Neutrons catch the shape-changing coronavirus protein complex in action

ORNL researchers found that the papain-like protease (in orange) can bind to human interferon-stimulated gene 15 protein (in blue) in different ways and in different forms. Photo credit: ORNL / Jill Hemman

While all viruses can fight the body’s immune system, scientists have been studying how the SARS-CoV-2 coronavirus – the cause of the global COVID-19 pandemic – can escape human immune systems.

Now, scientists at the US Department of Energy’s (DOE) Oak Ridge National Laboratory (ORNL) have revealed the molecular details of how a key protein (the papain-like protease or “PLpro”) from the virus combines to form a pair structure or “complex” with a human protein called interferon-stimulated gene 15 (ISG15). PLpro removes ISG15 from other human cellular proteins to help SARS-CoV-2 bypass the immune response. Understanding how the two proteins interact could help develop therapeutic drugs that prevent their formation and allow a person’s immune system to better fight the invading virus.

The research results entitled “Conformational Dynamics in the Interaction of SARS-CoV-2 Papain Like Protease with Human Interferon-Stimulated Gene 15 Protein” were published in the Journal of Physical Chemistry Letters. [ADD LINK]

“In human cells infected by the virus, the SARS-CoV-2 virus PLpro has a tendency to seek out and attach to the ISG15 protein, a key component of the cells’ immune response,” said Hugh O’Neill, Head of Bio-Facilities Group at ORNL and Director of the Laboratory’s Structural Molecular Biology Center. “If PLpro binds to ISG15, this changes the shape of the ISG15. The most important finding is that the ISG15 can take several forms when it binds to PLpro. “

With the help of small angle neutron scattering (SANS) at the High Flux Isotope Reactor (HFIR) of the ORNL, the researchers were able to study the changes in the complex during their occurrence.

“We improved the contrast between PLpro and ISG15 by making PLpro, in which many of the hydrogen atoms were replaced with deuterium atoms,” said Kevin Weiss, an expert in biodeuteration. “Neutrons interact differently with deuterium atoms, so that helped us to better differentiate the two proteins.

“We used neutrons to analyze the complex in solution, which better simulates the actual physiological environment of the human body,” said Leighton Coates, science and technology manager for instrument systems at the ORNL’s second target station. “This enabled us to examine the changing shapes of the complex that other techniques could not have captured.”

“The information we have gained from our experiments will expand our knowledge of how the virus works and will allow us to create more accurate computer models for other scientists,” said Wellington Leite, lead author and postdoctoral fellow at ORNL. “Researchers can use the model to quickly find sites on the ISG15 that the PLpro is connected to and then try to block those sites.”

Susan Tsutakawa, a biochemist at Lawrence Berkeley National Laboratory (Berkeley Lab), received small angle X-ray scattering (SAXS) data on the PLpro-ISG15 complex at the Berkeley Lab’s Advanced Light Source Synchrotron. “In the SAXS studies we were able to separate different complexes in the sample by coupling SAXS with size exclusion chromatography and at the same time obtain higher-resolution data on the overall configuration of the complex to complement the SANS studies that revealed the conformations of the individual components in the complex” said Tsutakawa.

The team plans to conduct additional experiments on this type of biological complex to investigate how small molecules can block the binding of PLpro to ISG15.

This research was supported by the DOE National Virtual Biotechnology Laboratory, a consortium of national DOE laboratories funded by the Coronavirus CARES Act. Additional support was provided by the DOE Office of Science, the Molecular Biophysics and Integrated Bioimaging Division at Berkeley Lab, and the Center for Structural Molecular Biology at ORNL.

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