The apo LETM1 F-EF-hand adopts a closed conformation that underlies a multi-modal sensory role in mitochondria.

Leucine zipper EF-hand containing transmembrane protein-1 (LETM1) plays a critical role in mitochondrial function, with haploinsufficiency linked to Wolf-Hirschhorn syndrome. Here, we present the solution NMR structure of the calcium (Ca)-depleted LETM1 EF-hand domain, revealing a closed conformation facilitated by a distinct F-helix pivot rather than decreased interhelical angle. Further, we observe regiospecific unfolding in response to hot and cold denaturation and show H662 has a pKa in-line with physiological pH fluctuations. Finally, we demonstrate Ca-dependent transient interactions between the EF-hand and other LETM1 or GHITM protein domains. Collectively, our data reveal the apo-to-holo structural dynamics and mechanisms underlying the multi-modal sensing by the LETM1 EF-hand domain, highlighting its role as an adaptable regulatory element within the mitochondrial matrix.