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Abstract
The emergence of the novel coronavirus SARS-CoV-2 has resulted in a worldwide pandemic not seen in generations. Creating treatments and vaccines to battle COVID-19, the disease caused by the virus, is of paramount importance in order to stop its spread and save lives. The viral main protease, 3CL Mpro, is indispensable for the replication of SARS-CoV-2 and is therefore an important target for the design of specific protease inhibitors. Detailed knowledge of the structure and function of 3CL Mpro is crucial to guide structure-aided and computational drug-design efforts. Here, the oxidation and reactivity of the cysteine residues of the protease are reported using room-temperature X-ray crystallography, revealing that the catalytic Cys145 can be trapped in the peroxysulfenic acid oxidation state at physiological pH, while the other surface cysteines remain reduced. Only Cys145 and Cys156 react with the alkylating agent N-ethylmaleimide. It is suggested that the zwitterionic Cys145-His45 catalytic dyad is the reactive species that initiates catalysis, rather than Cys145-to-His41 proton transfer via the general acid-base mechanism upon substrate binding. The structures also provide insight into the design of improved 3CL Mpro inhibitors.
Highlights
The coronavirus-induced disease COVID-19 has swiftly spread to every corner of the globe, causing a severe pandemic not seen in a century
In our first room-temperature X-ray structure of ligand-free SARS-CoV-2 3CL Mpro, structure I, obtained at 1.80 Aresolution, we observed that Cys145 is in the rarely seen peroxysulfenic (—S—O—OH) thiol oxidation state that is presumably formed by direct reaction of the cysteine S atom with the molecular oxygen dissolved in water [Figs. 2(a) and 2(b)]
We succeeded in trapping the SARS-CoV-2 3CL Mpro enzyme with Cys145 oxidized to the peroxysulfenic acid in the crystal and performed X-ray crystallographic experiments at a physiologically relevant temperature
Summary
The coronavirus-induced disease COVID-19 has swiftly spread to every corner of the globe, causing a severe pandemic not seen in a century. SARS-CoV-2 appears to be more contagious than similar viruses. The replication of such viruses is fully dependent on the function of a 3-chymotrypsin-like protease enzyme (3CL Mpro) that hydrolyzes large polyproteins to generate essential nonstructural proteins, which play a fundamental role in transcription/replication during infection The SARS-CoV-2 3CL Mpro enzyme operates at no fewer than 11 cleavage sites on two large polyproteins 1a and 1ab (replicase 1a, $450 kDa, and replicase 1ab, $790 kDa). Cleavage of the large polyprotein chains by 3CL Mpro occurs at the glutamine residue in the P1 position of the substrate via a Cys145–His dyad, in which the cysteine thiol functions as the nucleophile in the proteolytic process (Jin, Zhao et al, 2020)
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