bioRxiv2025
The mitochondrial Ca{superscript 2} uniporter (MCU) channel is essential for energy production, cytosolic Ca{superscript 2} signalling, and regulation of cell death. Its activity is regulated by the core proteins MICU1 and MICU2, which respond to intracellular Ca{superscript 2} levels. In cardiomyocytes, MICU1 inhibits mtMCU activity at basal Ca{superscript 2}, with Ca{superscript 2}-binding relieving this inhibition via a conformational change. However, the precise molecular basis for this dual regulation is unclear. While twelve MICU1 structures exist, each is approximately 30% of their structure missing, omitting key flexible regions and limits the understanding of the Ca{superscript 2}-sensing mechanism. Here, we provide structural and computational evidence to address this gap. Using structural modelling, molecular dynamics simulations, and large-scale sequence analysis, we investigate MICU1s Ca{superscript 2} binding sites from both conformational and evolutionary perspectives. Simulations based on human MICU1 models revealed a previously uncharacterized pseudo-EF-hand (pEF-h) motif. Our findings indicate that this motif functions as an early Ca{superscript 2} 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 MICU1s on-off regulation of the MCU. To link this mechanism to experimental observations, we simulated a series of point and double mutants targeting the pEF-h, EF-h1, and EF-h2 sites. Our simulations demonstrate that double mutants disrupt Ca{superscript 2} binding not only within the mutated site but also reduce the occupation of the other sites, reaffirming the cooperative nature of Ca{superscript 2} sensing in MICU1. 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)DEG(X)E motifs were exclusive to Opisthokonts, coinciding with the emergence of the auxiliary subunit EMRE. This pattern suggests that high-affinity Ca{superscript 2} binding evolved in parallel with increasing regulatory complexity in metazoans. Together, these findings support previous research linking EF-hand function as sensors to specialised Ca{superscript 2} gatekeepers in multicellular lineages. By integrating structural and evolutionary perspectives, our study provides mechanistic insight into how MICU1 can act as a Ca{superscript 2}-dependent molecular switch, clarifying the cooperative and threshold-setting behaviour underlying its regulatory role in mitochondrial Ca{superscript 2} uptake. HighlightsO_LIThe hierarchical activation mechanism refines MICU1s on-off regulation of the MCU. C_LIO_LIA novel Ca{superscript 2}-binding site in human MICU1, featuring a unique helix-loop-{beta}-sheet structure, was identified and functionally characterized. C_LIO_LIThis new Ca{superscript 2}-binding site in MICU1 acts as an early sensor, triggering structural changes key to its regulatory function. C_LIO_LICa{superscript 2}-binding motif distributions and phylogenetic constraints indicate possible functional divergence. C_LI