Please use this identifier to cite or link to this item: http://scholarbank.nus.edu.sg/handle/10635/15953
Title: Structural study revealing the unique enzymatic mechanism of the severe acute respiratory syndrome (SARS) coronavirus main protease highly mediated by the extra domain
Authors: SHI JIA-HAI
Keywords: SARS coronavirus main protease extra domain
Issue Date: 28-Aug-2008
Source: SHI JIA-HAI (2008-08-28). Structural study revealing the unique enzymatic mechanism of the severe acute respiratory syndrome (SARS) coronavirus main protease highly mediated by the extra domain. ScholarBank@NUS Repository.
Abstract: Severe acute respiratory syndrome (SARS) was the first pestilence in the 21stcentury, with more than 8,000 infectious cases including 774 fatalities in over 29countries. Because of its essential role in virus replication, the SARS coronavirusmain protease (Mpro) is considered to be one of the top targets for anti-SARS drugdesign. Although similar to picornavirus 3C proteases, SARS-CoV Mpro has achymotrypsin fold that hosts the entire catalytic dyad, and it has acquired a uniqueC-terminal extra domain with an unknown function.In this thesis, we aim at understanding the regulatory role of this extra domain inthe catalysis of the SARS-CoV main protease. We demonstrate that: 1) The extradomain contributes to the dimerization of SARS-CoV Mpro, switching the enzymefrom the inactive form (monomer) to the active form (dimer), as analyzed by proteindissection, Dynamic Light Scattering (DLS) and size-exclusion chamotography; 2)Four regions (residues 288-290, 291, 284-286 and 298-299) in the extra domain arecritical for the enzyme dimerization and catalysis of SARS-CoV Mpro, forming anano-scale channel passing through the central region of the enzyme, as revealed bysite-directed mutagenesis, DLS, nuclear magnetic resonance (NMR) spectroscopy andenzymatic activity assay; 3) Mutating the C-terminal residue Arg298 to Ala allows theswitching of SARS-CoV Mpro from dimer to monomer in solution, as measured byanalytical ultracentrifuge (AUC). A crystallography study further reveals that in themonomeric form, the SARS-CoV Mpro mutant is irreversibly inactivated because itscatalytic machinery becomes frozen in a collapsed state, characterized by the6formation of a short 310-helix within the chameleon catalytic loop; 4) Ala mutations inthe STI loop located at the dimer interface between the two extra domains are able toincrease the kcat value of SARS-CoV Mpro in the enzyme kinetics assay. Thecrystallographic study shows that these Ala mutations affect the interface inducing arigid-body re-orientation of the protomers if compared to that observed forSARS-CoV Mpro crystallized at low pH, mimicking the high-pH conformation ofSARS-CoV Mpro reported to have a higher catalytic potential than that at a low pH.Together, these results reveal a new and critical role of the C-terminal extra domain inthe dimerization and catalysis of the SARS-CoV Mpro. These may imply a generalfunction of the C-terminal extra domains in all coronavirus main proteases.The most important results of our study reveal a novel strategy for the design ofspecific inhibitors against coronavirus main protease. The ideal inhibitor would beone that can affect the conformation of the dimer interface of the proteases and at thesame time convert the main proteasesb active site into a catalytically incompetentconformation. Such a bifunctional inhibitor should be a highly competent drugcandidate for SARS and other coronavirus-related diseases. Last but not least, ourstudy sheds new light on the general principle of enzyme evolution, where thecatalytic machinery achieves improved regulation through oligomerization.
URI: http://scholarbank.nus.edu.sg/handle/10635/15953
Appears in Collections:Ph.D Theses (Open)

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