Only one protomer is active in the dimer of SARS 3C-like proteinase

Abstract

The severe acute respiratory syndrome coronavirus 3C-like protease has been proposed to be a key target for structurally based drug design against SARS. The enzyme exists as a mixture of dimer and monomer, and only the dimer was considered to be active. In this report, we have investigated, using molecular dynamics simulation and mutational studies, the problems as to why only the dimer is active and whether both of the two protomers in the dimer are active. The molecular dynamics simulations show that the monomers are always inactive, that the two protomers in the dimer are asymmetric, and that only one protomer is active at a time. The enzyme activity of the hybrid severe acute respiratory syndrome coronavirus 3C-like protease of the wild-type protein and the inactive mutant proves that the dimerization is important for enzyme activity and only one active protomer in the dimer is enough for the catalysis. Our simulations also show that the right conformation for catalysis in one protomer can be induced upon dimer formation. These results suggest that the enzyme may follow the association, activation, catalysis, and dissociation mechanism for activity control.

FIGURE 1

The r.m.s.d. values of the binding pocket in two typical trajectories. Shown are the heavy atom r.m.s.d. values of the binding pocket in trajectories fitted to the conformation of protomer A in the crystal structure (PDB code 1uk2). Here the binding pocket was defined by residues 41, 140, 142–145, 161, 163, 166, and 172. A, the trajectory 1 in the pseudodimer A simulation. B, the trajectory 3 in the pseudodimer B simulation.

FIGURE 2

The enzymatic activities of the hybrid SARS 3CL^pro and the wild-type enzyme.A □, adding C145A into the wild-type enzyme of 0.8 μm; ○, adding C145A into the wild-type enzyme of 0.6 μm; ▵, adding bovine serum albumin into the wild-type enzyme of 0.6 μm as the control. The concentration of C145A or bovine serum albumin ranged from 2.5 to 500 μm. B ▪, the wild-type enzyme concentration ranged from 0.1 to 3.0 μm; •, the wild-type enzyme plus the high concentration C145A mutant (200 μm when C_WT ranged from 0.1 to 0.6 μm; 500 μm when C_WT ranged from 0.8 to 3.0 μm). All of the experiments were repeated two times.