Subtle structural changes in virus ‘main protease’ over a range of temperatures may point to new targets for antiviral drugs


Scientists studying a COVID-19 coronavirus enzyme in temperatures ranging from the cold to the hot of the human body have discovered subtle structural changes that offer clues to how the enzyme works. The findings, published in IUCrJthe journal of the International Union of Crystallography, could inspire the design of new drugs to counter COVID-19 – and possibly help prevent future coronavirus pandemics.

“No previous study has examined this important coronavirus enzyme at physiological (or body) temperature,” said Daniel Keedy, a structural biologist at the City University of New York (CUNY), who conducted the study in collaboration with US Department scientists. from Brookhaven National Energy Laboratory.

Most structures to date come from frozen samples, far from the temperatures at which molecules operate in living cells. “If you work at physiological temperature, you should get a more realistic picture of what happens during an actual infection, because that’s where the biology happens,” Keedy said.

Additionally, he added, the team used temperature as a tool. “By turning this knob and seeing how the protein reacts, we can learn more about its mechanics – how it works physically.

Decipher the structure of Mpro

The protein in question is the major protease (Mpro) of SARS-CoV-2, the virus that causes COVID-19. Like all proteases, it is an enzyme that cuts other proteins. In many viral infections, including COVID-19, infected cells initially produce the functional proteins of a virus as a single chain of connected proteins. Proteases cut the pieces so individual proteins can make and assemble into new copies of the virus. Finding a drug to deactivate Mpro could curb COVID-19.

To study the structure of the enzyme, the researchers used a technique called X-ray crystallography at Brookhaven Lab’s National Synchrotron Light Source II (NSLS-II). NSLS-II is a DOE Office of Science user facility that produces light beams of X-rays. Shining these X-rays on a crystallized sample of a biological molecule can reveal the three-dimensional arrangement of the atoms that make up the molecule.

Studying samples that are not frozen can be a challenge.

“The higher the temperature, the more likely the X-rays are to damage the crystal,” explained study co-author Babak Andi, who operates NSLS-II’s Frontier Macromolecular Crystallography (FMX) beamline.

“To minimize damage, we rotate and move the crystal in a linear fashion as it moves through the x-rays. This distributes the x-ray dose along the length of the crystal,” he said. -he declares.

He noted that the small size of the X-ray beam at NSLS-II helps keep the beam focused on even the smallest dimension of the crystal – an edge measuring 10 to 20 millionths of a meter or less – as it rotates.

“Additionally, the FMX detector and other systems work so quickly that we can collect a full dataset in just 10-15 seconds per sample, with quality good enough to resolve a structure before significant X-ray damage occurs. do not occur.”

From frozen to physiological

Scientists used FMX to collect the first-ever set of Mpro crystallographic data at five different temperatures, ranging from cryogenic (-280 degrees Fahrenheit) to what is often referred to as “room temperature” in X-ray crystallography (~39 ° F ) to physiological (98°F). They also studied the crystal at room temperature under high humidity. Then they fed the data into a special type of computer simulation to identify many possible arrangements at the atomic level under each set of conditions.

The results revealed subtle conformational changes, including increased flexibility in parts of the protein at higher temperatures. The team also observed some unique characteristics of the enzyme under physiological conditions.

Most of the changes were not directly in the enzyme’s “active site” – the part that is directly involved in cutting other proteins. Instead, they were in parts of the enzyme farther from this location. But the data suggests that these remote sites could affect the active site through some sort of remote control mechanism common in biological systems, Keedy said. Disabling even these remote locations could potentially block the function of the enzyme.

“You can think of Mpro as a sort of folded ribbon, made up of two identical halves (forming dimers) that bind together symmetrically, much like a handshake,” Keedy said. The center of this handshake region (the “dimer interface”) binds to the active site via a flexible loop region of the protein.

As Keedy explained, the scientists found that at higher temperatures “the grip of the ‘handshake’ changes – both components readjust their grip a bit. This tells us that when the virus infects us , there may be some sort of communication through this loop between the dimer interface and the active site,” Keedy said.

The path to drug design

“We see subtle changes in the structure of this study, but drug design depends on subtle changes – less than a billionth of a meter here, less than a billionth of a meter there,” Keedy noted.

Other studies have shown that small drug-like molecules box bind to the enzyme at some remote locations identified in this new work.

“If we could perfect these molecules, optimize them, engineer them, modify them, we could potentially have a new foothold to alter the function of the enzyme – not at the active site, as virtually all antivirals currently target this protein, but at a different site via a different mechanism,” Keedy said. “Our findings inspired the exploration of this idea.”

The role of water

Exploring the enzyme at high humidity also mimics physiological conditions in water-filled cells – and can provide additional clues to guide drug design.

“For these studies, after selecting the crystal we want to study, we put a special sleeve on it to prevent it from drying out,” said Babak Andi of NSLS-II. “Then when we place the sample in the beamline for X-ray data collection, we remove the sleeve and blow a continuous stream of air at 99.5% humidity over the crystal while we collect the data.”

The results revealed specific water molecules that bind loosely to the enzyme, including one near the active site, only under high humidity conditions.

“These water molecules give you a clue where inhibitors can bind,” Andi said.

His group investigated a range of potential drug molecules that appear to replace loosely bound water molecules when they bind near the active site of Mpro. This work is reported in an article recently published in Scientific reports from the Nature magazine portfolio.

Both scientists were grateful for the collaborative spirit of the entire team, which included other scientists from Brookhaven Lab and CUNY, as well as Diamond Light Source in the UK.

“It was very important to be able to have remote collaboration to make this project work, where we had people onsite at the beamline and people offsite who could do the computer modeling,” Keedy said. “This is just one example of many during the pandemic of the scientific community coming together.”

This research was supported by the DOE Office of Science through the National Virtual Biotechnology Laboratory (NVBL) with funding from the Coronavirus CARES Act and funding from the Laboratory-led Research and Development Program for COVID Research -19 at Brookhaven Lab. The FMX beamline (17-ID-2) of NSLS-II is funded by the Office of Science (BES). Daniel Keedy is supported by the National Institutes of Health (NIH, R35 GM133769). The Center for BioMolecular Structure (CBMS) at Brookhaven Lab’s NSLS-II, which includes the FMX beamline, is funded by the National Institutes of Health, the National Institute of General Medical Sciences, and the DOE Office of Science (BER) .


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