Lin, Q. T.; Colussi, D. M.; Lake, T.; Stathopulos, P.

AlphaFold2 can accurately predict high-resolution protein structure from sequence but does not account for solvent conditions, ligands, post-translational modifications, lowly populated states or rare folds. Human leucine zipper EF-hand transmembrane protein-1 (LETM1) has one sequence-identifiable EF-hand but whether and how calcium (Ca2+) binding affects structure and function remains enigmatic. Here, we developed an approach that used highly confident AlphaFold2 Calpha positions 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 domain with non-canonical Ca2+ coordination but enhanced hydrophobicity for protein interactions compared to calmodulin. The structure also reveals the basis for pH sensing by His662, linking the canonical and partial EF-hands. Functionally, mutations that augmented or weakened Ca2+ binding led to increased and decreased matrix Ca2+ levels, respectively, establishing F-EF as a two-way mitochondrial Ca2+ potentiator. Collectively, we show how AlphaFold2 can be synergized with NMR data to produce solution-specific structures, revealing here an extraordinary LETM1 F-EF-hand sensor.