Abstract Fri130: Defining the molecular calcium control points of cellular metabolism

Mitochondrial matrix calcium concentration (mCa2+) is thought to regulate key dehydrogenases in the TCA cycle, making it a crucial regulator of cellular metabolism. Current dogma states that MICU1, MICU2, and MICU3 regulate mCa2+ by interacting with and altering the open-probability of the mitochondrial calcium uniporter channel (mtCU). MICU2 and MICU3 independently associate with MICU1 to form heterodimers that modulate mitochondrial calcium uptake in a tissue-dependent manner. Interestingly, knockdown or deletion of MICU1 results in organism lethality that is not rescued by the ablation of mtCU function. This indicates that MICU1—and by extension, MICU2 and MICU3—have broader mitochondrial functions beyond regulating the mtCU. MICU heterodimers localize to the mitochondrial inner membrane space (IMS) and therefore can interact with metabolic complexes in the inner mitochondrial membrane (IMM). Our current working hypothesis is that the MICU heterodimers and their respective interactomes serve as a fundamental calcium-responsive system at the IMS/IMM to control mitochondrial processes. Here, we developed an in-silico protein docking and scoring pipeline to characterize the interactomes of MICU1-MICU2 and MICU1-MICU3 heterodimers to determine their functional roles in calcium control of cellular metabolism. Template-free protein docking was performed to test the interaction of each heterodimer with all known mitochondrial proteins. Theoretical complexes were analyzed and ranked by their stability using free-energy values from molecular dynamics simulations. A hit threshold was established by calculating free-energy values of heterodimers docked to known interactors, e.g. mtCU components. This novel screening approach delivered an extensive in silico characterization of the MICU1-MICU2 and MICU1-MICU3 heterodimer interactomes, providing context for their respective roles in calcium-dependent regulation of mitochondrial function. Future hypothesis-based experiments will validate high-priority interactors using genetic approaches in cell lines and mouse models and define their functional significance in mitochondrial metabolism. These insights have the potential to reshape our understanding of calcium regulation of mitochondrial bioenergetics and reveal novel targets for metabolic disorders and cardiovascular diseases.