Despite utilizing the same chymotrypsin fold to host the catalytic machinery, coronavirus 3C-like proteases (3CLpro) noticeably differ from picornavirus 3C proteases in acquiring an extra helical domain in evolution. Previously, the extra domain was demonstrated to regulate the catalysis of the SARS-CoV 3CLpro by controlling its dimerization. Here, we studied N214A, another mutant with only a doubled dissociation constant but significantly abolished activity. Unexpectedly, N214A still adopts the dimeric structure almost identical to that of the wild-type (WT) enzyme. Thus, we conducted 30-ns molecular dynamics (MD) simulations for N214A, WT, and R298A which we previously characterized to be a monomer with the collapsed catalytic machinery. Remarkably, three proteases display distinctive dynamical behaviors. While in WT, the catalytic machinery stably retains in the activated state; in R298A it remains largely collapsed in the inactivated state, thus implying that two states are not only structurally very distinguishable but also dynamically well separated. Surprisingly, in N214A the catalytic dyad becomes dynamically unstable and many residues constituting the catalytic machinery jump to sample the conformations highly resembling those of R298A. Therefore, the N214A mutation appears to trigger the dramatic change of the enzyme dynamics in the context of the dimeric form which ultimately inactivates the catalytic machinery. The present MD simulations represent the longest reported so far for the SARS-CoV 3CLpro, unveiling that its catalysis is critically dependent on the dynamics, which can be amazingly modulated by the extra domain. Consequently, mediating the dynamics may offer a potential avenue to inhibit the SARS-CoV 3CLpro.
Severe acute respiratory syndrome (SARS) is the first emerging infectious disease of the 21st century which has not only caused rapid infection and death, but also triggered a dramatic social crisis. Its 3C-like protease is crucial for reproducing virus and thus represents a top target for drug design. Interestingly, unlike 3C protease such as from picorovirus, the SARS protease evolutionarily acquired a C-terminal extra domain with previously-unknown function. Immediately after SARS outbreak, we revealed that the extra domain was able to regulate the catalysis by controlling the dimerization essential for activity. Here, we studied one mutant with only slightly-weakened dimerization but almost completely abolished activity. We determined its three-dimensional structure but very unexpectedly it is almost identical to that of the wild-type enzyme. Therefore, we initiated 30-ns molecular dynamic simulations for five forms of the enzyme and the results demonstrate that the dynamical changes in this mutant are responsible for its inactivation. Therefore, the extra domain can also control the catalysis by modulating the enzyme dynamics. This is not only of fundamental significance to understanding how enzymes evolve, but also implies a novel avenue for design of anti-SARS molecules.