"We have provided high-resolution structural images with a resolution of up to 2.9 angstroms, which have explained the doubts about Escherichia coli cytidine triphosphate synthetase, and provided ideas for the development of species-specific small molecule inhibitors and agonists targeting this enzyme, as well as the development of new antibiotics," said Professor Liu Jilun from the Shanghai University of Science and Technology.
In the study, he and his team demonstrated the 2.9 angstrom cryo-electron microscopy structure of Escherichia coli cytidine triphosphate synthetase fibers, along with Escherichia coli cytidine triphosphate, reduced coenzyme I, and covalent inhibitor 6-diazo-5-oxo-L-norleucine (DON).
They conducted a comprehensive analysis of its structure, providing an evolutionary perspective on the formation of Escherichia coli cytidine triphosphate synthetase fibers.
Through structural analysis and biochemical assays, the research group also revealed the synergistic inhibitory effect of reduced coenzyme I and adenine on Escherichia coli cytidine triphosphate synthetase.
Escherichia coli cytidine triphosphate synthetase is a potential drug target for many different diseases. The team's analysis indicates that alpha-helix 12 is a more characteristic feature of eukaryotic Escherichia coli cytidine triphosphate synthetase.
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It also indicates that when this helix is inserted into Escherichia coli cytidine triphosphate synthetase, it will form fibers in the substrate binding state like human Escherichia coli cytidine triphosphate synthetase.
In addition, the research group also obtained the cryo-electron microscopy structure of Escherichia coli cytidine triphosphate synthetase with covalently bound DON, Escherichia coli cytidine triphosphate, and reduced coenzyme I.
In terms of application prospects:
In the future, broad-spectrum inhibition can be carried out for prokaryotes, which will not affect eukaryotes, so new antibiotics can be designed.In addition, they have now generated high-resolution structural diagrams, which allows for activation or inhibition targeting specific species. At the same time, based on the characteristics of the metabolic enzyme cells known as "cell snakes," modifications and processing of the metabolic enzymes can be carried out. That is, by utilizing their ability to form cell snakes and fibers, the development of new materials can be expanded. Currently, several members of this laboratory are already conducting research in this area.
The term "cell snake" was first coined in biology, and after returning to the country, they actively embraced cryo-electron microscopy.
To elaborate, the main subject of this study is the structure of a metabolic enzyme in Escherichia coli, a synthetic enzyme. As previously mentioned, it is called Escherichia coli cytidine triphosphate synthase (CTPS, Cytidine triphosphate synthase). It is understood that deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are both long-chain molecules, both composed of a few nucleotides arranged in combinations.The four basic nucleotides that make up RNA are:
- Adenosine triphosphate (ATP);
- Uridine triphosphate (UTP);
- Guanosine triphosphate (GTP);
- Cytidine triphosphate (CTP).
CTPS is responsible for catalyzing the last step in the de novo synthesis of the nucleotide CTP, which is able to generate CTP from UTP and ATP as substrates.In this catalytic reaction, another nucleotide—guanosine triphosphate (GTP)—can exert a strong and effective regulatory effect on the aforementioned reaction. As a result, all four basic nucleotides can bind to CTPS.
Therefore, CTPS is also an enzyme that has been thoroughly studied in the field of biochemistry. Since its initial study in the 1950s, it has become one of the classic enzymes in biochemistry textbooks.
Through the study of CTPS, many classic concepts in biochemistry have been derived. Taking the concept of mutual regulation between enzymes and substrates as an example, CTPS is used as a paradigm.
Although there has been much biochemical research in this area, the academic community has not yet paid attention to the distribution of CTPS within cells.
In 2007, Jilong Liu began teaching at the University of Oxford and started using fruit flies as model organisms for research.
In May 2010, Jilong Liu reported in a paper that CTPS in fruit flies could form elongated structures.
Due to the structure's resemblance to a snake under an optical microscope, Jilong Liu named it "cytoophidium" (plural is "cytoophidia").
This term comes from Greek, where "cyto" means "cell," and "ophidium" means "snake," making the Chinese meaning of "cytoophidium" "cell snake."
Between July and August 2010, two other research groups from the United States reported that CTPS could form similar fibrous structures in bacteria and Saccharomyces cerevisiae (brewer's yeast).
Since then, more and more scientists have begun to focus on the following two scientific questions: How do metabolic enzymes like CTPS form cell snakes? What role do cell snakes play inside cells?Since Liu Jilun published the first Cytoophidium paper in 2010, the field of Cytoophidium has gradually entered its early stages.
By May 2024, searching for the keyword "cytoop*" (including the singular and plural forms of Cytoophidium in English) in the biomedical literature database PubMed yielded 95 papers, of which 62 papers came from Liu Jilun's research group and collaborators.
In 2016, Liu Jilun's laboratory moved from the University of Oxford to the Shanghai University of Science and Technology. After returning to China, an important direction for him and his team was to use cryo-electron microscopy technology to explore the near-atomic-level high-resolution structures of CTPS after binding with different ligands.
