## 5 A New Level of Regulation: Role of the GAT Domain in Forming Filamentous Structures
and its regulation of catalytic activity in the GAT domain remain unclear. In budding yeast, the GAT domains of URA7 and URA8 are bound with different strengths and interact via different amino acids at this insertion [121]. Therefore, regulation of the activity of the GAT domain through the insertion in this region may vary between species. CTPS with Bound GTP: Confirmation of the Location of the GTP-Binding Site and Relationship to Other Cryo-EM Structures  
Despite the various kinetics and site-directed mutagenesis experiments conducted to delineate the structural and functional aspects of the GTP-dependent regulation of CTPS activity, specific structural information on the GTP binding site and the conformational changes that accompanied GTP binding remained elusive. In 2021, the first structure of CTPS with bound GTP was captured using cryo-EM (Figure 6) [66]. This structure of \(Dm\)CTPS was covalently modified at the GAT domain by the Gln analogue DON and revealed a tightly closed state with the CTPS tetramers forming a filamentous structure through the amino acid insertion mentioned above. As predicted by [PERSON] and co-workers [32], GTP is bound in a cleft between the GAT domain and the synthase domain (Figure 3). The GTP molecule is tightly surrounded by the gate (which forms a part of the NH\({}_{3}\) tunnel), lid L11, Phe 373 (which encapsulates the catalytic triad of the GAT domain), and the L4 loop 105-110 of an adjacent protomer (Figure 7). Compared with other CTPS structures in the tightly closed state, the DON-modified tetramer with bound GTP exhibits an even tighter conformation, which may be related to the formation and tight connection of the NH\({}_{3}\) tunnel. Intriguingly, [PERSON] and co-workers also observed the presence of 4-phosphorylated UTP intermediate in their cryo-EM structure of \(Dm\)CTPS [66]. This observation suggests that the first step of the catalytic reaction (i.e., ATP-dependent phosphorylation of UTP) has been completed in the synthase domain, and the intermediate is waiting for NH\({}_{3}\) to be transferred from the GAT domain to complete the final animation reaction. Figure 6: The location of GTP-binding site. Atom model display for \(Dm\)CTPS in a closed state with GTP bound (**A**) and in an open state without GTP bound (**B**) [66]. The GAT domain, interdomain linker, and the synthase domain are colored pink, light purple, and white, respectively. Regions interacting with GTP are colored as orange for the gate, dark green for F373 and the lid L11, light green for loop 105–110 (L4), purple for the “wing” (L13). GTP and the 4-phospho-UTP intermediate are colored blue and red in panel A, respectively. CTP is colored red in panel B.  
GTP also plays an important role in the formation of the NH\({}_{3}\) tunnel. Upon simulating the removal of GTP from their structure, [PERSON] and co-workers found that there is a hole in the NH\({}_{3}\) tunnel adjacent to where GTP binds, as originally suggested by [PERSON] and co-workers [32]. This observation indicates that GTP can indeed prevent leakage of NH\({}_{3}\) from the ammonia tunnel and appears to account for why GTP inhibits the reaction with exogenous NH\({}_{3}\) when CTPS is covalently modified by DON, as shown in previous studies [36; 64; 86]. Interestingly, the NH\({}_{3}\) tunnel formed in GTP-bound _Dm_CTPS is narrower than that in CTPS bound with the products or some other substrates. Compared with carbamoyl-phosphate synthetase 2, which is also a class I Gln-dependent amidotransferase, there is an insertion of about eight amino acids between \(\beta\) sheets 5 and 6 in the GAT domain of CTPS, which has been referred to as the \"wing\" (residues 440-448 in _Dm_CTPS and 431-439 in _Ec_CTPS; L13 purple region in Figure 2) [66]. Many of the previous crystal and cryo-EM structures of CTPSs have shown that this wing is very flexible, making it very challenging to determine its precise structure and thereby limiting our understanding of its role in GTP-dependent activation.