## 7 A Model for GTP-Dependent Regulation of CTPS  
With the report of the GTP-bound structure of _Dm_CTPS [66], combined with the results from the site-directed mutagenesis and kinetics studies described in Sections 2 and 3, a more comprehensive model for the role of GTP in regulating CTPS catalysis emerges. While the model described here incorporates features observed for CTPSs from different organisms, subtle differences likely exist between various CTPSs. As [PERSON] and co-workers noted, the  
Figure 7: Atomic model of interaction between GTP and _Dm_CTPS. (**A**) _Dm_CTPS and GTP-Mg\({}^{2+}\) are shown in space-filling representation from the cryo-EM structure solved by [PERSON] and co-workers [66]. Shown in stick representation are residues implicated in GTP-dependent activation of CTPS, which are highlighted in light green (L4), dark green (L11), and purple (L13 or “wing”) (_cf._ coloring in Figure 2), and orange (gate region). (**B**) GTP-binding pocket of _Dm_CTPS is depicted by a transparent surface. GTP and DON are shown in ball and stick representation, as are residues that interact with GTP or play a role in GTP-dependent conformational effects: F50, H55, L107, F373, R376, L444, M448, R429, and R481 (colored as in Figure 2). The Mg\({}^{2+}\) ion is shown as a light green sphere in both panels. initial binding of UTP and ATP effects the open-to-closed state transition that alters the conformation of the GTP-binding cleft to facilitate GTP binding [66]. Concomitantly, ATP and UTP binding cause a rotation of the GAT and synthase domains to align their active sites for formation of the NH\({}_{3}\) tunnel [56]. In _LI_CTPS, [PERSON] and co-workers proposed that GTP binding not only closes a lid over the active site to effect proficient hydrolysis of Gln, but there is also concomitant formation of the 4-phospho-UTP intermediate, which causes allosteric activation of the glutaminase activity [36]. This reciprocal activating effect of GTP and the intermediate is consistent with the observed presence of the 4-phospho-UTP intermediate in the GTP-bound structure of DON-modified _Dm_CTPS [66]. Within the GAT domain, a series of interactions contribute to altering the conformation of the GAT domain. Gln binding orders Phe 373 (_Dm_CTPS numbering, 353 in _E_c_CTPS) of the L11 lid to afford an additional stabilizing interaction with bound GTP. In addition to interacting with GTP, Leu 107 (109 in _E_c_CTPS), from the L4 region of an adjacent protomer, interacts with Leu 444 (435 in _E_c_CTPS) located in the unique wing structure (L13) in the GAT domain to propagate the conformational change accompanying GTP binding. Arg 376 (Arg 359 in _L_c_CTPS) interacts with GTP and appears to act as a lever to alter the conformation of the lid L11 to favor Gln hydrolysis [84]. The interaction between Phe 50 and GTP appears to play a role in decreasing the flexibility of the L2 region, thereby ensuring the integrity of the NH\({}_{3}\) tunnel while GTP is bound. The hole from which the nascent NH\({}_{3}\) could be lost to bulk solvent is effectively \"capped\" by the bound GTP, ensuring the fidelity of the transfer of NH\({}_{3}\) to the synthase domain. Thus, GTP not only promotes the hydrolysis of Gln through its multiple interactions with the GAT domain of CTPS, but also participates in the assembly and maintenance of the NH\({}_{3}\) tunnel, and thereby coordinates the reactions catalyzed by the GAT and synthase domains of the enzyme. Although direct experimental evidence on the specific mechanism for GTP-dependent regulation of catalysis at the GAT domain of CTPS is still lacking, we posit that the GAT domain in CTPS forms a unique regulatory system around GTP binding that is both efficient and sensitive. [PERSON], [PERSON] and [PERSON] wrote, reviewed, and edited the paper. Funding acquisition, S.L.B. and J.-L.L. All authors have read and agreed to the published version of the manuscript. This work was funded by the Ministry of Science and Technology of the People's Republic of China (grant no.