Researchers create first maps of two melatonin receptors essential for sleep

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An international team of researchers has used an X-ray laser at SLAC, the National Accelerator Laboratory, of the Ministry of Energy, to create the first detailed maps of two melatonin receptors that tell our body when to sleep or get up and who lead other biological processes. A better understanding of how they work could enable researchers to design better drugs to combat sleep disorders, cancer and type 2 diabetes. Their findings were published in two articles today in Nature.

The team, led by the University of Southern California, used the X-ray of SLAC's coherent Linac light source (LCLS) to map the receptors, MT1 and MT2, bound to four different compounds that activate them: a Insomnia drug, a mixture melatonin with antidepressant serotonin and two melatonin analogues.

They discovered that both melatonin receptors contain narrow channels anchored in the fat membranes of our body cells. These channels only allow melatonin – which can exist in both water and fat – to pass through, blocking serotonin, which has a similar structure but is only available in aqueous environments. They also discovered how much larger compounds could only target MT1 and non-MT2, despite the structural similarities between the two receptors. This should inform the design of drugs that selectively target MT1, which has been difficult so far.

"These receptors perform extremely important functions in the human body and are major therapeutic targets of great interest to the pharmaceutical industry," said Linda Johansson, a USC postdoctoral researcher, who said directed the structural work on MT2. "Through this work, we have been able to gain a very detailed understanding of how melatonin is able to bind to these receptors."

Time to go to bed

People do it, birds do it, fish do it. Almost all living things in the animal kingdom sleep, and for good reason.

"It is essential for the brain to rest, process and store the memories we have accumulated during the day," said co-author Alex Batyuk, a scientist at SLAC. "Melatonin is the hormone that regulates our sleep-wake cycles. When it's daylight, melatonin production is inhibited, but when darkness comes in, it's the signal that our brain sleeps. "

Melatonin receptors belong to a group of membrane receptors called G protein-coupled receptors (GPCRs), which regulate almost all the physiological and sensory processes of the human body. MT1 and MT2 are present in many parts of the body, including the brain, retina, cardiovascular system, liver, kidneys, spleen, and intestine.

These receptors monitor our clock genes, the timekeepers of the body's internal clock or circadian rhythm. In a perfect world, our internal clocks would synchronize with sunrise and sunset. But when people travel through time zones, work at night or spend too much time in front of screens or other artificial sources of blue light, these timekeepers are shaken.

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<img src = "https://3c1703fe8d.site.internapcdn.net/newman/csz/news/800/2019/5cc0256617416.jpg" alt = "The first maps of two melatonin receptors essential for sleep” title=”Overall view of the MT2 structure. Credit: Nature (2019). DOI: 10.1038 / s41586-019-1144-0″/>

Control the rhythm

When our circadian rhythms are disrupted, this can lead to psychiatric, metabolic, oncological and many other disorders. MT1 in particular plays an important role in controlling these rhythms, but the design of drugs capable of selectively targeting this receptor has proven difficult. Many people take over-the-counter melatonin supplements to combat sleep problems or change their circadian rhythm, but the effects of these medications often disappear within hours.

By deciphering these receptors' plans and mapping how the ligands bind to and activate them, the researchers urged others to design safer, more effective drugs that can selectively target each receptor.

"Since the discovery of melatonin 60 years ago, many significant discoveries have led to this moment," said Margarita L. Dubocovich, professor emeritus of pharmacology and toxicology at the University of Buffalo, who was pioneering in the identification of functional receptors for melatonin. in the early 1980s and provided an outside perspective on this research. "Despite remarkable progress, the discovery of selective MT1 drugs remains challenging for my team and researchers around the world.The elucidation of the crystal structures of the MT1 and MT2 receptors opens a new exciting chapter for the development of drugs for the treatment sleep or circadian stress known to cause psychiatric, metabolic, oncological and many other conditions. "

Harvesting crystals

To map biomolecules such as proteins, researchers often use a method called X-ray crystallography, scattering X-rays of crystallized versions of these proteins and using the patterns thus created to obtain a three-dimensional structure. Until now, the challenge of mapping MT1, MT2 and similar receptors was the difficulty of growing crystals of sufficient size to obtain high resolution structures.

"With these melatonin receptors, we really had to make an extra effort," said Benjamin Stauch, who led the structural work on MT1. "Many people had tried to crystallize them without success, so we had to be a little inventive."

A key element of this research was the unique method used by researchers to grow their crystals and collect X-ray diffraction data from them. For this research, the team expressed these receptors in insect cells and extracted them with the help of a detergent. They mutated these receptors to stabilize them, allowing crystallization. After purifying the receptors, they placed them in a membrane-type gel, which promotes the growth of crystals directly from the membrane environment. After obtaining microcrystals in suspension in this gel, they used a special injector to create a narrow flow of crystals that they zapped with LCLS X-rays.

"Because of the tiny size of the crystals, this work could only be done by LCLS," said Vadim Cherezov, a professor at USC, who oversaw both studies. "Such small crystals diffract badly from the sources of the synchrotron because they are quickly damaged by radiation, and X-ray lasers can solve the problem of radiation damage by the principle of" diffraction before destruction. "

The researchers collected hundreds of thousands of scattered X-ray images to understand the three-dimensional structure of these receptors. They also tested the effects of dozens of mutations to deepen their understanding of how receivers work.

In addition to discovering tiny channels of melatonin control in receptors, researchers were able to map the mutations associated with type 2 diabetes on the MT2 receptor, first discovering the exact location of these mutations in the receiver.

Lay the foundation

In these experiments, the researchers only examined the compounds that activate the receptors, called agonists. To follow, they hope to map receptors linked to antagonists, which block receptors. They also hope to use their techniques to study other GPCR receptors in the body.

"As a structural biologist, it was exciting to see the structure of these receptors for the first time and to analyze them to understand how these receptors selectively recognize their signaling molecules," Cherezov said. "We have known them for decades, but until now no one could tell what they looked like, we can now analyze them to understand how they recognize specific molecules, which we hope will throw the basics for better, more effective medicines. "