The leucine zipper EF‐hand containing transmembrane protein‐1 EF‐hand is a tripartite calcium, temperature, and pH sensor

Abstract

Leucine Zipper EF‐hand containing transmembrane protein‐1 (LETM1) is an inner mitochondrial membrane protein that mediates mitochondrial calcium (Ca2+)/proton exchange. The matrix residing carboxyl (C)‐terminal domain contains a sequence identifiable EF‐hand motif (EF1) that is highly conserved among orthologues. Deletion of EF1 abrogates LETM1 mediated mitochondrial Ca2+ flux, highlighting the requirement of EF1 for LETM1 function. To understand the mechanistic role of this EF‐hand in LETM1 function, we characterized the biophysical properties of EF1 in isolation. Our data show that EF1 exhibits α‐helical secondary structure that is augmented in the presence of Ca2+. Unexpectedly, EF1 features a weak (~mM), but specific, apparent Ca2+‐binding affinity, consistent with the canonical Ca2+ coordination geometry, suggested by our solution NMR. The low affinity is, at least in part, due to an Asp at position 12 of the binding loop, where mutation to Glu increases the affinity by ~4‐fold. Further, the binding affinity is sensitive to pH changes within the physiological range experienced by mitochondria. Remarkably, EF1 unfolds at high and low temperatures. Despite these unique EF‐hand properties, Ca2+ binding increases the exposure of hydrophobic regions, typical of EF‐hands; however, this Ca2+‐induced conformational change shifts EF1 from a monomer to higher order oligomers. Finally, we showed that a second, putative EF‐hand within LETM1 is unreactive to Ca2+ either in isolation or tandem with EF1. Collectively, our data reveal that EF1 is structurally and biophysically responsive to pH, Ca2+ and temperature, suggesting a role as a multipartite environmental sensor within LETM1.

An AI-informed NMR structure reveals a LETM1 F-EF-hand for two-way mitochondrial calcium regulation.

“…This pKa and fully solvent-exposed location within the short linker, suggest a mechanism for the robust structural and biophysical attribute sensitivity of the domain to pH changes in the 6.0-8.0 range. Indeed, as previously reported (Lin et al, 2021), we found apo WT pH 6.0 showed significantly greater negative ellipticity at ~222 nm (i.e. more -helicity) compared to apo WT at pH 7.8; moreover, no such pH-dependent difference was observed for holo WT, likely because of the increased -helicity mediated by Ca 2+ binding (Figure 7b and Table 2).…”

“…Sequence comparison of the 12-residue loop shows strong conservation with other canonical EF-hand loop sequences such as calmodulin EF2 (CaM), parvalbumin EF3 and calbindin D9k EF2 with the exception of the Asp residue at the 12 th position, which commonly confers Mg 2+ binding affinity to non-canonical EF-hands (Figure 1b) (Tan et al , 2022; Yanyi et al , 2010). Nevertheless, our previous research indicates that the LETM1 EF-hand selectively binds Ca 2+ (Lin et al ., 2021), which is consistent with the high sequence conservation at the other liganding loop residue positions when compared to ∼1,700 canonical Ca 2+ binding loops (Figure 1c) (Sigrist et al , 2013).…”

“…Furthermore, this helix-helix-loop-helix region is flanked both up and downstream by coiled-coils. Studies from our laboratory previously showed that the LETM1 EF-hand displays tripartite sensitivity to temperature, pH and Ca 2+ binding selectively (Lin et al , 2021). Sequence comparison of the 12-residue loop shows strong conservation with other canonical EF-hand loop sequences such as calmodulin EF2 (CaM), parvalbumin EF3 and calbindin D9k EF2 with the exception of the Asp residue at the 12 th position, which commonly confers Mg 2+ binding affinity to non-canonical EF-hands (Figure 1b) (Tan et al , 2022; Yanyi et al , 2010).…”

The Interplay between Dysregulated Ion Transport and Mitochondrial Architecture as a Dangerous Liaison in Cancer

“…While this transporter has been a topic of controversy, the mitochondrial Ca 2+ /H + exchanger is most probably encoded by LETM1 [ 80 , 81 ]. LETM1 appears to directly sense both H + and Ca 2+ [ 82 ] and to play transport-unrelated roles in signaling, including its physical anchoring to the mitochondrial ribosome protein L36 [ 83 ]. The nature of the mitochondrial Na + /H + exchanger (NHE) is also controversial.…”

Mitochondrial Ca2+efflux controls neuronal metabolism and long-term memory across species

“…Re-expression of an shRNA-resistant variant of wild-type Letm1 in Letm1 KD neurons rescued mitochondrial Ca 2+ efflux rates, validating the specificity of the Letm1 shRNA (Figure 1F). Letm1 presents a Ca 2+ binding EF-hand domain oriented towards the mitochondrial matrix 46,47 (Figure 1A). We hypothesized that such a domain would be ideally positioned to activate Letm1 during neurotransmission, as it could sense mitochondrial Ca 2+ increases during firing.…”