Original researchDrosophila CTP synthase can form distinct substrate- and product-bound filaments
Introduction
Intracellular compartmentation appears to involve regulatory machineries that adjust various protein properties. In past decades, oligomerization and higher order aggregates have been demonstrated as a common feature of many metabolic enzymes, including acetyl-CoA carboxylase (Kleinschmidt et al., 1969; Hunkeler et al., 2018), glutamine synthase (Petrovska et al., 2014), inosine monophosphate dehydrogenase (Ji et al., 2006), CTP synthase (CTPS) (Ingerson-Mahar et al., 2010; Liu, 2010; Noree et al., 2010; Carcamo et al., 2011; Chen et al., 2011), and more unveiled by the budding yeast-based genome-wide survey (Noree et al., 2010; Shen et al., 2016).
CTPS has been studied biochemically for almost 70 years. This enzyme contains an N-terminal ammonia ligase (AL) domain and a C-terminal glutamine amidotransferase (GAT) domain (Weng and Zalkin, 1987). It catalyzes the rate-limiting step of de novo CTP synthesis, in which a UTP is converted into a CTP with the consumption of ATP and ammonium, which is derived from the hydrolysis of glutamine by the GAT domain (Lewis and Villafranca, 1989). Because the biochemical properties and the regulatory mechanisms of CTPS were thoroughly studied in the past decades, it came as a surprise when a new feature of CTPS was revealed in 2010. CTPS can form a filamentous macrostructure, also termed the cytoophidium, in many organisms across biological kingdoms, implying that the underlying significance of polymerization is conserved through evolution (Ingerson-Mahar et al., 2010; Liu, 2010; Noree et al., 2010; Chen et al., 2011; Gou et al., 2014; Zhang et al., 2014; Daumann et al., 2018).
In 2014, several groups tried to understand whether and how the filamentation of CTPS affects the metabolism of CTPS, specifically with regard to its enzymatic activity (Aughey et al., 2014; Barry et al., 2014; Noree et al., 2014; Strochlic et al., 2014). However, several different views were derived from these studies, as follows: 1) A study in Escherichia coli suggested that tetramers are the basic units of the CTPS filaments/cytoophidia, although the conformation of tetramers changed into an inactive form in the filaments. 2) Dimers are considered to be the basic units of CTPS filaments/cytoophidia in yeast, fruit fly, and human, and CTPS is also in an inactive state. 3) An additional study in humans showed that filamentation of CTPS can increase the enzymatic activity. Obviously, the conclusions of these studies are inconsistent, which prompted us to further investigate the structure of the filamentous form of CTPS.
Previous studies have shown that E. coli CTPS tetramers (ecCTPS) pile up to form filaments while bound with CTP. The interaction between tetramers within the polymer is suggested to maintain ecCTPS in an inactive state, and the mutations that interfere with ecCTPS polymerization disrupt E. coli growth and metabolic regulation (Barry et al., 2014; Lynch et al., 2017). Surprisingly, the opposite scenario has been observed in human CTPS1 (hCTPS1) recently. Although the hCTPS1 polymer is built up by tetramers in a similar fashion, hCTPS1 molecules that comprise the filament are in the substrate-bound state rather than the CTP-bound state and may enhance hCTPS1 catalytic activity, suggesting differences in the regulation of CTPS polymers between species (Lynch et al., 2017). The authors concluded that the conformation of CTPS filaments in prokaryotes and eukaryotes differs, with the filaments in prokaryotes being inactive whereas in eukaryotes they are activated.
For our research, we decided to investigate the structure of Drosophila melanogaster CTPS (dmCTPS). The fruit fly serves as an excellent model for CTPS cytoophidium study, as cytoophidia have been reported as dynamically present in many tissues of D. melanogaster depending on the developmental stage and metabolic status (Liu, 2010; Aughey et al., 2014; Strochlic et al., 2014; Wang et al., 2015; Wu and Liu, 2019). However, lack of understanding of the structural basis of Drosophila CTPS has obstructed investigation into its physiological importance.
Section snippets
Drosophila CTPS can form both substrate-bound and product-bound filaments
Our initial aim was to determine whether CTPS in Drosophila is capable of forming filaments in the presence of products or substrates. In fact, we found by negative staining under a 120 kV transmission electron microscope that there were filaments present in both states, whereas no CTPS filaments were observed in the absence of nucleotides (Fig. 1, Fig. 3A). This result cannot be explained by the previous model.
Structure of substrate-bound CTPS filaments
We used cryo-electron microscopy (EM) for structural analysis, which showed that the
Discussion
Through our work, we have found that CTPS in Drosophila, as in a eukaryotic organism, can form substrate-bound and product-bound filaments. Our study indicates that cytoophidia in two different conformations can coexist in the same species. In addition, it is possible that a single cytoophidium could accommodate dmCTPS in both states.
These results challenge a previous model that “the inactive product-bound conformation is stabilized in bacterial filaments, while the active substrate-bound
dmCTPS expression and purification
The full-length Drosophila wild-type and H355A dmCTPS sequences were fused with a C-terminal 6× His-tag and driven by T7 promoter. Wild-type dmCTPS and dmCTPSH355A constructs were transformed into DH5α cells and Transetta E. coli cells, respectively. The expression of dmCTPS was induced by incubation with 1 mM IPTG at 16 °C overnight. Cells were collected and pelleted by centrifugation at 4000 rpm for 20 min. Cell pellets were subsequently resuspended in cold lysis buffer containing 500 mM
Acknowledgments
This work was supported by grants from the National Natural Science Foundation of China (No. 31771490) to J.-L.L. We thank Kang Ding, Dijun Du, Brenda Gonzalez, Yu Guo, Wen Jiang, Feng Song, Xinzhen Zhang and Suwen Zhao for technical assistance, and the Cryo-EM Imaging Facilities at ShanghaiTech University and the National Center for Protein Science Shanghai (NCPSS) for providing the imaging facility.
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These authors contributed to this work equally.