The binding of the protease with shikonin does not affect the structure of the complex formed with previously described inhibitors. Shikonin has an inhibitor head group at the 1,4-naphthoquinone part, and a ligand tail at the six-carbon side chain. The binding pocket is a narrow cleft with four subsites around it, S1 to S4.
Shikonin has a threefold interaction with the protease at the substrate-inhibitor binding site. The S1 subsite has a triad of residues that forms hydrogen bonds with the shikonin. The aromatic head group of the shikonin interacts with His41 on S2, the imidazole group of the histamine residue flipping outward from its inward-pointing orientation, and thus allowing access to shikonin. The ligand tail has two hydrogen bonds with two S3 residues.
The difference in the shikonin-bound complex lies in the catalytic paired residues, as well as in Phe140 and Glu166. With covalently bonded inhibitors, they bind to the Cys 145 at the Sγ atom. With shikonin, the Cys 145 side-chain changes its configuration to form a hydrogen bond.
The Cys145 and the His41 have a longer distance between them than with any other main protease. The Phe140 loses its former π-π bond with His163, with the phenyl ring changing its conformation to move outward to the solvent. The Glu166 has a flexible side chain, and becomes inactive in both the apo and the shikonin-protease complexes, whereas it is ordered in the other inhibitor complexes. Since this residue is important to keep the shape of the substrate-binding pocket intact through hydrogen bonding with inhibitors that mimic the binding peptides, and with the N-terminal residue of the other protomer, it is highly conserved within the main protease family.
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