Abstract
The SARS 3C-like proteinase (SARS-3CLpro), which is the main proteinase of the SARS coronavirus, is essential to the virus life cycle. This enzyme has been shown to be active as a dimer in which only one protomer is active. However, it remains unknown how the dimer structure maintains an active monomer conformation. It has been observed that the Ser139-Leu141 loop forms a short 310-helix that disrupts the catalytic machinery in the inactive monomer structure. We have tried to disrupt this helical conformation by mutating L141 to T in the stable inactive monomer G11A/R298A/Q299A. The resulting tetra-mutant G11A/L141T/R298A/Q299A is indeed enzymatically active as a monomer. Molecular dynamics simulations revealed that the L141T mutation disrupts the 310-helix and helps to stabilize the active conformation. The coil-310-helix conformational transition of the Ser139-Leu141 loop serves as an enzyme activity switch. Our study therefore indicates that the dimer structure can stabilize the active conformation but is not a required structure in the evolution of the active enzyme, which can also arise through simple mutations.
Highlights
The first outbreak of severe acute respiratory syndrome (SARS) occurred over 10 years ago in 2003
By comparing the active protomer of the wild-type SARS-3C-like proteinase (3CLpro) with both the inactive protomer of the wild-type SARS-3CLpro (PDB Code 1UK2)[6] and the inactive monomer R298A (PDB Code 2QCY)[12], we found that the most distinguishable differences between the active and inactive structures were in the conformational changes of residues Ser139-Phe140-Leu[141]
Previous studies using molecular dynamics simulations and hybrid protein experiments have shown that only one protomer of the 3CLpro dimer is active[17]
Summary
The first outbreak of severe acute respiratory syndrome (SARS) occurred over 10 years ago in 2003. The N-finger α -helix A’ of Domain I (residues 10-15) and helix Domain III have been recognized as the main components of the dimer formation Changes in these components are known to disrupt the monomer-dimer equilibrium. Ser[139] and Phe[140] are two key residues that contribute to interactions between the two protomers in the parent dimer and maintain the correct conformation of the S1 subsite in the substrate-binding pocket. Other residue mutations, which are neither on the dimer interface nor key to catalysis, can influence enzyme activity and dimer association-dissociation of SARS-3CLpro via long-range interactions[15,16]. To the best of our knowledge, no active monomer mutant has been reported previously for SARS3CLpro or the main proteinases of other CoVs. In the present study, we successfully designed an active monomer of SARS-3CLpro by comparing the active and inactive structures of the proteinase. We studied the mechanism that regulates this active monomer using mutational and enzymatic studies, as well as molecular dynamics simulations
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