Deciphering the structure of cad: Structural and functional characterization of the human aspartate transcarbamoylase domain

• Autores: Alba Ruiz Ramos
• Directores de la Tesis: Santiago Ramon Maiques (dir. tes.)
• Lectura: En la Universidad Autónoma de Madrid ( España ) en 2016
• Idioma: español
• Tribunal Calificador de la Tesis: Vicente Rubio Zamora (presid.), Esteban Montejo de Garcini Guedas (secret.), Daniel Lietha (voc.), Marta Martinez Julve (voc.), Juan Hermoso (voc.)
• Programa de doctorado: Programa Oficial de Doctorado en Bioquímica, Biología Molecular, Biomedicina y Biotecnología (Biociencias Moleculares)
• Materias:
  • Química
    • Bioquímica
      • Biología molecular
  • Ciencias tecnológicas
    • Procesos tecnológicos
      • Cristalización
• Enlaces
  • Tesis en acceso abierto en: Biblos-e Archivo
• Resumen

  • Aspartate transcarbamoylase (ATCase) catalyzes the synthesis of N-carbamoyl-L-aspartate from carbamoyl phosphate and aspartate in the second step of the de novo biosynthesis of pyrimidines. In prokaryotes and plants, the first three activities of the pathway, namely carbamoyl phosphate synthetase (CPSase), ATCase and dihydroorotase (DHOase), are encoded as distinct proteins that function independently or in non-covalent association. In turn, in animals, CPSase, ATCase and DHOase are part of a 243 kDa multifunctional polypeptide named CAD whose up-regulation is essential for normal and tumor cell proliferation. Although the structures of numerous prokaryotic ATCases have been determined, there is no structural information about any eukaryotic ATCase. In fact, the only detailed structural information about CAD is that it self-assembles through its ATCase domain forming hexameric particles of 1.5 MDa. In this study, we report the cloning, expression and purification of the ATCase domain of human CAD. The recombinant protein, expressed in bacteria and purified with good yields, forms homotrimers in solution. Crystallization experiments, both in the absence and presence of the inhibitor PALA, yielded small crystals that diffracted X-rays to 2.1 Å resolution using synchrotron radiation. We have determined the structure of the human ATCase domain, the first example of a eukaryotic ATCase, confirming the overall similarity with bacterial homologues. Surprisingly, the enzymatic and biochemical characterization of human ATCase showed that this domain exhibits cooperativity effects that reduce the affinity for the anti-tumoral drug PALA. These results were unexpected since the prokaryotic ATCase catalytic trimers lack cooperativity between active sites. Combining structural, mutagenic and biochemical analysis we identified key structural elements in human ATCase for the necessary regulation and transmission of conformational changes leading to cooperativity between subunits. Mutation of one of these elements, the active site residue R2024, was recently found to cause the first non-lethal CAD-deficit. We reproduced this mutation in the recombinant human ATCase domain and measured its effect. Our data suggest that this arginine is part of a molecular switch that regulates the conformational equilibrium between low and high affinity states of the subunits for the ligands. To further understand the role of ATCase trimers in the assembly of CAD particles, we produced a bifunctional construct harbouring the DHOase and ATCase domains of human CAD together with the 91 amino acid interlinking sequence. We demonstrated that this construct assembles into hexamers in solution and that site-specific mutations preventing either the dimerization of DHOase or the trimerization of ATCase, results in DHOase-ATCase trimers and dimers respectively. These results indicate that the ATCase and DHOase domains form the structural core of CAD particles.