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 (Ca2+) 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 Ca2+. 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 Ca2+ 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 Ca2+ binding increased or decreased matrix Ca2+, respectively, establishing F-EF as a two-way mitochondrial Ca2+ regulator. Thus, we show how to synergize AI prediction with NMR data, elucidating a solution-specific and extraordinary LETM1 F-EF-hand. [Display omitted] • An AI-informed NMR structure elucidation method reveals a unique LETM1 F-EF-hand • The Ca2+ bound LETM1 F-EF-hand adopts an open conformation with high hydrophobicity • High affinity LETM1 F-EF-hand Ca2+ binding increases mitochondrial matrix Ca2+ • Abrogated LETM1 F-EF-hand Ca2+ binding decreases mitochondrial matrix Ca2+ AI has revolutionized structural biology but must be applied responsibly. Lin et al. present a method for synergizing AlphaFold predictions with solution NMR data, using the approach to expose an unprecedented F-EF-hand domain in LETM1. This LETM1 F-EF-hand functions as a weak Ca2+ binding sensor that modulates mitochondrial matrix Ca2+. [ABSTRACT FROM AUTHOR]