Functional Specialization of Ca²⁺-Binding Motifs in Human MICU1

The mitochondrial Ca²⁺ 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 mtMCU activity at basal Ca²⁺, with Ca²⁺-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 Ca²⁺-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 MICU1’s Ca²⁺ 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 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. 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 Ca²⁺ binding not only within the mutated site but also reduce the occupation of the other sites, reaffirming the cooperative nature of Ca²⁺ 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)₃DEG(X)₇E motifs were exclusive to Opisthokonts, coinciding with the emergence of the auxiliary subunit EMRE. This pattern suggests that high-affinity Ca²⁺ 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 Ca²⁺ uptake.

Highlights

• The hierarchical activation mechanism refines MICU1’s on–off regulation of the MCU.

• A novel Ca²⁺-binding site in human MICU1, featuring a unique helix–loop–β-sheet structure, was identified and functionally characterized.

• This new Ca²⁺-binding site in MICU1 acts as an early sensor, triggering structural changes key to its regulatory function.

• Ca²⁺-binding motif distributions and phylogenetic constraints indicate possible functional divergence.