Conformational analysis of the riboflavin-responsive ETF:QO-p.Pro456Leu variant associated with mild multiple acyl-CoA dehydrogenase deficiency

• Published in: Biochimica et biophysica acta. Proteins and proteomics 2020-06, Vol.1868 (6), p.140393-140393, Article 140393
• Main Authors: Lucas, Tânia G., Henriques, Bárbara J., Gomes, Cláudio M.
• Format: Article
• Language: English
• Subjects: Flavin; Flavoprotein; Glutaric aciduria type II; Inborn errors metabolism; Protein folding
• ISSN: 1570-9639; 1878-1454
• DOI: 10.1016/j.bbapap.2020.140393

Multiple-CoA dehydrogenase deficiency (MADD) is an inborn disorder of fatty acid and amino acid metabolism caused by mutations in the genes encoding for human electron transfer flavoprotein (ETF) and its partner electron transfer flavoprotein:ubiquinone oxidoreductase (ETF:QO). Albeit a rare disease, extensive newborn screening programs contributed to a wider coverage of MADD genotypes. However, the impact of non-lethal mutations on ETF:QO function remains scarcely understood from a structural perspective. To this end, we here revisit the relatively common MADD mutation ETF:QO-p.Pro456Leu, in order to clarify how it affects enzyme structure and folding. Given the limitation in recombinant expression of human ETF:QO, we resort to its bacterial homologue from Rhodobacter sphaeroides (Rs), in which the corresponding mutation (p.Pro389Leu) was inserted. The in vitro biochemical and biophysical investigations of the Rs ETF:QO-p.Pro389Leu variant showed that, while the mutation does not significantly affect the protein α/β fold, it introduces some plasticity on the tertiary structure and within flavin interactions. Indeed, in the p.Pro389Leu variant, FAD exhibits a higher thermolability during thermal denaturation and a faster rate of release in temperature-induced dissociation experiments, in comparison to the wild type. Therefore, although this clinical mutation occurs in the ubiquinone domain, its effect likely propagates to the nearby FAD binding domain, probably influencing electron transfer and redox potentials. Overall, our results provide a molecular rational for the decreased enzyme activity observed in patients and suggest that compromised FAD interactions in ETF:QO might account for the known riboflavin responsiveness of this mutation. [Display omitted] •MADD is a rare disease of fatty acid and amino-acid metabolism affecting ETF/ETF:QO.•Over 160 mutations known for ETF:QO but structure-function relations are illusive.•The variant ETF:QO-p.Pro456Leu was studied using a bacterial homologue as model.•Protein folding is unaffected but tertiary interactions and FAD lability are compromised.•Findings contribute to uncover basis for MADD patient's riboflavin-responsiveness.