Figure 1.
Enzymatic activity and dissociation constant of the dimer-monomer equilibrium.
(a). Enzymatic activity of STI/A vs. substrate concentrations as measured by monitoring the increase of the emission fluorescence intensity at a wavelength of 538 nm. The Km and kcat values are presented. (b). The ITC dilution profiles for measuring the dissociation constant of the dimer-monomer equilibrium for STI/A. The Kd and ΔH values were obtained by fitting the ITC data with the built-in Microcal ORIGIN software. (c). Dimeric structure of the SARS 3CLpro with the catalytic folds colored in blue (protomer 1) and cyan (protomer 2); the extra domains in red (protomer 1) and lightpink (protomer 2), as well as the connecting loops in black (protomer 1) and purple (protomer 2). The catalytic dyad residue His41 is displayed as green sphere while Cys145 as yellow sphere. Mutation residues S284T285I286 and N214 are displayed as spheres and the N-finger residues Ser1-Fhe8 are displayed as dots.
Figure 2.
Crystal structure of the STI/A mutant.
(a). Overall superimposition of the dimeric STI/A (violet) and WT (cyan; PDB code of 2H2Z (18) structures. (b) Superimpositions of the catalytically critical residues of STI/A (violet) and WT (cyan; PDB code of 2H2Z (18). (c). Diagram showing the distance (d) between the mass centers of two extra domains; and the angle (Θ) by the mass centers of two extra domains as well as the mass center of two chymotrypsin folds together (see Material and Methods for more details). The cavity volumes (V) of the nano-channels of STI/A (d) and WT (e) as represented by violet dots, which were calculated with the program: POVME (38).
Figure 3.
Overall dynamic behaviors in the MD simulations.
Root-mean-square deviations (RMSD) of the Cα atoms (from their positions in the energy minimized structures) for three independent MD simulations of the dimeric STI/A (black) and WT (red) for the whole enzyme (a–c); catalytic fold (d–f); and extra domain (g–i). Root-mean-square fluctuations of the Ca atoms computed for three independent simulations for protomer A (j-l) and protomer B (m-o) of STI/A (black) and WT (red). Protomer A and B are denoted as P1 and P2 respectively.
Figure 4.
Changes of the packing interaction between two extra domains.
Three separate time-trajectories of the distance d (a-c); angle Θ (see Figure 2d) (d-f) and volume of nano-channel (residue 284–286), V (g-i) of STI/A (black) and WT (red).
Table 1.
Hydrogen Bond Occupancy for STI/A and WT with Significant Differences.
Figure 5.
Dynamic behavior of the catalytic dyad.
Three separate time-trajectories of the distance between NE2 of His41 and SG of Cys145 atoms of protomer A (a–c) and protomer B (d-f) for STI/A (black) and WT (red). Three separate time-trajectories of the Chi2 dihedral angle of His41 of protomer A (g-i) and protomer B (j-l) for STI/A (black) and WT (red). Protomer A and B are denoted as P1 and P2 respectively.
Figure 6.
Dynamic behavior of the Glu166-His172 interaction.
Three separate time-trajectories of the Chi1 dihedral angle of Glu166 of protomer A (a-c) and protomer B (d-f) for STI/A (black) and WT (red). Three separate time-trajectories of the distance between Glu166 and His172 of protomer A (g-i) and protomer B (j-l) for STI/A (black) and WT (red). Protomer A and B are denoted as P1 and P2 respectively.
Figure 7.
Three separate time-trajectories of the Chi1 dihedral angle of Asn28 of protomer A (a-c) and protomer B (d-f) for STI/A (black) and WT (red). Three separate time-trajectories of the Chi2 dihedral angle of Asn28 of protomer A (g-i) and protomer B (j-l) for STI/A (black) and WT (red). Protomer A and B are denoted as P1 and P2 respectively.
Figure 8.
Dynamic behavior of Thr25 and Cys44.
Three separate time-trajectories of Phi (a-c) and Psi (d-f) dihedral angles of Thr25 of protomer A; and Phi (g-i) and Psi (j-l) dihedral angles of Thr25 of protomer B for STI/A (black) and WT (red). Three separate time-trajectories of the distance between Thr25 and Cys44 of protomer A (m-o) and protomer B (p-r) for STI/A (black) and WT (red). Three separate time-trajectories of the volume enclosed by Thr25 and Cys44 of protomer A (s-u) and protomer B (v-x) for STI/A (black) and WT (red). Protomer A and B are denoted as P1 and P2 respectively.
Figure 9.
Networks of correlated motions in WT, STI/A and N214A.
Mutual information matrixes calculated from the MD simulation data of WT, STI/A and N214A by MutInf (39); as well as the SARS 3CL protease structures with residues having significant correlation motion displayed in spheres which are colored in red if in the protomer 1; and brown in the protomer 2. The catalytic dyad residue His41 is displayed as green sphere while Cys145 as yellow sphere. The STI/A and N214A mutation residues are colored in splitpea. Yellow boxes in WT highlight the highly correlated motions between Phe3-Ser62 and: CII-BII (Leu115-Cys156), oxyanion loop (Ser139-Leu141), as well as extra domain (Asn214-Asn238, Ile281-Phe305). Yellow boxes in P1 of STI/A highlight the highly correlated motions of N-fingers with the other regions of the enzyme, similar to the WT while in P2 of STI/A highlight the expanded, highly correlated motions between the Domain III (Asn214-Asn238, Ile281-Phe305) and: substrate pocket (LeuP1), CII-BII (Leu115-Cys156), oxyanion loop (Ser139-Leu141), pocket 2 (Ser147-Thr175), loop region (Val186-Thr198). Yellow boxes in P1 of N214A highlight the correlated motions amongst residues in internal (Ile59-Arg105), CII-BII (Leu115-Cys156), oxyanion loop (Ser139-Leu141), catalytic dyad (Cys145) and the A Helix (Ser10-Glu14); while in P2 of N214A highlight the correlated motions within Domain III.
Figure 10.
Transmission of the STI/A mutation effects on the extra domains to the catalytic machinery by the dynamically-driven allostery.
(a)-(e). The cavity volumes of the nano-channel of STI/A in the first simulation at 0, 25, 50, 75 and 100 ns. (f). The crystal structure of STI/A with key residues having relevant dynamical changes displayed and labeled. The cavity is represented by the violet mesh.