Structural basis for ligand binding modes of CTP synthase

Abstract

Cytidine triphosphate synthase (CTPS), which comprises an ammonia ligase domain and a glutamine amidotransferase domain, catalyzes the final step of de novo CTP biosynthesis. The activity of CTPS is regulated by the binding of four nucleotides and glutamine. While glutamine serves as an ammonia donor for the ATP-dependent conversion of UTP to CTP, the fourth nucleotide GTP acts as an allosteric activator. Models have been proposed to explain the mechanisms of action at the active site of the ammonia ligase domain and the conformational changes derived by GTP binding. However, actual GTP/ATP/UTP binding modes and relevant conformational changes have not been revealed fully. Here, we report the discovery of binding modes of four nucleotides and a glutamine analog 6-diazo-5-oxo-L-norleucine in Drosophila CTPS by cryo–electron microscopy with near-atomic resolution. Interactions between GTP and surrounding residues indicate that GTP acts to coordinate reactions at both domains by directly blocking ammonia leakage and stabilizing the ammonia tunnel. Additionally, we observe the ATP-dependent UTP phosphorylation intermediate and determine interacting residues at the ammonia ligase. A noncanonical CTP binding at the ATP binding site suggests another layer of feedback inhibition. Our findings not only delineate the structure of CTPS in the presence of all substrates but also complete our understanding of the underlying mechanisms of the allosteric regulation and CTP synthesis.

Cryo-EM Structures of CTP Synthase Filaments Reveal Mechanism of pH-Sensitive Assembly During Budding Yeast Starvation

“…3a, table 1). In the Ura8 structures all ligands were clearly visible and bound as previously described for other CTPS homologs, including an iminophosphate reaction intermediate recently observed in drosophila CTPS, and the presence of two distinct CTP binding sites per monomer recently reported in human and drosophila CTPS (Endrizzi et al, 2004;Goto et al, 2004;Lynch et al, 2017;Zhou et al, 2021, in review) (Fig3 Supp 1c). Ura7 filaments reached lower resolutions, 7.3 Å (substrates) and 3.7 Å (products), but the overall structures were indistinguishable from Ura8 at these resolutions (Fig3 Supp 2, Table 2).…”

“…Although at lower resolution, the Ura7 structures appear to make the same lateral contacts as substrate-bound Ura8 (Fig6 Supp 1). Similar filament bundles have not been reported for other species, despite extensive structural characterization of human, drosophila, and E. coli CTPS filaments (Barry et al, 2014;Lynch et al, 2017;Zhou et al, 2021), suggesting that the unique yeast insert promotes lateral assemblies that are specific to yeast.…”

“…Cellular CTPS filament assembly is widespread across the domains of life, having been observed in bacteria, archaea, and eukaryotes (Carcamo et al, 2011;Ingerson-Mahar et al, 2010;Liu, 2010;Noree et al, 2010;Zhou et al, 2021). We have previously shown, however, that there are striking differences between the assembly mechanisms and filament architectures among species (Fig 1d-f).…”

“…Maintenance of balanced nucleotide pools is essential for all organisms, and CTPS plays a critical, conserved role in directly balancing pyrimidine levels. The polymerization of CTPS into cellular filamentous polymers occurs in bacteria, archaea, and in eukaryotes (Carcamo et al, 2011;Ingerson-Mahar et al, 2010;Liu, 2010;Noree et al, 2010;Zhou et al, 2021). Bacterial and animal CTPS filaments assemble with completely different interfaces and functional consequences for enzyme regulation, inhibiting activity in the bacterial enzymes and increasing activity or enhancing cooperative regulation in the animal enzymes (Barry et al, 2014;Lynch et al, 2017;.…”

“…We have previously shown, however, that there are striking differences between the assembly mechanisms and filament architectures among species (Fig 1d-f). These differences give rise to differences in function, with bacterial CTPS filaments providing a mechanism to allosterically inhibit the enzyme, while animal CTPS filaments act to increase activity and cooperativity of regulation (Barry et al, 2014;Lynch et al, 2017;Zhou et al, 2021Zhou et al, , 2019.…”