Folding and assembly defects of pyruvate dehydrogenase deficiency-related variants in the E1 alpha subunit of the pyruvate dehydrogenase complex

The pyruvate dehydrogenase complex (PDC) bridges glycolysis and the citric acid cycle. In human, PDC deficiency leads to severe neurodevelopmental delay and progressive neurodegeneration. The majority of cases are caused by variants in the gene encoding the PDC subunit E1 alpha. The molecular effects of the variants, however, remain poorly understood. Using yeast as a eukaryotic model system, we have studied the substitutions A189V, M230V, and R322C in yeast E1 alpha (corresponding to the pathogenic variants A169V, M210V, and R302C in human E1 alpha) and evaluated how substitutions of single amino acid residues within different functional E1 alpha regions affect PDC structure and activity. The E1 alpha A189V substitution located in the heterodimer interface showed a more compact conformation with significant underrepresentation of E1 in PDC and impaired overall PDC activity. The E1 alpha M230V substitution located in the tetramer and heterodimer interface showed a relatively more open conformation and was particularly affected by low thiamin pyrophosphate concentrations. The E1 alpha R322C substitution located in the phosphorylation loop of E1 alpha resulted in PDC lacking E3 subunits and abolished overall functional activity. Furthermore, we show for the E1 alpha variant A189V that variant E1 alpha accumulates in the Hsp60 chaperonin, but can be released upon ATP supplementation. Our studies suggest that pathogenic E1 alpha variants may be associated with structural changes of PDC and impaired folding of E1 alpha.