An Evolutionarily Conserved TUSC2-OSCP Interface Suggests a Mitochondrial Ca2⁺-Sensing Mechanism for ATP Synthase Regulation.

Mitochondrial ATP synthase (mATPS) has the capacity to regulate the permeability transition pore (mPTP) during Ca2⁺ fluctuations. Dysregulation of this mechanism is implicated in neurodegenerative and other diseases; however, the endogenous mechanisms that couple calcium sensing to mATPS function remain poorly defined. We recently demonstrated that loss of TUSC2, a mitochondrial Ca2⁺-binding protein, leads to mitochondrial Ca2⁺ overload and sustained mPTP opening. The oligomycin sensitivity-conferring protein (OSCP), a regulatory subunit of mATPS, responds to Ca2⁺ and oxidative stress, positioning it as a potential integrator of bioenergetic and stress signals. With age, the ability of OSCP to maintain regulatory interactions and resist stress-induced conformational perturbations declines, contributing to multiple pathologies. Here, we combine AI-assisted structural modeling with phylogenetic analysis to nominate TUSC2 as a previously unrecognized candidate OSCP-interacting partner. Across evolution-from basal eukaryotes to higher metazoans-TUSC2 exhibits near-invariant conservation of its central Ca2⁺-binding motif (CBM), in contrast to pronounced divergence of its N-terminal region. AlphaFold3 modeling provides a structural framework for this conservation, predicting Ca2⁺-dependent engagement of the CBM with a conserved C-terminal region of OSCP with established regulatory function. The predicted interface is preserved across phylogenetically distant species, indicating co-conservation of interacting surfaces. The model further suggests coordinated engagement of Ca2⁺ and a second metal ion by TUSC2 and OSCP, as well as accommodation of a nucleotide. The deep conservation of these features across eukaryotes is consistent with a functionally constrained, Ca2⁺-sensing regulatory role for TUSC2 in mATPS function, with potential implications for mPTP regulation.