Fig 1.
Structure of PLpro and location of the mutations.
Upper panel: cartoon representation of the protein with inhibitor S43 shown in sticks and zinc ion as a sphere (based on PDB ID: 7e35). Teal region of the protein indicates N-terminal ubiquitin-like domain (Ubl). Lower panel: cartoon representation of the protein with residues colored according to their mutation frequency (percentage of sequences with a given mutation out of all mutated sequences).
Table 1.
Selected amino acid variants of papain-like protease from SARS-CoV-2.
Frequent mutations are those with mutation rate >0.002. Residues from inhibitor binding site were selected based on residue-ligand distance (no more than 6 Å) calculated for all available PLpro-ligand crystal structures. Amino acids in Variants column are sorted from most to least frequent. All found variants are available in Supplementary Material.
Fig 2.
Structure of PLpro and variants within noncovalent inhibitor binding site.
The rates of mutations of these residues are presented in Table 1, along with the residues they are replaced to. It is worth noting that the residues forming the BL2 also can be mutated (aa 267–270). Based on PLpro-S43 complex from PDB ID: 7e35.
Table 2.
Results of the estimated impact of the mutations at the PLpro binding site on the inhibitor binding.
For each PLpro-inhibitor complex, the first column shows mutation energy, which is the difference of the binding energies between the respective mutant and the wild type (WT) protein, estimated using Calculate Binding Energies protocol in Discovery Studio. The next two columns include negatives of the CDOCKER interaction energies from molecular docking conducted to the PLpro mutants prepared with two methods in Discovery Studio. The first one utilized the Build Mutant protocol. The second one consisted of the mutation of specific residues with a subsequent side chain conformation refinement.
Fig 3.
Selected variants found in PLpro.
(A) Location of amino acids subject to mutation relative to the ligand (S43) binding site (based on PDB ID: 7d7t). (B) Representative S43 conformations bound to PLpro. Calculations are based on frames sampled every 100 ps from joint Molecular Dynamics trajectories. Conformations found in less than 5% of the frames are not shown for clarity. (C) Frequency of protein-ligand hydrogen bonds in different trajectories. Left: GRL-0617, right: S43. Calculations are based on frames sampled every 100 ps from Molecular Dynamics trajectories. Dashed rectangle points to the amino acid interacting via hydrogen bond with GRL-0617 in the crystal structure (PDB ID: 7jrn), the one with solid line with S43 (PDB ID: 7d7t). In the case of Q269 the ligand can bind to either its main chain (M) or side chain (S). The red asterisk indicates the trajectories in which the ligand dissociated from the binding site. (D) Ligands MMGBSA binding energy (kcal/mol) to PLpro. Left: GRL-0617, right: S43. Calculations are based on frames sampled every 100 ps from Molecular Dynamics trajectories—the averages and standard errors are shown. The trajectories in which the ligand dissociated from the binding site are marked with red asterisk.
Fig 4.
Characterization of activity and inhibitor binding properties of recombinant SARS-CoV-2 PLpro variants.
Assay conditions: [E]=100 nM, [S]=10 μM. (A) Determination of activity of the PLpro mutants toward tetrapeptide fluorogenic substrate. Experiment was repeated at least two times. (B) IC50 determination for variants that possessed enzymatic activity of GRL-0617 and S43 inhibitors. Enzyme was incubated in assay buffer for 10’ followed by enzyme incubation with inhibitor for 30’ prior to the measurement. Measurements were performed three times.