G. Montgomery, G.W. Leslie, J.E. Walker, 2007). To explore its role in the different steps of IF1 binding, kinetics of inhibition of the isolated and membrane-bound enzymes were investigated using Saccharomyces cerevisiae IF1 derivatives modified in N-terminal extremity. First, we studied peptides truncated in Nter up to the amino acid immediately preceding Phe17, a well-conserved residue thought to play a key role. These deletions did not affect or even improve the access of IF1 to its target. They decreased the stability of the inhibited complex but much less than previously proposed. We also mutated IF1-Phe17 and found this amino acid not mandatory for the inhibitory effect. The most striking finding came from experiments in which PsaE, a 8 kDa globular-like protein, was attached in Nter of IF1. Unexpectedly, such a modification did not appreciably affect the rate of IF1 binding. Taken together, these data show that IF1-Nter plays no role in the recognition step but contributes to stabilize the inhibited complex. Moreover, the data obtained using chimeric PsaE-IF1 suggest that before binding IF1 presents to the enzyme with its middle part facing a catalytic interface and its Nter extremity folded in the opposite direction.