The leucine zipper EF-hand containing transmembrane protein-1 EF-handis an atypical tripartite calcium, temperature, and pH sensor.

Abstract

Mitochondrial calcium (mtCa2+) is integral to ATP production; however, excessive Ca2+ leads to cell death. Both these processes are crucial to cellular function, and thus mtCa2+ homeostasis is tightly regulated. Leucine zipper EF-hand containing transmembrane protein-1 (LETM1) is an essential inner-mitochondrial membrane protein that regulates mtCa2+ through exchange of Ca2+ for protons (H+). LETM1 haploinsufficiency is associated with Wolf-Hirschhorn syndrome, characterized by microcephaly, growth retardation, and epileptic seizures, indicating a fundamental function for LETM1 in humans. LETM1 contains a conserved Ca2+ binding EF-hand motif (EF1). Deletion of EF1 abrogates LETM1 mediated mtCa2+ flux, but the precise mechanism by which EF1 regulates exchanger function remains unknown. To begin understanding the role of EF1 in LETM1 function, we biophysical and structurally characterized isolated EF1. Our data show that EF1 exhibits augmented α-helical secondary structure, solvent exposed hydrophobicity and oligomerization in the presence of Ca2+ due to a weak but specific, Ca2+-binding affinity. The low affinity is, in part, due to a terminal Asp within the binding loop, where mutation to Glu increases the affinity ∼4-fold. EF1 is structurally sensitive to pH changes within the physiological range for mitochondria, and remarkably, unfolds at high and low temperatures. Using solution NMR spectroscopy, we reveal that EF1 adopts an atypical EF-hand arrangement by pairing with an upstream loop-helix motif. Collectively, our data suggest that EF1 functions as a divergent multipartite environmental sensor within the EF-hand protein family by adopting an inimitable three-helix EF-hand domain arrangement in LETM1.