X-rays increase the structure of the protein in the “heart” of the COVID-19 virus

A team of researchers from the Oak Ridge and Argonne National Laboratories of the Department of Energy have made the first X-ray measurements at room temperature on the main protease of SARS-CoV-2, the enzyme that allows the virus to reproduce.

X-ray measurements mark an important first step in the researchers’ ultimate goal of building a comprehensive three-dimensional model of the enzyme protein. The model will be used to advance supercomputing simulations aimed at finding drug inhibitors to block the virus’s replication mechanism and help end the COVID-19 pandemic. The results of his research are publicly available and have been published in the journal. Nature’s Communications.

SARS-CoV-2 is the virus that causes COVID-19 disease. The virus reproduces by expressing long chains of proteins that the protease enzyme must cut into smaller pieces.

“Protease is indispensable to the life cycle of the virus. The protein is shaped like a Valentine’s heart, but it really is the heart of the virus that allows it to replicate and spread. If it inhibits protease and stops the heart, the virus it cannot produce the proteins that are essential for its replication. That is why protease is considered such an important drug target, “said Andrey Kovalevsky, corresponding author of ORNL. Although the structure is known from cryogenically preserved crystals, “this is the first time that the structure of this enzyme has been measured at room temperature, which is significant because it is close to the physiological temperature where cells operate.”

Building a complete model of the protein structure requires identifying each element within the structure and how they are organized. X-rays are ideal for detecting heavy elements such as carbon, nitrogen, and oxygen atoms. Due to the intensity of X-ray rays in most large-scale synchrotron facilities, biological samples generally must be cryogenically frozen at around 100 K, or approximately minus 280 degrees Fahrenheit, to withstand radiation long enough to the data is collected.

To extend the lifespan of crystallized protein samples and measure them at room temperature, ORNL researchers grew the crystals larger than necessary for cryo synchrotron studies and used an internal X-ray machine that has a less intense beam.

“Protein crystal cultivation and data collection is a tedious and time consuming process. In the time it usually takes to prepare and send the sample to a synchrotron, we were able to cultivate the crystals, take measurements, and start analyzing the data.” said Daniel Kneller of ORNL, the study’s first author. “And when there is a pandemic with many scientists mobilizing to study this problem, there is no day off.”

The protease enzyme consists of chains of amino acids with a repeating pattern of nitrogen-carbon-carbon atoms that make up the protein’s backbone. The side groups of the amino acid building blocks, or “residues,” extend from each of the central central carbon atoms. The enzyme folds into a specific three-dimensional shape, creating special pockets where a drug molecule would bind.

The study revealed significant structural disparities between the orientations of the spine and some of the residues in cryogenic samples at room temperature. Research suggests that freezing crystals may introduce structural artifacts that could result in a less accurate understanding of the protease structure.

The team’s results are shared with researchers, led by ORNL-University of Tennessee Governor President Jeremy Smith, who are conducting drug docking simulations using Summit at ORNL, the nation’s fastest supercomputer.

“What the researchers are doing at Summit is taking known drug compounds and trying to computationally bind them to the main protease for drug reuse, as well as looking for new clues in other possible drug candidates,” said corresponding ORNL author Leighton Coates. . “Our room temperature data is being used to build a more accurate model for those simulations and to improve drug design activities.”

The researchers’ next step in completing the 3-D model of the main SARS-CoV-2 protease is to use neutron scattering in the ORNL High-Flow Isotope Reactor and Spallation Neutron Source. Neutrons are essential for locating hydrogen atoms, which play a critical role in many of the catalytic functions and drug design efforts.

The DNA from the protease plasmid used to make the enzyme was provided by the Argonne Center for Structural Biology at the Advanced Photon Source. The crystallization of the proteins used in the X-ray scattering experiments was carried out at the ORNL Center for Structural and Molecular Biology.