A low‐populated helical region of the mitochondrial inhibitor IF1 drives interaction with the ATP synthase OSCP subunit

The upregulation of mitochondrial IF1 is associated with the progression of several cancer types. In HeLa cells, naturally overexpressing this protein, IF1 binds to the ATP synthase OSCP subunit, resulting in inhibition of permeability transition‐dependent apoptosis. This newly identified protein–protein interaction represents a promising therapeutic target in tumors characterized by IF1 overexpression. However, the rational design of targeted molecules is currently limited by a lack of structural data on the binding mechanism. Here we employed native mass spectrometry and NMR spectroscopy to identify the minimal IF1 segment required to target the OSCP subunit. NMR revealed that the interaction is weak in solution, with an affinity K D in the low millimolar range, consistent with a binding event occurring exclusively when IF1 is overexpressed in cancer cells. These findings guided the design of a model peptide containing the IF1 binding epitope, which was used in molecular dynamics simulations (MD) aimed at investigating its binding mechanism to the OSCP subunit. NMR and MD converged to show that the IF1 binding epitope requires a helical folding for OSCP engagement, consistent with a helical Molecular Recognition Feature (αMoRF) mechanism. Helicity of the epitope is strictly necessary for productive binding, with key contacts involving IF1 residues E30, E31, Y33, F34, and Q37 packing against OSCP helix H3. Structural analysis of the binding complex provided a first molecular framework to inspire the design of molecules capable of displacing IF1 from the OSCP subunit and potentially exerting anticancer activity.