Structure of the synthetase domain of human CTP synthetase, a target for anticancer therapy

Abstract

Cytidine triphosphate synthetase (CTPS) is a key enzyme in nucleic acid and phospholipid biosynthesis and its activity is increased in certain human cancers, making it a promising drug target. The crystal structure of the synthetase domain of human CTPS, which represents the first structure of a CTPS from an eukaryote, has been determined. The structure is homotetrameric and each active site is formed by three different subunits. Sulfate ions bound to the active sites indicate the positions of phosphate-binding sites for the substrates ATP and UTP and the feedback inhibitor CTP. Together with earlier structures of bacterial CTPS, the human CTPS structure provides an extended understanding of the structure-function relationship of CTPS-family members. The structure also serves as a basis for structure-based design of anti-proliferative inhibitors.

Figure 1

The overall reaction catalysed by CTPS. Phosphate is transferred from ATP to the O4 of UTP and the ammonium ion obtained from glutamine is then used to replace the phosphate to generate CTP. R = ribose-5′-triphosphate.

Figure 2

The structure of human CTPS synthetase domain. (a) Structure of the monomer. The seven-stranded β-sheet in the middle is covered by helices on both sides. (b) Assembly into a tetramer. Surfaces of the tetramer are shown with the bound sulfates at the active site; four such active sites are present in the tetramer. Monomers are coloured as follows: A, white; B, blue; C, pink; D, orange. The view has been rotated by 90° on the right. The synthetase domains form a tetramer with 222 symmetry; the glutaminase domains would be expected to point out from the tetramer core like knobs. (c) Sequence alignment of human and E. coli CTPS synthetase domains. The secondary structures are from human CTPS and the underlined segments are disordered in human CTPS.

Figure 3

The active site. (a) Detailed interactions of the sulfates with CTPS. Hydrogen bonds are shown by green lines. (b) Superposition of the sulfates with ADP and UTP from E. coli CTPS. Human CTPS is indicated in light grey (A subunit) and orange (D subunit) and E. coli CTPS in dark grey. ADP is in green and CTP in cyan. Note the superposition of the observed sulfates in the human CTPS structure with one phosphate of each nucleotide ligand.