Functional Specialization of Ca²⁺-Binding Motifs in Human MICU1

Abstract

The mitochondrial calcium uniporter (MCU) channel is essential for energy production, cytosolic Ca²⁺ signalling, and regulation of cell death. Its activity is regulated by the core proteins MICU1 and MICU2, which respond to intracellular Ca²⁺ levels. In cardiomyocytes, MICU1 inhibits calcium uptake under basal conditions, while Ca²⁺ binding during contraction induces a conformational change that relieves this inhibition, allowing calcium entry. However, the molecular basis of MICU1’s dual regulatory effect, mediated by its two known conformations, remains unclear. Although twelve x-ray crystal and cryo-EM structures of MICU1 have been resolved, each has approximately 30% of their structure missing, leaving key flexible regions uncharacterised. This structural incompleteness limits our understanding of the molecular basis underlying MICU1’s dual regulatory effect on calcium sensing. Here, we provide structural and computational evidence to address this gap further. Using structural modelling, molecular dynamics simulations, and large-scale sequence analysis, we investigate MICU1’s calcium binding sites from both conformational and evolutionary perspectives. Simulations based on human MICU1 models revealed a previously uncharacterised pseudo-EF-hand (pEF-h) motif. Our findings indicate that this motif functions as an early Ca²⁺ sensor, triggering conformational transitions– including shifts in surface charge distribution and isoelectric point– that prime the canonical EF-hand sites for subsequent binding. This hierarchical activation mechanism refines MICU1’s on–off regulation of the MCU. The biological relevance of the EF-hand motifs would be supported by its evolutionary conservation. Therefore, we analysed the evolutionary shaping of MICU1 EF-hand motifs across major eukaryotic lineages using clustering analysis and found strong lineage-specific segregation: canonical DXN/DXD-type motifs predominated in EF-h1 and EF-h2 in plants and protists, while non-canonical EXE(X)₃DEG(X)₇E motifs were exclusive to Opisthokonts, coinciding with the emergence of the auxiliary subunit EMRE. This pattern suggests that high-affinity calcium binding evolved in parallel with increasing regulatory complexity in metazoans. Together, these findings support previous research linking EF-hand function as sensors to specialised Ca²⁺ gatekeepers in multicellular lineages. By integrating structural and evolutionary perspectives, our study provides mechanistic insight into how MICU1 can act as a Ca²⁺-dependent molecular switch, clarifying the cooperative and threshold-setting behaviour underlying its regulatory role in mitochondrial calcium uptake.