Structure and Functional Characterization of Human Aspartate Transcarbamoylase, the Target of the Anti-tumoral Drug PALA

CAD, the multienzymatic protein that initiates and controls de novo synthesis of pyrimidines in animals, associates through its aspartate transcarbamoylase (ATCase) domain into particles of 1.5 MDa. Despite numerous structures of prokaryotic ATCases, we lack structural information on the ATCase domain of CAD. Here, we report the structure and functional characterization of human ATCase, confirming the overall similarity with bacterial homologs. Unexpectedly, human ATCase exhibits cooperativity effects that reduce the affinity for the anti-tumoral drug PALA. Combining structural, mutagenic, and biochemical analysis, we identified key elements for the necessary regulation and transmission of conformational changes leading to cooperativity between subunits. Mutation of one of these elements, R2024, was recently found to cause the first non-lethal CAD deficit. We reproduced this mutation in human ATCase and measured its effect, demonstrating that this arginine is part of a molecular switch that regulates the equilibrium between low- and high-affinity states for the ligands. [Display omitted] •Crystal structures of human ATCase domain free and bound to CP or PALA were determined•Human ATCase is a catalytic homotrimer with three cooperative active sites•Only two active sites show high affinity for the binding of the anti-tumoral drug PALA•R2024 and loop H1-S2 regulate and transmit conformational changes between subunits CAD, the multifunctional protein controlling the synthesis of pyrimidines, associates through its aspartate transcarbamoylase domain (ATCase) into 1.5-MDa particles. Ruiz-Ramos et al. determine the structure and characterize the functioning of the ATCase domain of human CAD, revealing a mechanism of cooperativity between the subunits within the trimer.