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Leucine zipper-EF-hand containing transmembrane protein 1 (LETM1) encodes an inner mitochondrial membrane protein with an osmoregulatory function controlling mitochondrial volume and ion homeostasis. The putative association of LETM1 with a human disease was initially suggested in Wolf-Hirschhorn syndrome, a disorder that results from de novo monoallelic deletion of chromosome 4p16.3, a region encompassing LETM1. Utilizing exome sequencing and international gene-matching efforts, we have identified 18 affected individuals from 11 unrelated families harboring ultra-rare bi-allelic missense and loss-of-function LETM1 variants and clinical presentations highly suggestive of mitochondrial disease. These manifested as a spectrum of predominantly infantile-onset (14/18, 78%) and variably progressive neurological, metabolic, and dysmorphic symptoms, plus multiple organ dysfunction associated with neurodegeneration. The common features included respiratory chain complex deficiencies (100%), global developmental delay (94%), optic atrophy (83%), sensorineural hearing loss (78%), and cerebellar ataxia (78%) followed by epilepsy (67%), spasticity (53%), and myopathy (50%). Other features included bilateral cataracts (42%), cardiomyopathy (36%), and diabetes (27%). To better understand the pathogenic mechanism of the identified LETM1 variants, we performed biochemical and morphological studies on mitochondrial K+/H+ exchange activity, proteins, and shape in proband-derived fibroblasts and muscles and in Saccharomyces cerevisiae , which is an important model organism for mitochondrial osmotic regulation. Our results demonstrate that bi-allelic LETM1 variants are associated with defective mitochondrial K+ efflux, swollen mitochondrial matrix structures, and loss of important mitochondrial oxidative phosphorylation protein components, thus highlighting the implication of perturbed mitochondrial osmoregulation caused by LETM1 variants in neurological and mitochondrial pathologies. [Display omitted] Kaiyrzhanov et al. describe 18 affected individuals with bi-allelic variants in the leucine zipper-EF-hand containing transmembrane protein 1 gene presenting with clinical features suggestive of a mitochondrial disease. Functional studies showed defective mitochondrial K+ efflux, swollen mitochondrial matrix structures, and loss of mitochondrial oxidative phosphorylation protein components. [ABSTRACT FROM AUTHOR]

AlphaFold can accurately predict static protein structures but does not account for solvent conditions. Human leucine zipper EF-hand transmembrane protein-1 (LETM1) has one sequence-identifiable EF-hand but how calcium (Ca 2+ ) affects structure and function remains enigmatic. Here, we used highly confident AlphaFold Cα predictions to guide nuclear Overhauser effect (NOE) assignments and structure calculation of the LETM1 EF-hand in the presence of Ca 2+ . The resultant NMR structure exposes pairing between a partial loop-helix and full helix-loop-helix, forming an unprecedented F-EF-hand with non-canonical Ca 2+ coordination but enhanced hydrophobicity for protein interactions compared to calmodulin. The structure also reveals the basis for pH sensing at the link between canonical and partial EF-hands. Functionally, mutations that augmented or weakened Ca 2+ binding increased or decreased matrix Ca 2+ , respectively, establishing F-EF as a two-way mitochondrial Ca 2+ regulator. Thus, we show how to synergize AI prediction with NMR data, elucidating a solution-specific and extraordinary LETM1 F-EF-hand., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)