Three-dimensional domain swapping as a mechanism to lock the active conformation in a super-active octamer of SARS-CoV main protease

Proteolytic processing of viral polyproteins is indispensible for the lifecycle of coronaviruses. The main protease (M-pro) of SARS-CoV is an attractive target for anti-SARS drug development as it is essential for the polyprotein processing. M-pro is initially produced as part of viral polyproteins and it is matured by autocleavage. Here, we report that, with the addition of an N-terminal extension peptide, M-pro can form a domain-swapped dimer. After complete removal of the extension peptide from the dimer, the mature M-pro self-assembles into a novel super-active octamer (AO-M-pro). The crystal structure of AO-M-pro adopts a novel fold with four domain-swapped dimers packing into four active units with nearly identical conformation to that of the previously reported M-pro active dimer, and 3D domain swapping serves as a mechanism to lock the active conformation due to entanglement of polypeptide chains. Compared with the previously well characterized form of M-pro, in equilibrium between inactive monomer and active dimer, the stable AO-M-pro exhibits much higher proteolytic activity at low concentration. As all eight active sites are bound with inhibitors, the polyvalent nature of the interaction between AO-M-pro and its polyprotein substrates with multiple cleavage sites, would make AO-M-pro functionally much more superior than the M-pro active dimer for polyprotein processing. Thus, during the initial period of SARS-CoV infection, this novel active form AO-M-pro should play a major role in cleaving polyproteins as the protein level is extremely low. The discovery of AO-M-pro provides new insights about the functional mechanism of M-pro and its maturation process.