Overview

The coronavirus disease 2019 (COVID-19) pandemic is caused by the novel severe acute respiratory syndrome (SARS) coronavirus 2 (CoV-2). Due to the highly contagious nature of SARS-CoV-2, the pandemic caused almost 7 million deaths worldwide, making it one of the deadliest infectious diseases in history. Several vaccines are available, that have contributed immensely to stop the spread of the virus. Few antiviral compounds are also approved for the treatment of mild to moderate COVID-19 in people who are more likely to get very sick. During the time pandemic was prevalent, the vaccine and treatment costs have been free of charge. However recently, the WHO declared an end to COVID-19 pandemic. The government will soon stop supplying the COVID vaccines for free. Still, there are thousands of people who get infected daily and are at risk of severe illness. This calls for a need for an effective and inexpensive treatment and prevention of COVID-19. A prerequisite is to understand the structure and function of vital enzymes to the virus. The replication of the SARS CoV-2 is dependent on the activity of two cysteine proteases, a papain like protease (PLpro), and the 3C like protease (3CLpro). In its functional form the 3CLpro (also known as the main protease, Mpro) is a homo dimer with Cys/His dyad in the catalytic site. The 3CLpro tailor cuts various essential virus proteins out of a long poly peptide translated from virus RNA. These proteins are responsible for the replication and transcription of the viral genome, and thus, the infectivity of the virus. Here, we investigate the structure of 3CLpro at cryogenic and room temperatures and assess the binding of small molecules that may affect the activity of the 3CLpro.

Intellectual Merit

Structures were determined at cryogenic temperatures from several, not previously reported crystal forms (Fig. 1). This includes several orthorhombic crystal forms with new cell parameters. The 3CLpro was investigated at cryogenic temperatures during multiple beamtimes at sector 19 of the Advanced Photon Source. The beamtimes were fully remote with robotic sample exchange.

The binding of small molecules such as ascorbate, isopropanol and trifluoroethanol was observed (Fig. 2). Soaking overnight with the strong 3CLpro inhibitor ebselen destroys the crystals. However, the binding of ascorbate is interesting as it might provide an easy way to affect the activity of the 3CLpro. Binding of ascorbate has been corroborated by a molecular docking simulation. Nevertheless, the binding of ascorbate is weak and may not be physiologically relevant. Still, ascorbate and other small molecules point to potential sites that can be exploited to identify a binding pattern for more potent effectors. The unit cell and space group plasticity (more than half a dozen of different crystal forms are known) indicates that the structure of the 3CLpro reacts strongly to ligand (and substrate) binding by conformational changes. A room temperature structure was determined with serial crystallography at the Linac Coherent Light Source (LCLS). Mix and inject experiments with ebselen are not successful since the ebselen concentration in the aqueous solution is too low to generate measurable occupancy in the active sites. At room temperature the flexible loop near the active site is more open compared to that observed cryo temperatures. This seems to be important for the enzymes activity, since the crystal form that results from ascorbate binding also features this open loop configuration.

Broader Impacts

The project sparked institutional awareness that meaningful COVID-19 research is possible at UWM even during difficult times. We were able to return to work in person and maintain an open laboratory during the course of the pandemic whereas most labs at UWM were closed. This project sparked great enthusiasm of the graduate students in the lab. Several undergraduate and graduate students from Northeastern Illinois (NEIU) University were also included in the project to assist with protein overexpression and purification. NEIU is a public, federally designated Hispanic Serving Institution (HSI). At NEIU this project was very motivational for the students, who were present in person and worked on a meaningful project during challenging times.

X-ray data collected on SARS CoV-2 3CLpro microcrystals produced a treasure trove of new crystallographic data. The data will provide a comprehensive foundation to train undergraduate and graduate students interested in solving structure-function relationships. In partnership with NEIU underrepresented STEM students (undergraduate and graduate students) were introduced to effective ways to solve protein structures. All students were trained in protein purification and microcrystal preparation for data collection at XFEL and synchrotron facilities. Overall, the project benefited a group of scientists with a broad and diverse portfolio of research skills.

Last Modified: 10/05/2023
Modified by: Marius Schmidt