h163a mutant  (BPS Bioscience)

Journal: Nature communications

Article Title: The H163A mutation unravels an oxidized conformation of the SARS-CoV-2 main protease.

doi: 10.1038/s41467-023-40023-4

Figure Lengend Snippet: Fig. 1 | Structure of the SARS-CoV-2 Mpro highlights the importance of the lateral pocket in inhibitor design. Mpro is an obligate homodimeric cysteine protease (PDB 7BB2).a Each monomer can be broken up into three regions: Domain I (residues 1–101; yellow/orange); Domain II (102–184; light violet/magenta); and Domain III (201–301; pale green/forest green). b The active site in each monomer is created from the interface between Domains I and II, whereby the catalytic dyad’s H41 and C145 are derived from Domains I and II, respectively. In the WT structure, the active-site cysteine (C145) is located ~12 Å from C117, the cysteine involved in the disulfide bond in the H163A Mpro structure. c Surface representation of the WT Mpro with a focus on the active-site cleft. The enzyme’s S2 to S4 pockets are denoted by the black line. The key residue of interest, H163, is located in the S1 pocket, laterally connected to this active site groove (denoted by *). d The surface representation from (c) is rotated 90° counterclockwise to show this H163 lateral pocket from a head-on perspective. Side chains that make up the lateral pocket and the catalytic dyad are rendered as cylinders in both (c) and (d). All molecular representations in this paper were generated in CCP4MG (version 2.10.11)77.

Article Snippet: Toobtain crystals of theH163AMpro in complexwithGC376, 5mg/mL H163A mutant (~148μM) was incubated with 400μM GC376 (BPS Bioscience; San Diego, CA, USA) at room temperature for 2 h prior to setting up the crystallization experiment.

Techniques: Derivative Assay, Residue, Generated

Journal: Nature communications

Article Title: The H163A mutation unravels an oxidized conformation of the SARS-CoV-2 main protease.

doi: 10.1038/s41467-023-40023-4

Figure Lengend Snippet: Fig. 4 | Structural comparisons between wildtype and H163A Mpro structures distal to the active site. In addition to the local restructuring of the active site, structural changes are also seen distally in Domain I. A NOS bridge between C22 and K61 is captured in chain B (a) (2Fo-Fc density shown at 1.2 σ) but not in chain A (b) (2Fo-Fc density shown at 1.0 σ). c This structural asymmetry is also seen when comparing the N-termini of the two monomers, where 2Fo-Fc density is only seen for the N-terminus of chain B (shown at 1.4 σ) but not chain A. d When comparing the positions of the N-termini between the WT (gray) and H163A mutant, the four most N-terminal residues are drastically rotated approximately 90° to fit into an alternate pocket. This is due to the movement of the F140 loop in the H163A mutant structure, which occupies the space previously held by the WT N-terminus. There is no density to support a single conformation of the N-terminus in the other protomer.

Article Snippet: Toobtain crystals of theH163AMpro in complexwithGC376, 5mg/mL H163A mutant (~148μM) was incubated with 400μM GC376 (BPS Bioscience; San Diego, CA, USA) at room temperature for 2 h prior to setting up the crystallization experiment.

Techniques: Mutagenesis