Nature | Xu Huaqiang/Zhang Yan/Jiang Yi collaborated to reveal the drug action of the neurotransmitter serotonin receptor and the molecular mechanism of phospholipid regulation

Editor | Xi In our brain, there is a happy neurotransmitter-serotonin (also known as serotonin, 5-HT), which helps us feel pleasure, relaxation and self-confidence, and makes us feel satisfied and happy.

Serotonin can regulate our cognition and emotions.
When its content increases, we feel happy; when its content decreases, we feel anxious, depressed, and even depression.

Most of the physiological functions of serotonin are mediated by G protein-coupled receptors (GPCR) on the cell membrane.
In the human body, there are 12 kinds of serotonin G protein-coupled receptors.
These receptors are used to treat schizophrenia.
It is an important drug target for diseases such as disease, depression, migraine and so on.

Scientists have long been committed to the structure and function of serotonin family receptors, but due to technical difficulties, the structure of most serotonin family receptors has not been resolved.

At present, there are three major scientific problems in the research on the functional mechanism of serotonin family receptors.
First, serotonin receptors are regulated by phospholipids and cholesterol on the cell membrane like other membrane receptors, but they are not yet available.
It is clear how these molecules regulate their physiological functions; second, many serotonin receptors have constitutive activation, that is, there is still a certain activity without agonist binding, but the activation mechanism is unknown; third, as more The molecular mechanism of the binding of serotonin receptors with different drugs is still unknown.

On March 24, 2021, the team of Xu Huaqiang/Jiang Yi from the Shanghai Institute of Materia Medica, Chinese Academy of Sciences, and the team of researcher Zhang Yan from Zhejiang University, jointly published the latest research results in the journal Nature.
Structural insights into the lipid and ligand regulation of serotonin receptors, this work has successfully achieved multiple technological breakthroughs in this field, successfully resolved the five structures of three different types of serotonin receptors, and answered the above three scientific questions.

This work reveals how phospholipid PI4P (PtdIns4P) and cholesterol regulate receptor functions, and the molecular mechanism of how the drug Aripiprazole recognizes serotonin receptors.

Aripiprazole is a first-line drug used clinically to treat schizophrenia, and it is also used to treat depression, bipolar disorder, autism and other important mental diseases.

The research team used single-particle cryo-electron microscopy technology to analyze for the first time the complex structure of a total of 5 serotonin receptors and Gi protein (inhibitory G protein) (2.
9-3.
1 angstroms) with near-atomic resolution (Figure 1): respectively It is the structure of the 5-HT1A receptor in the state of unbound ligand, the structure of the 5-HT1A receptor that binds to the endogenous small molecule ligand 5-HT, the structure of the 5-HT1A receptor that binds to the drug aripiprazole, and 5 -HT 5-HT1D receptor structure and 5-HT1E receptor structure that binds to the selective small molecule agonist BRL-54443.

Figure 1.
Cryo-EM structure of 5-HT1A, 5-HT1D, and 5-HT1E receptors with ligands and Gi protein complexes.
This study was the first to find phospholipids at the interface of 5-HT1A receptor complex receptors and Gi protein.
And identify the molecule as PtdIns4P.

The two fatty chains of 14-16 carbon atoms in the PtdIns4P structure form a wide range of hydrophobic interactions with the receptor transmembrane helices TM6 and TM7 and the receptor's eighth helix H8.
The polar head is inserted into the receptors MT3 and TM6.
And the pocket formed by the α5 helix of TM7 and the α subunit of Gi protein (Figure 2a).

The researchers then used the peptidisc technology to find that a variety of cell membrane lipids have different degrees of G protein activation activity, of which PtdIns4P has the most significant effect on G protein activation (Figure 2b).

At the same time, PtdIns4P can play a positive allosteric regulatory role.

In addition to ptdIns4P, the researchers found at least 10 cholesterol molecules at the interface between the receptor and the cell membrane, of which 2 cholesterol molecules sandwiched a fatty chain of PtdIns4P to form a "sandwich"-like spatial structure (Figure 2c), and verified Cholesterol molecule plays a key role in the regulation of receptor function.

Figure 2.
Phospholipid and cholesterol regulation of the 5-HT1A receptor.
In the structure of the 5-HT1A receptor where the ligand is not bound, the researchers found the density of multiple water molecules in the ligand's forward binding pocket.

Some water molecules are arranged on a plane similar to the structure of 5-HT and form stable hydrogen bond interactions with the receptor, including the conserved hydrogen bonds in the binding of 5-HT to the receptor (Figure 3).

The researchers further used molecular dynamics simulations to confirm that the water molecule can simulate the formation of hydrogen bonds between the endogenous ligand 5-HT and the receptor, thereby mediating the constitutive activation of the 5-HT1A receptor.

