Essential covalent linkage between the chymotrypsin-like domain and the extra domain of the SARS-CoV main protease.
The main protease of the coronavirus causing severe acute respiratory syndrome performs proteolytic processing of the viral polyproteins. The active form of the enzyme is a homodimer with each subunit consisting of three structural domains. Domains I and II, hosting the complete catalytic machinery, constitute the N-terminal chymotrypsin-like folding scaffold and connect to the extra C-terminal domain III by a long loop. Previously, the domain III-truncated enzyme was demonstrated to fold independently into an intact chymotrypsin-like fold, but it showed no enzyme activity. To further delineate the structure-function relationships of the domain III and the long loop, we generated some truncated and mutated M(pro) forms bearing various combinations of the loop with other structural parts of the enzyme. Their conformational and association properties were investigated in detail. Far-ultraviolet circular dichroism (CD) measurements revealed that these fragments could fold independently. The secondary, tertiary and quaternary structures of these mixtures were monitored by CD, fluorescence spectroscopy and analytical ultracentrifugation. However, no enzyme activity was observed for any mutant or mixtures. These observations indicate that the covalent linkage between the chymotrypsin like and the extra domain is essential for enzymatic activity of the main coronavirus protease and for the integrity of its quaternary structure.
Tsai
Meng-Ying
MY
Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, 155 Li-Nong St., Section 2, Taipei 112, Taiwan.
Chang
Wei-Hsin
WH
Liang
Jin-Yi
JY
Lin
Long-Liu
LL
Chang
Gu-Gang
GG
Chang
Hui-Ping
HP
D002918:Chymotrypsin; D000086782:Coronavirus 3C Proteases; D003546:Cysteine Endopeptidases / Q000737:chemistry*; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D045473:Severe acute respiratory syndrome-related coronavirus / Q000201:enzymology*; D014764:Viral Proteins / Q000737:chemistry*
D021122:Protein Subunits; D014764:Viral Proteins; D002918:Chymotrypsin; D003546:Cysteine Endopeptidases; D000086782:Coronavirus 3C Proteases