Mitochondrial disorders causing neurodegeneration in childhood are genetically heterogeneous, and the underlying genetic etiology remains unknown in many affected individuals. We identified biallelic variants in PMPCB in individuals of four families including one family with two affected siblings with neurodegeneration and cerebellar atrophy. PMPCB encodes the catalytic subunit of the essential mitochondrial processing protease (MPP), which is required for maturation of the majority of mitochondrial precursor proteins. Mitochondria isolated from two fibroblast cell lines and induced pluripotent stem cells derived from one affected individual and differentiated neuroepithelial stem cells showed reduced PMPCB levels and accumulation of the processing intermediate of frataxin, a sensitive substrate for MPP dysfunction. Introduction of the identified PMPCB variants into the homologous S. cerevisiae Mas1 protein resulted in a severe growth and MPP processing defect leading to the accumulation of mitochondrial precursor proteins and early impairment of the biogenesis of iron-sulfur clusters, which are indispensable for a broad range of crucial cellular functions. Analysis of biopsy materials of an affected individual revealed changes and decreased activity in iron-sulfur cluster-containing respiratory chain complexes and dysfunction of mitochondrial and cytosolic Fe-S cluster-dependent enzymes. We conclude that biallelic mutations in PMPCB cause defects in MPP proteolytic activity leading to dysregulation of iron-sulfur cluster biogenesis and triggering a complex neurological phenotype of neurodegeneration in early childhood.

Graphical Abstract Highlights d The LisH motif of muskelin acts as a dimerization interface d The generic binding site in the discoidin domain mediates a head-to-tail interaction d Muskelin forms a tetramer as a dimer of dimers via both interfaces d An impaired LisH dimerization relocates muskelin from the cytoplasm to the nucleus In Brief Muskelin organizes the retrograde transport of certain GABA A receptors. In the crystal structure of the N-terminal discoidin domain and LisH motif, Delto et al. observe a LisH-mediated dimer. They demonstrate that this interaction is required for muskelin tetramerization and determines cellular localization of muskelin. Accession Numbers 4OYU Delto et al., 2015, Structure 23, 364-373 February 3, SUMMARYNeurons regulate the number of surface receptors by balancing the transport to and from the plasma membrane to adjust their signaling properties. The protein muskelin was recently identified as a key factor guiding the transport of a1 subunit-containing GABA A receptors. Here we present the crystal structure of muskelin, comprising its N-terminal discoidin domain and Lis1-homology (LisH) motif. The molecule crystallized as a dimer with the LisH motif exclusively mediating oligomerization. Our subsequent biochemical analyses confirmed that the LisH motif acts as a dimerization element in muskelin. Together with an intermolecular head-to-tail interaction, the LisH-dependent dimerization is required to assemble a muskelin tetramer. Intriguingly, our cellular studies revealed that the loss of this dimerization results in a complete redistribution of muskelin from the cytoplasm to the nucleus and impairs muskelin's function in GABA A receptor transport. These studies demonstrate that the LisH-dependent dimerization is a crucial factor for muskelin function.

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Ligand-binding of Cys-loop receptors is determined by N-terminal extracellular loop structures from the plus as well as from the minus side of two adjacent subunits in the pentameric receptor complex. An aromatic residue in loop B of the glycine receptor (GlyR) undergoes direct interaction with the incoming ligand via a cation-π interaction. Recently, we showed that mutated residues in loop B identified from human patients suffering from hyperekplexia disturb ligand-binding. Here, we exchanged the affected human residues by amino acids found in related members of the Cys-loop receptor family to determine the effects of side chain volume for ion channel properties. GlyR variants were characterized in vitro following transfection into cell lines in order to analyze protein expression, trafficking, degradation and ion channel function. GlyR α1 G160 mutations significantly decrease glycine potency arguing for a positional effect on neighboring aromatic residues and consequently glycine-binding within the ligand-binding pocket. Disturbed glycinergic inhibition due to T162 α1 mutations is an additive effect of affected biogenesis and structural changes within the ligand-binding site. Protein trafficking from the ER toward the ER-Golgi intermediate compartment, the secretory Golgi pathways and finally the cell surface is largely diminished, but still sufficient to deliver ion channels that are functional at least at high glycine concentrations. The majority of T162 mutant protein accumulates in the ER and is delivered to ER-associated proteasomal degradation. Hence, G160 is an important determinant during glycine binding. In contrast, T162 affects primarily receptor biogenesis whereas exchanges in functionality are secondary effects thereof.

Ligand-binding of Cys-loop receptors results in rearrangements of extracellular loop structures which are further translated into the tilting of membrane spanning helices, and finally opening of the ion channels. The cryo-EM structure of the homopentameric α1 glycine receptor (GlyR) demonstrated an involvement of the extracellular β8–β9 loop in the transition from ligand-bound receptors to the open channel state. Recently, we identified a functional role of the β8–β9 loop in a novel startle disease mouse model shaky. The mutation of residue GlyRα1Q177 to lysine present in shaky mice resulted in reduced glycine potency, reduced synaptic expression, and a disrupted hydrogen network at the structural level around position GlyRα1Q177. Here, we investigated the role of amino acid volume, side chain length, and charge at position Q177 to get deeper insights into the functional role of the β8–β9 loop. We used a combined approach of in vitro expression analysis, functional electrophysiological recordings, and GlyR modeling to describe the role of Q177 for GlyR ion channel function. GlyRα1Q177 variants do not disturb ion channel transport to the cellular surface of transfected cells, neither in homomeric nor in heteromeric GlyR configurations. The EC50 values were increased for all GlyRα1Q177 variants in comparison to the wild type. The largest decrease in glycine potency was observed for the variant GlyRα1Q177R. Potencies of the partial agonists β-alanine and taurine were also reduced. Our data are further supported by homology modeling. The GlyRα1Q177R variant does not form hydrogen bonds with the surrounding network of residue Q177 similar to the substitution with a basic lysine present in the mouse mutant shaky. Among all investigated Q177 mutants, the neutral exchange of glutamine to asparagine as well as the introduction of the closely related amino acid glutamic acid preserve the hydrogen bond network. Introduction of amino acids with small side chains or larger volume resulted in a loss of their hydrogen bonds to neighboring residues. The β8–β9 loop is thus an important structural and functional determinant of the inhibitory GlyR.