Thesis Format

Integrated Article

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

Author

Qi-Tong Lin Mr., Western UniversityFollow

Degree

Doctor of Philosophy

Program

Physiology and Pharmacology

Supervisor

Stathopulos, Peter B.

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.

Summary for Lay Audience

Mitochondria, the powerhouses of the cell, use calcium (Ca²⁺) to produce energy. However, too much Ca²⁺ can cause cell death. To maintain a healthy balance, Ca²⁺ levels inside the mitochondria must be carefully controlled. LETM1 has been implicated in mitochondrial Ca²⁺ regulation; however, the precise mechanisms with atom-specific detail by which LETM1 regulates mitochondrial Ca²⁺ remains a major knowledge gap and a topic of debate.

LETM1 is a transmembrane protein residing in the inner mitochondrial membrane and has been shown to directly mediate the bidirectional exchange of Ca²⁺ for protons (H⁺). Conversely, other studies have implicated an indirect mechanism by which LETM1 mediates mitochondrial Ca²⁺ regulation through interaction with other mitochondrial proteins.

LETM1 contains a conserved Ca²⁺ binding EF-hand motif. Upon binding Ca²⁺, EF-hands have been shown in nature to facilitate self-association and act as an interaction site for other proteins. Experiments monitoring mitochondrial Ca²⁺ transport in mammalian cells have shown deletion of the LETM1 EF-hand decreases mitochondrial Ca²⁺ transport, mirroring the effect of decreased LETM1 protein expression, and thus, highlighting the importance of the EF-hand in LETM1 mediated mitochondrial Ca²⁺ regulation.

In this work we sought to determine how the LETM1 EF-hand plays a role in the mechanism or regulation of LETM1 function with respect to mitochondrial Ca²⁺ regulation. We characterized the biophysical and structural properties of the isolated LETM1 EF-hand, solved the three-dimensional (3D) structure unbound and bound to Ca²⁺ and probed the effects of augmenting and weaking EF-hand Ca²⁺ binding mutations on full length LETM1 function. We reveal the LETM1 EF-hand possesses sensitivity not only to Ca²⁺ binding, but also changes in pH and temperature within the physiological range. Furthermore, the EF-hand undergoes conformational changes that expose a large protein interaction site and acts as a two-way regulatory domain for LETM1 mediated mitochondrial Ca²⁺. This thesis also delineates an approach to apply the accuracy of artificial intelligence (AI)/AlphaFold-based protein structure prediction to experimental protein structure determination. Collectively, the research described in the thesis provides new atom-level insights into how the LETM1 EF-hand to senses Ca²⁺, pH, temperature to modulate LETM1 function in the regulation of mitochondrial Ca²⁺.

Recommended Citation

Lin, Qi-Tong Mr., "Structural And Mechanistic Elucidation Of The EF-hand In LETM1 Ca2+/H+ Antiporter Function" (2024). Electronic Thesis and Dissertation Repository. 10527.
https://ir.lib.uwo.ca/etd/10527

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