Open the "black box" of glycoprotein hormone mechanism

Open the "black box" of glycoprotein hormone mechanism

Open the "black box" of the mechanism of glycoprotein hormones Open the "black box" of the mechanism of glycoprotein hormones

■Tian Ruiying, trainee reporter of our newspaper

■Our newspaper trainee reporter Tian Ruiying ■Our newspaper trainee reporter Tian Ruiying

Glycoprotein hormones are key drugs for assisted reproduction and treatment of thyroid diseases
.

In recent decades, although the clinical application of glycoprotein hormone has achieved great success, how it activates the receptor mechanism in human cells is a "black box" that has been difficult for researchers to open for a long time

In a study published in "Nature" on September 22, the Shanghai Institute of Materia Medica, Chinese Academy of Sciences (hereinafter referred to as Shanghai Institute of Materia Medica) researchers Xu Huaqiang, Jiang Yi, Jiang Hualiang, etc.
joined Zhejiang University professor Zhang Yan team, Peking Union Medical College Hospital Chang Zhang Shuyang's team opened this "black box"
.

They analyzed for the first time the glycoprotein hormone receptor, that is, the four structures of the full-length luteinizing hormone/chorionic gonadotropin receptor (LHCGR) in the inactive state and multiple activated states, and how the glycoprotein hormone recognizes and The activation of its receptors provides detailed molecular mechanisms that will help clinical development of small molecule drugs that replace hormone therapy

The receptor structure in the "activated state" from the beginning

The receptor structure in the "activated state" from the beginning

Glycoprotein hormones are mainly divided into two categories
.

One type is gonadotropin, including luteinizing hormone (LH), follicle stimulating hormone (FSH) and chorionic gonadotropin (CG); the other is thyroid stimulating hormone (TSH)

The reason why the structure of glycoprotein hormone receptor is difficult to understand is closely related to its particularity
.

The glycoprotein hormone receptor is a type A G protein-coupled receptor (GPCR), but unlike most type A GPCRs, it contains a huge N-terminal extracellular domain (ECD) composed of 340 to 420 amino acids

Xu Huaqiang told the China Science News: “Because of its special structure, glycoprotein hormone receptors are difficult to express recombinantly
.

In addition, in addition to the traditional transmembrane region, it also has a large extracellular region, which also causes its expression to be very different.

Obtaining full-length hormone receptors in vitro can be described as difficult
.

Duan Jia, the first author of the paper and a doctoral student at the Shanghai Institute of Pharmaceutical Sciences, told the China Science News: “The first problem facing us is CG ligands

When checking information on the Internet, she was pleasantly surprised to find that the hospital had ready-made CG prescription drugs, "If you use prescription drugs, it may be effective
.

" Subsequent experiments proved this conjecture, and they then solved the problem of CG ligands

However, the amount of protein prepared by conventional methods is not only small, but the result is also unstable
.

By consulting the literature, Duan Jia found that there are many point mutations in the glycoprotein hormone receptor, and these mutations can cause a series of diseases

With the "blessing" of single-particle cryo-electron microscopy technology, the research team finally analyzed the structure of 3 full-length LHCGR with near-atom resolution in an activated state, including the LHCGR (wild-type) receptor structure that binds to the endogenous hormone CG ( 4.
3 Angstroms), LHCGR (containing persistent activating mutation S277I) receptor structure (3.
8 Angstroms) that binds endogenous hormone CG and LHCGR (containing persistent activating mutation S277I) receptor structure that binds endogenous hormone CG and small molecule compound Org43553 (3.
2 Angstroms)
.

The "deactivated state" that cannot be absent

The "deactivated state" that cannot be absent

Solving the mystery of the full-length LHCGR structure in the activated state is only the first step
.
"Many phenomena are not enough to analyze the structure in the activated state
.
" Duan Jia said, "In the past, when analyzing GPCRs, the interaction between the ligand and the transmembrane region of the receptor caused a conformational change in the transmembrane region, and then Activate the receptor
.
But for the activation of the glycoprotein hormone receptor, the ligand only interacts with the extracellular domain of the receptor, and can still cause a conformational change in the transmembrane region without contacting the transmembrane region of the receptor
.
"

This means that only by continuing to analyze the structure of the glycoprotein hormone receptor in the inactivated state can the activation mechanism of this type of receptor be fully revealed
.
Although the analytical work in the activated state laid an important foundation for subsequent research, it still encountered many difficulties
.

"Before this study, there was no report on the cryo-EM structure of a separate GPCR
.
The glycoprotein hormone receptor is very unstable and may be degraded during the purification process,
" said Duan Jia
.

The team tried many methods, but the effect was not obvious
.
They thought that maybe adding small molecule inhibitors in the purification process could make the protein more stable
.
In this way, the team overcomes the technical problem of highly unstable conformation of the full-length inactivated receptor, and by expanding the amount of data, finally analyzed the electron microscope structure of the full-length LHCGR in the inactivated state with a resolution of 3.
8 angstroms
.

By comparing the activated LHCGR structure, the team found that the receptor's ECD was deflected by about 45 degrees
.
Further through structural analysis and functional test verification, they finally proposed a "push-pull" model of glycoprotein hormone receptor activation
.
"This is the first electron microscope structure of a full-length single GPCR
.
" Xu Huaqiang said
.

Assist in the development of small molecule drugs

Assist in the development of small molecule drugs

In recent years, the research and development of small molecule drugs has gradually shown great advantages, and it has become more and more favored by patients and the market
.
Xu Huaqiang said: "On the one hand, small molecule drugs can be taken orally, which reduces the physical pain and psychological burden of patients; on the other hand, small molecule drugs are more controllable and more complying, and can be quantified regularly
.
"

To this end, the researchers also analyzed the molecular details of the interaction between the small molecule compound Org43553 and LHCGR in the phase 1 clinical trial, and revealed the binding pocket of Org43553, which is a selective small molecule drug replacement for the clinical development of LHCGR, FSHR and TSHR.
Hormone therapy provides an important structural template
.

In fact, there has been a long history of attention to glycoprotein hormones at home and abroad
.
Ten years ago, Xu Huaqiang's team began to study the structure of glycoprotein hormone receptor, but limited by the structure analysis technology at that time, the research was only carried out for two years and was forced to shelve
.

Two years ago, Xu Huaqiang's team and collaborators restarted the project
.
In his opinion, the success of the research is not only due to the advancement of cryo-electron microscopy technology, but also from the technical and platform support of the Shanghai Institute of Medicine and the sincere cooperation of teams from different directions
.

"The structural analysis of the glycoprotein hormone receptor is only the first step, and it is also the most critical step
.
Although the mechanism of glycoprotein hormone is common to the entire family, there is also a specificity with the receptor, and structural biology is still needed to answer.

In addition, we also hope to further analyze the structure of receptors such as FSH and TSH, reveal the molecular mechanisms of diseases such as infertility, hyperthyroidism or hypothyroidism, and provide a structural basis for the development of drugs for the treatment of related diseases
.
" Xu Huaqiang said
.

Related paper information:

Related paper information:

https://doi.
org/10.
1038/s41586-021-03924-2

https://doi.
org/10.
1038/s41586-021-03924-2