Functional specialization of Ca²⁺-binding motifs in human MICU1

The mitochondrial Ca²⁺ uniporter (mtMCU) channel is essential for energy production, cytosolic Ca²⁺ signaling, and cell death regulation. Calcium dependent proteins MICU1 and MICU2 regulate its activity. To date, twelve MICU1 structures have been experimentally determined. In each, about 30% of the residues are missing, excluding key disordered regions and limiting a complete molecular-level description of the Ca²⁺-sensing mechanism. Using structural modeling, molecular dynamics simulations, large-scale sequence analysis, and in silico mutagenesis, we investigate MICU1's Ca²⁺-binding sites from both conformational and evolutionary perspectives. We identified a previously uncharacterized pseudo-EF-hand (pEF-h) motif that acts as an early Ca²⁺ sensor, triggering structural transitions that prime the canonical EF-h1 and EF-h2 sites for subsequent binding. Occupation of the pEF-h induces coordinated changes in surface charge distribution, a decrease in isoelectric point, and enhanced flexibility of the EF-hand regions, facilitating high-affinity Ca²⁺ binding. In addition, in silico single and double mutagenesis targeting the pEF-h, EF-h1, and EF-h2 demonstrated that mutations at the pEF-h markedly reduced Ca²⁺ occupancy and delayed conformational transitions. Evolutionary analysis highlighted the relevance of the EF-hand motifs, supported by the evolutionary shaping of MICU1 EF-hand motifs across major eukaryotic lineages using clustering analysis, and revealed strong lineage-specific segregation. We observed patterns suggesting that high-affinity Ca²⁺ binding evolved in parallel with increasing regulatory complexity in metazoans. Together, these findings provide a unified structural and evolutionary framework explaining how MICU1 functions as a Ca²⁺-dependent molecular switch that sets the threshold and precision of mitochondrial Ca²⁺ uptake through a hierarchical and cooperative activation mechanism.