Figure 3.
Regulation of constitutive activation of 5-HT1A receptor by water molecules.
In addition, this study provides an important structural basis for revealing the selectivity of the anti-schizophrenia and antidepressant drug aripiprazole.

As a highly selective ligand for 5-HT1A receptors, aripiprazole is 10-1000 times weaker than 5-HT1B, 5-HT1D and 5-HT1E receptors [1].

The study found that the extracellular end of TM7 that binds to the 5-HT1A receptor of aripiprazole is 3 angstroms out of other low-selectivity receptor subtypes, thereby forming a relatively larger ligand binding pocket to accommodate aripiprazole.
The quinolinone group of the azole (Figure 4c).

At the same time, the researchers discovered that a cholesterol molecule bound around the 5-HT1A receptor is involved in the formation of the aripiprazole ligand binding pocket and the maintenance of the TM1 and TM7 conformations near the quinolinone group of the ligand.

Together, these findings revealed the highly selective mechanism of aripiprazole for 5-HT1A receptors (Figure 4d).

Figure 4.
The molecular mechanism of 5-HT1A, 5-HT1D, and 5-HT1E receptor ligand recognition selectivity.
In summary, this study analyzed for the first time the 5-HT1A receptors that were not bound to ligands, as well as 5-HT1A, 5-HT1A and 5-HT1A receptors.
Five near-atom resolution cryo-electron microscopy structures of HT1D and 5-HT1E receptors and small molecule ligands or therapeutic drugs and their Gi protein complexes.

Combined with structure-based functional analysis, this study expounds for the first time the key role of PtdIns4P and cholesterol molecules in 5-HT receptor ligand recognition and G protein coupling, and reveals the constitutive activation mechanism of 5-HT receptor.
And the molecular mechanism by which ligands, including the anti-schizophrenia drug aripiprazole, selectively recognize 5-HT receptor subtypes.

Due to the importance of the neurotransmitter serotonin, Xu Huaqiang's group at the Shanghai Institute of Materia Medica, Chinese Academy of Sciences has long been committed to the structure and function of serotonin receptors.

In 2013, Xu Huaqiang's team used X-ray crystallography to analyze the structure of serotonin receptor 1B combined with anti-migraine drugs for the first time [2].

In 2018, the Xu Huaqiang/Jiang Yi team analyzed the crystal structure of serotonin receptor 1B binding inverse agonist for the first time [3].

Combined with the cryo-EM structure of the five serotonin receptors published by the team on Nature this time, it will greatly enrich our understanding of the structure and function of the serotonin system, and provide important information for the development of drugs for the serotonin system.
Structural basis.

In addition, Xu Huaqiang's team and Zhang Yan's team have recently made important progress in the structure and function of another important neurotransmitter dopamine and its receptor.
In 2021, the joint team will continue to conduct research in Cell, Molecular Cell and Cell Research.
The magazine published the cryo-EM structure of dopamine receptors D1R, D2R and D3R (see BioArt report for details: Mol Cell | Xu Huaqiang/Zhang Yan/Cheng Xi, etc.
, cooperated to reveal the structural basis of dopamine receptor D3 ligand selectivity and activation of Gi; Cell | Xu Huaqiang/Zhang Cheng/Zhang Yan collaborate to reveal the mechanism of activated dopamine receptor D1R and D2R ligand selectivity and G protein selectivity; Cell Research | Xu Huaqiang/Zhang Yan and others collaborate to reveal the binding of dopamine receptor D1R to dopamine Characteristics and potential allosteric regulation mechanism) [4-6], revealing the mechanism of action and allosteric regulation of dopamine receptors and drugs.

These results provide a systematic structural model for future drug design for central nervous system diseases.

This project was developed by the research team of Xu Huaqiang/Jiang Yi of Shanghai Institute of Materia Medica, Chinese Academy of Sciences and Zhang Yan team of Zhejiang University.
It was completed with the assistance of Professor Gloriam, the team of Professor Karsten Melcher from the Wen'anluo Institute in the United States and Professor Carol V.
Robinson from the University of Oxford in the United Kingdom.

Shanghai Institute of Materia Medica and Zhejiang University School of Basic Medicine jointly trained doctoral student Xu Peiyu, Shanghai Institute of Materia Medica and Shanghai University of Science and Technology jointly trained doctoral student Huang Sijie, Zhejiang University School of Basic Medicine doctoral student Zhang Huibing and postdoctoral student Mao Chunyou (now Shao, affiliated to Zhejiang University School of Medicine) Researcher of Yifu Hospital), X.
Edward Zhou of Wen'anluo Institute of the United States and associate researcher of Shanghai Institute of Materia Medica Cheng Xi are the co-first authors of this article.

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