Expression, purification, crystallization and preliminary X-ray diffraction analysis of the aspartate transcarbamoylase domain of human CAD

Abstract

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, 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 noncovalent association. In animals, CPSase, ATCase and DHOase are part of a 243 kDa multifunctional polypeptide named CAD. Up-regulation of CAD is essential for normal and tumour 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 into hexamers and trimers through interactions of the ATCase domains. Here, the expression, purification and crystallization of the ATCase domain of human CAD is reported. The recombinant protein, which was expressed in bacteria and purified with good yield, formed homotrimers in solution. Crystallization experiments both in the absence and in the presence of the inhibitor PALA yielded small crystals that diffracted X-rays to 2.1 Å resolution using synchrotron radiation. The crystals appeared to belong to the hexagonal space group P6(3)22, and Matthews coefficient calculation indicated the presence of one ATCase subunit per asymmetric unit, with a solvent content of 48%. However, analysis of the intensity statistics suggests a special case of the P21 lattice with pseudo-symmetry and possibly twinning.

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

“…huATCase Exhibits Positive Cooperativity for Substrate Binding huATCase was purified as previously reported (Ruiz-Ramos et al, 2013). Based on thermal shift assays, huATCase was stable at pH 6-9, with a melting temperature (T M ) of 54 C ( Figure S1).…”

“…Purification was as reported by Ruiz-Ramos et al (2013) with one additional step. Prior to the final size-exclusion chromatography step, the sample was loaded onto a HiTrap Heparin column (GE Healthcare) in 20 mM Tris-HCl (pH 7), 75 mM NaCl, 5% glycerol, and 2 mM b-mercaptoethanol, and eluted in a linear NaCl gradient.…”

“…Crystallization, Data Collection, and Structure Determination Crystals were obtained as reported by Ruiz-Ramos et al (2013). huATCaseapo crystals were soaked for 5 min in mother liquor supplemented with 5 mM CP and transferred to the same solution plus 30% ethylene glycol prior to freezing.…”

Crystal structure of truncated aspartate transcarbamoylase fromPlasmodium falciparum

“…Nucleotide BLAST analysis also shows 37% identity (86% query cover) to the ATC domain of human CAD (carbamoyl phosphate synthetase 2, aspartate transcarbamylase and dihydroorotase) protein. The crystal structure of this domain is not yet available, although preliminary data have been reported (Ruiz-Ramos et al, 2013). Further analysis with the PlasmoAP tool (Foth et al, 2003) showed a strong prediction for the N-terminal region to contain an apicoplast-targeting sequence, with a possible cleavage site between residues 27 and 28 (IRT-KK; Supplementary Table S1).…”

Cellular Serpents and Dreaming Spires: New Frontiers in Arginine and Pyrimidine Biology

“…Santiago Ramón-Maiques (Spanish National Cancer Research Centre, Spain) described the crystal structure and function of the DHOase and ATCase domains of human CAD using site directed mutagenesis in combination with MALS (multi-angle static light scattering) (Ruiz-Ramos et al, 2013;GrandeGarcia et al, 2014). Sergio de Cima and Carmen Díez Fernandez from Vicente Rubio's lab (Instituto de Biomedicina de Valencia) further enlightened us about CPS1 (carbamoyl phosphate synthetase I).…”