Structural And Mechanistic Elucidation Of The EF-hand In LETM1 Ca2+/H+ Antiporter Function

Qi-Tong Lin Mr., Western University

Abstract

Mitochondrial calcium (Ca²⁺) is integral cellular energy production; however, excessive Ca²⁺ leads to cell death. Both these processes are crucial to cellular function, and thus Ca²⁺ homeostasis is tightly regulated. Leucine zipper EF-hand containing transmembrane protein-1 (LETM1) is an essential inner-mitochondrial membrane protein that regulates Ca²⁺ through exchange for protons (H⁺). LETM1 is an essential gene where haploinsufficiency is associated with Wolf-Hirschhorn syndrome (WHS), characterized by microcephaly, growth retardation, and epileptic seizures. LETM1 contains a conserved Ca²⁺ binding EF-hand motif (EF1). Deletion of EF1 abrogates LETM1 mediated Ca²⁺ flux, but the precise mechanism by which EF1 regulates exchanger function remains unclear. To begin understanding the role of EF1 in LETM1 function, we biophysically and structurally characterized isolated EF1 to probe the effects of Ca²⁺ binding and changes in pH and temperature on secondary, tertiary and quaternary structure, thermal stability and high-resolution structure. We discovered the LETM1 EF1 features weak (~mM) but specific Ca²⁺ binding affinity that is responsive to both changes in pH and temperature within the physiological range experienced by mitochondria. Furthermore, Ca²⁺ binding increases the exposure of hydrophobic regions shifting its oligomerization state from a monomer to higher order oligomers. Determining the nuclear magnetic resonance (NMR) structure of the LETM1 EF-hand in the presence of Ca²⁺ exposed a pairing between a partial loop-helix and full helix-loop-helix, forming a unique “F-EF”-hand pairing with non-canonical Ca²⁺ coordination. Mutations that augmented or weakened Ca²⁺ binding increased or decreased matrix Ca²⁺, respectively, establishing F-EF as a two-way mitochondrial Ca²⁺ regulator. Finally, we determined the Ca²⁺-depleted LETM1 F-EF-hand domain structure to reveal the conformational changes associated with binding Ca²⁺. We found regiospecific unfolding in response to hot and cold denaturation above 0 °C and a pKa in the physiological range of pH fluctuations. Further, this thesis defines an approach that leverages the accuracy of AlphaFold to guide nuclear Overhauser effect (NOE) assignments during NMR-based protein structure determination. Overall, these findings provide the first atomic resolution view of the structural dynamics and mechanisms underlying the multi-modal sensing of the LETM1 F-EF-hand domain, highlighting a role as an adaptable regulatory element within the mitochondrial matrix.