An AI-informed NMR structure reveals an extraordinary LETM1 F-EF-hand domain that functions as a two-way regulator of mitochondrial calcium.

AlphaFold can accurately predict static protein structures but does not account for solvent conditions. Human leucine zipper EF-hand transmembrane protein-1 (LETM1) has one sequence-identifiable EF-hand but how calcium (Ca ²⁺ ) affects structure and function remains enigmatic. Here, we used highly confident AlphaFold Cα predictions to guide nuclear Overhauser effect (NOE) assignments and structure calculation of the LETM1 EF-hand in the presence of Ca ²⁺ . The resultant NMR structure exposes pairing between a partial loop-helix and full helix-loop-helix, forming an unprecedented F-EF-hand with non-canonical Ca ²⁺ coordination but enhanced hydrophobicity for protein interactions compared to calmodulin. The structure also reveals the basis for pH sensing at the link between canonical and partial EF-hands. Functionally, mutations that augmented or weakened Ca ²⁺ binding increased or decreased matrix Ca ²⁺ , respectively, establishing F-EF as a two-way mitochondrial Ca ²⁺ regulator. Thus, we show how to synergize AI prediction with NMR data, elucidating a solution-specific and extraordinary LETM1 F-EF-hand.
Competing Interests: Declaration of interests The authors declare no competing interests.
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