Structural and functional significance of the amino acid differences Val35Thr, Ser46Ala, Asn65Ser, and Ala94Ser in 3C-like proteinases from SARS-CoV-2 and SARS-CoV

Abstract

Three dimensional structures of (chymo)trypsin-like proteinase (3CL^pro) from SARS-CoV-2 and SARS-CoV differ at 8 positions. We previously found that the Val₈₆Leu, Lys₈₈Arg, Phe₁₃₄His, and Asn₁₈₀Lys mutations in these enzymes can change the orientation of the N- and C-terminal domains of 3CL^pro relative to each other, which leads to a change in catalytic activity. This conclusion was derived from the comparison of the structural catalytic core in 169 (chymo)trypsin-like proteinases with the serine/cysteine fold. Val₃₅Thr, Ser₄₆Ala, Asn₆₅Ser, Ala₉₄Ser mutations were not included in that analysis, since they are located far from the catalytic tetrad. In the present work, the structural and functional roles of these variable amino acids at positions 35, 46, 65, and 94 in the 3CL^pro sequences of SARS-CoV-2 and SARS-CoV have been established using a comparison of the same set of proteinases leading to the identification of new conservative elements. Comparative analysis showed that, in addition to interdomain mobility, which could modulate catalytic activity, the 3CL^pro(s) can use for functional regulation an autolytic loop and the unique Asp₃₃-Asn₉₅ region (the Asp₃₃-Asn₉₅ Zone) in the N-terminal domain. Therefore, all 4 analyzed mutation sites are associated with the unique structure-functional features of the 3CL^pro from SARS-CoV-2 and SARS-CoV. Strictly speaking, the presented structural results are hypothetical, since at present there is not a single experimental work on the identification and characterization of autolysis sites in these proteases.

Keywords: (Chymo)trypsin-like proteinases; Autolysis; COVID-19; Catalytic tetrad; Interdomain loop; SARS-CoV-2; Structural catalytic core.

Graphical abstract

Fig. 1

(A) and (B) show the “Val₄₂-Leu₂₇ Zone” and “Cys₅₈-Cys₄₂ Zone” of the SARS-CoV-2 3CL^pro and Trypsin Bos Taurus, respectively. 58_T–42_T Zone is the representative zone for 148 (chymo)trypsin-like proteinases with serine/cysteine fold. 58_T–42_T Zone consists of the two part: well-structured (four hydrogen bonds) part, which consists of the residues in positions 42_T, 33_T, 34_T and 64_T, and the 58_T–64_T loop, variable in length. The positions of the C_α-atoms of the amino acids Ser₄₆ and Asn₆₅ of the SARS-CoV-2 3CL^pro (A), which have changed in comparison with the SARS-CoV 3CL^pro, are marked with large green circles. The location of the 58_T–42_T Zone in relation to the NBCZone formed by amino acids: 42_T, 43_T, 57_T (catalytic base), 58_T, 195_T (catalytic nucleophile), 196_T, 197_T and 213_T is also shown. Some variations in the organization of the well-structured part of the 58_T–42_T Zone: (C) shows 3D complex of the NS3 protease and NS4A cofactor (brown); (D) 3C-like viral cysteine protease shows another variant of structural organization of the well-structured part of the 58_T–42_T Zone. The second and third β-strands are connected by aromatic residues; (E) Instead of four hydrogen bonds, only two remained in this variant of the 58_T–42_T Zone; and (F) The 2A proteinase has no structural analogue of the 58_T–42_T Zone at all. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

(A) Hydrophobic interactions (small green and orange circles) between Val₄₂-Leu₆₇ loop and Cys₈₅-Zone in the SARS-CoV-2 3CL^pro. The positions of the C_α-atoms of the amino acids Ser₄₆ and Asn₆₅ of the SARS-CoV-2 3CL^pro (A), which have changed in comparison with the SARS-CoV 3CL^pro, are marked with large green circles. Polar contacts (small blue and green circles) between the conserved parts of the interdomain loop (IDL): Val₁₈₆-Gln₁₉₂, Horse Shoe-Shaped Region (HSSR) and Val₄₂-Leu₆₇ loop. (B) In the complex between ligand and SARS-CoV-2 3CL^pro hydrophobic amino acids Cys₄₄, Met₄₉ and Tyr₅₄ of the Val₄₂-Leu₆₇ loop and the Leu₄ residue of the ligand interact with each other and maintain the 3D position of the catalytic histidine in position 41. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

The structure-based multiple sequence alignment of Val₄₂-Leu₆₇ loop of SARS-CoV-2 3CL^pro, ten corresponding coronavirus proteases and one 3Cl protease from Cavally virus loops.

Fig. 4

(A) and (B) show the “Asp₃₃-Asn₉₅ Zone” and “Ser₄₉-Ala₁₁₂ Zone” of the SARS-CoV-2 3CL^pro and Trypsin Bos Taurus, respectively. 49_T–112_T Zone is the representative zone for 104 (chymo)trypsin-like proteinases with serine/cysteine fold: the code name of this zone is “(5 + 2)”. The positions of the C_α-atoms of the amino acids Val₃₅ and Ala₉₄ of the SARS-CoV-2 3CL^pro (A), which have changed in comparison with the SARS-CoV 3CL^pro, are marked with large green circles. The location of the 49_T–112_T Zone in relation to the catalytic base and catalytic acid is also shown. (C) a zone with the code name is “(8 + 2)”; (D) a zone with the code name “(9 + 2)” and disulfide bond Cys₁₁₁-Cys₅₀; (E) a zone with the code name: “(11 + 2)”, and the lack of 49_T–111_T contact or disulfide bond Cys₅₀-Cys₁₁₁; and (F) a zone with the code name: “(21 + 3)”, a special version of the zone in which the second fragment of the zone has 3 amino acids residues instead of two. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

Superposition of three-dimensional (3D) structures of the 3CLpro(s) from SARS-CoV (PDB ID 1UJ1; shown in gray) and SARS-CoV-2 (PDB ID 6LU7; shown in blue). (A) Shows the location within the entire structure of the V42-L27 Zone (for reference, see Fig. 1A), the catalytic triad (H41, C85 and C145 in SARS-CoV-2), and the variable amino acids at the positions 35, 46, 65 and 94. (B) Shows the location of the D33-N95 Zone (for reference, see Fig. 4A), the catalytic triad (H41, C85 and C145 in SARS-CoV-2), and the variable amino acids at the positions 35, 46, 65 and 94. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)