Molecular determinants of inhibition of UCP1-mediated respiratory uncoupling

Brown adipose tissue expresses uncoupling protein 1 (UCP1), a mitochondrial transporter that uncouples respiration from ATP synthesis and dissipates energy as heat, making it a target for treating obesity and related metabolic disorders. Here, we combine molecular dynamics simulations with mitochondrial respiration assays to investigate how purine nucleotides inhibit respiration uncoupling by UCP1. Simulations predict that GDP binds UCP1 in the common substrate binding site in an upright orientation, where the base moiety interacts with a pair of charged residues (R92/E191) that are specifically conserved in the subfamily of UCPs. E191, among others, interacts with purine but not pyrimidine bases, suggesting a rationale for nucleotide specificity in UCP1 inhibition. We also identify a triplet of uncharged residues involved in hydrophobic contacts with GDP. Site-directed mutagenesis of either I187 or W281 to alanine increases lauric acid-induced uncoupling activity of UCP1 and partially suppresses inhibition of UCP1 activity by GDP in yeast spheroplasts. The triple mutant (F88, I187, W281) to alanine is overactivated by lauric acid even in a high concentration of purine nucleotides. Variants at these positions may help increase energy expenditure in a cellular and therapeutic context.