Key features of inhibitor binding to the human mitochondrial pyruvate carrier hetero-dimer

Fifty years of the mitochondrial pyruvate carrier: New insights into its structure, function, and inhibition

“…The substrate and inhibitors differ by the presence of one or more hydrophobic moieties, which in turn contribute to the inhibitory potency through the size and number of additional groups. In support of the binding site forming in the hetero-dimer interface, both human hetero-dimers bind known MPC inhibitors with high affinities, 15 whereas the individual protomers do not bind any of the ligands with the expected affinity or at all. 55,61 The longstanding theory that binding of cyanocinnamates and derivatives is mediated by a reversible covalent bond with an MPC cysteine has been disputed by in vitro mass spectrometry data and alanine scanning mutagenesis on purified MPC hetero-dimers.…”

“…This is consistent with our proposal that both protomers are involved in inhibitor binding. 15 We have performed comparative analysis of the structural models from AlphaFold and trRosetta for the yeast and human MPC protomers (Figure 4). The comparison shows that the agreement on the three-transmembrane-helical topology of each protomer is very high.…”

“…We followed a biophysical approach to determine the molecular mass, oligomeric state, and subunit stoichiometry of MPC by using the purified yeast Mpc1/Mpc3 and human MPC1L/MPC2 hetero‐complexes, analyzed via size‐exclusion chromatography linked to multi‐angle laser light scattering (SEC‐MALLS) 14,15 . This technique can determine the mass of the detergent–lipid complex and the protein component 56,57 and therefore, is considered the golden standard for sizing of membrane proteins.…”

“…13 Recently, it was demonstrated that the functional MPC is hetero-dimeric. 14,15 Despite its name, the MPC is unrelated to SLC25 members and was assigned to a new protein family, named SLC54. 16 Given its central role in metabolism, MPC has been proposed as a pharmacological target for different pathologies, including diabetes, non-alcoholic steatohepatitis (NASH), [17][18][19][20][21] neurodegenerative disorders, [22][23][24] and specific cancers.…”

“…These proteins were originally shown to form large hetero‐complexes of ~150 KDa 12 on blue native electrophoresis, but this approach leads to artifacts for small membrane proteins 13 . Recently, it was demonstrated that the functional MPC is hetero‐dimeric 14,15 . Despite its name, the MPC is unrelated to SLC25 members and was assigned to a new protein family, named SLC54 16 …”