In 2016, Guo Chenjun entered the Shanghai University of Science and Technology as an undergraduate student, and Liu Jilun was his mentor at that time. In the second semester of his sophomore year, Guo Chenjun came to Liu Jilun's laboratory for an internship.
At that time, Liu Jilun assigned him a task: to use cryo-electron microscopy to understand the structure of Cytoophidium and metabolic fibers.
In 2019, the team discovered that the fruit fly CTPS could bind to substrates or products to form two different conformations of fibers, and published the first structural biology paper of the research group.
This also gave them a preliminary understanding of the power of cryo-electron microscopy, but the resolution obtained was still not good enough. At that time, their goal was to achieve a resolution of 4 angstroms.
In 2021, they resolved the structure of fruit fly CTPS binding with different ligands, with a resolution of 2.5 angstroms.
Previously, the team's research on fruit fly CTPS and some peers' research on yeast and E. coli CTPS held different views.
As a result, some people in the industry proposed that CTPS has different regulatory modes in prokaryotic and eukaryotic organisms.However, such an explanation did not seem perfect to Liu Jilun, so he wanted to be more meticulous and began to choose E. coli CTPS as the research subject, thus starting this study.
It has been more than 70 years, and the "treasure" of CTPS is still waiting to be unearthed.
In general, this study has three highlights, which explain the phenomena that have been discovered by biochemical methods before but could not be clearly explained.
The first highlight of this achievement is the combination of CTPS with the covalent inhibitor DON. That is, CTPS can covalently bind to an inhibitor called DON (DON is an analog of glutamine).When DON is combined with CTPS, one of the two structural domains of the enzyme can be locked.
As early as 1971, scholars found that after the prokaryotic CTPS combined with DON, it could still react with ammonia.
However, where does the ammonia enter the ammonia channel inside the enzyme structural domain? People have always been unclear about this issue.
Liu Jilun said: "We often say that seeing is believing. For metabolic enzymes, although the academic community has done a lot of biochemical experiments, if there is no structure, there will be a feeling of looking at flowers in the fog."
In this study, the team obtained the structure of E. coli CTPS combined with DON.
Due to the resolution reaching 2.9 angstroms in this study, they found that the structure has a hole, but it is different from the classic hole in the field.
Specifically: this hole can allow ammonia to enter the ammonia channel, and this channel can allow ammonia to diffuse to another structural domain for the reaction.
In other words, this study can explain from the structural aspect, how CTPS after DON binding, allows ammonia to enter the ammonia channel and how the reaction occurs.
The second highlight of this achievement is the combination of CTPS with CTP.
CTP is the reaction product of CTPS catalysis. This research group was the first to find that CTPS has two CTP binding sites in eukaryotic fruit flies. Other research groups found that there is only one CTP binding site in prokaryotes.The team has confirmed through this work that E. coli CTPS has only one binding site. The high-resolution electron density maps owned by the research group enable them to carry out the relevant calculations. This discovery reveals that the second binding site for eukaryotic CTP in E. coli CTPS is too small to accommodate a second CTP. The third highlight of this achievement is the binding of CTPS with reduced coenzyme I. In 2016, scholars found that reduced coenzyme I could bind to CTPS, but how and where it binds was still unclear. In this study, the team successfully resolved the binding structure of CTPS and reduced coenzyme I. During this process, they combined the three different ligands, DON, CTP, and reduced coenzyme I, with E. coli CTPS. This has achieved a high-resolution structure close to the atomic level, providing a structural basis for some unresolved key issues. In addition, they also compared the metabolic fiber interface of prokaryotic and eukaryotic CTPS from an evolutionary perspective. Through this, they found that the alpha-helix 12 in eukaryotic CTPS is missing in most species of prokaryotic CTPS.For this purpose, they also conducted some experiments and found that alpha-helices can indeed affect the fiber structure.
It is also reported that in this study, they also used AlphaFold2 to design a prokaryotic CTPS protein that fuses with human protein fragments.
Compared with traditional methods, AlphaFold2 is not only easy to use, but also provides more accurate results.
Finally, the related paper was published as a cover article in the academic journal mLife in the field of microbiology, titled "Filamentation and inhibition of prokaryotic CTP synthase with ligands"[1].
ShanghaiTech University doctoral student Guo Chenjun and undergraduate student Wang Zixuan are co-first authors, and Professor Liu Jilun from ShanghaiTech University serves as the corresponding author.
In addition, Liu Jilun said, "I believe that studying cellular snakes and metabolic fibers will provide many insights."
Guo Chenjun of the team also said, "CTPS is a treasure trove, although it has been studied for more than 70 years, there are still many key issues that have not been accurately answered."
Subsequently, they plan to further explore the mechanism and use existing knowledge to design some specific inhibitors and agonists with practical value.
