Abstract
SARS-CoV-2 emerged at the end of 2019 to cause an unprecedented pandemic of the deadly respiratory disease COVID-19 that continues to date. The viral main protease (Mpro) is essential for SARS-CoV-2 replication and is therefore an important drug target. Understanding the catalytic mechanism of Mpro, a cysteine protease with a catalytic site comprising the noncanonical Cys145-His41 dyad, can help in guiding drug design. Here, a 2.0 Å resolution room-temperature X-ray crystal structure is reported of a Michaelis-like complex of Mpro harboring a single inactivating mutation C145A bound to the octapeptide Ac-SAVLQSGF-CONH2 corresponding to the nsp4/nsp5 autocleavage site. The peptide substrate is unambiguously defined in subsites S5 to S3' by strong electron density. Superposition of the Michaelis-like complex with the neutron structure of substrate-free Mpro demonstrates that the catalytic site is inherently pre-organized for catalysis prior to substrate binding. Induced fit to the substrate is driven by P1 Gln binding in the predetermined subsite S1 and rearrangement of subsite S2 to accommodate P2 Leu. The Michaelis-like complex structure is ideal for in silico modeling of the SARS-CoV-2 Mpro catalytic mechanism.
Highlights
Severe acute respiratory syndrome coronavirus 2 (SARSCoV-2) causes the deadly coronavirus disease 2019 (COVID19) that engulfed the globe in a pandemic in early 2020 and remains a significant health threat despite the availability of several vaccines and antiviral therapies (Gavor et al, 2020; Meo et al, 2021; Doroftei et al, 2021; Bidram et al, 2021)
A DNA insert encoding a 6ÂHis tag followed by a TEV protease cleavage site and SARS CoV-2 main protease (Mpro) (306 amino acids) bearing an active-site C145A mutation was cloned into the pJ414 vector (ATUM) and transformed into Escherichia coli BL21(DE3) cells (Agilent)
P1 Gln is driven into the S1 subsite via a 2.7 Ahydrogen bond to the His163 imidazole and a water-mediated interaction with Asn142, whereas its main-chain carbonyl O atom is centered into the characteristic oxyanion hole [Fig. 2(b)]
Summary
Severe acute respiratory syndrome coronavirus 2 (SARSCoV-2) causes the deadly coronavirus disease 2019 (COVID19) that engulfed the globe in a pandemic in early 2020 and remains a significant health threat despite the availability of several vaccines and antiviral therapies (Gavor et al, 2020; Meo et al, 2021; Doroftei et al, 2021; Bidram et al, 2021). The main protease of SARS-CoV-2 (3CLpro or Mpro) is virally encoded and its precise function is essential for virus replication. Pp1a and pp1ab ($450 and $790 kDa, respectively), into individual functional proteins that build the virus genome replication/transcription machinery (Wu et al, 2020; Xu et al, 2020). The substrate specificity of Mpro and its active-site architecture are dissimilar relative to human proteases. Mpro is considered an important drug target for the design and development of specific antivirals with potentially minimal nonspecific binding to human proteins
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