Molecular architecture of the human citrate synthase-malate dehydrogenase 2 metabolon

Metabolons are transient biomolecular complexes that enhance the efficiency of metabolic pathways through substrate channeling. These complexes are difficult to study because of their transient nature, thus limiting our understanding of how they are formed and regulated. The citric acid cycle is proposed to contain many such complexes, although few have been characterized structurally. Here, we provide direct structural evidence for the complex of human citrate synthase (hCS) and human mitochondrial malate dehydrogenase 2 (hMDH2), which is part of the larger proposed citric acid cycle metabolon. Our structural model supports previous cross-linking studies and suggests that hMDH2 can interact with each subunit of the hCS dimer, forming up to a hexameric complex. However, this complex appears to be transient, as titration of hMDH2 into hCS in activity assays does not saturate. We further show that the interaction site with hCS is nonspecific, as hCS could also stimulate oxaloacetate formation by cytosolic and plant MDH enzymes. This structural model will provide a basis for understanding the structure and regulation of the broader citric acid cycle metabolon.