Title
Quaternary Structure of the Severe Acute Respiratory Syndrome (SARS) Coronavirus Main Protease
Author
Chi-Yuan Chou, Hui-Chuan Chang, Wen-Chi Hsu, Tien-Zheng Lin, Chao-Hsiung Lin, Gu-Gang Chang
Year
2004
Journal
Biochemistry
Abstract
SARS (severe acute respiratory syndrome) has been one of the most severe viral infectious diseases last year and still remains as a highly risky public health problem around the world. Exploring the types of interactions responsible for structural stabilities of its component protein molecules constitutes one of the approaches to find a destabilization method for the virion particle. In this study, we performed a series of experiments to characterize the quaternary structure of the dimeric coronavirus main protease (Mpro, 3CLpro). By using the analytical ultracentrifuge, we demonstrated that the dimeric SARS coronavirus main protease exists as the major form in solution at protein concentration as low as 0.10 mg/mL at neutral pH. The enzyme started to dissociate at acidic and alkali pH values. Ionic strength has profound effect on the dimer stability indicating that the major force involved in the subunit association is ionic interactions. The effect of ionic strength on the protease molecule was reflected by the drastic change of electrostatic potential contour of the enzyme in the presence of NaCl. Analysis of the crystal structures indicated that the interfacial ionic interaction was attributed to the Arg-4···Glu-290 ion pair between the subunits. Detailed examination of the dimer−monomer equilibrium at different pH values reveals apparent pKa values of 8.0 ± 0.2 and 5.0 ± 0.1 for the Arg-4 and Glu-290, respectively. Mutation at these two positions reduces the association affinity between subunits, and the Glu-290 mutants had diminished enzyme activity. This information is useful in searching for substances that can intervene in the subunit association, which is attractive as a target to neutralize the virulence of SARS coronavirus.
Full Article
Instrument
J-810
Keywords
Circular dichroism, Secondary structure, Biochemistry