Characterization of the cysteine protease domain of Semliki Forest virus replicase protein nsP2 by in vitro mutagenesis

被引:31
作者
Golubtsov, A
Kääriäinen, L
Caldentey, J
机构
[1] COST Off, European Sci Fdn, B-1050 Brussels, Belgium
[2] Univ Helsinki, Bioctr Viikki, Inst Biotechnol, FIN-00014 Helsinki, Finland
关键词
alphavirus; Semliki Forest virus; nsP2; protease; temperature-sensitive mutants; glycine specificity motif; cysteine protease;
D O I
10.1016/j.febslet.2006.01.071
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The function of Semliki Forest Virus nsP2 protease was investigated by site-directed mutagenesis. Mutations were introduced in its protease domain, Pro39, and the mutated proteins were expressed in Escherichia coli, purified and their activity in vitro was compared to that of the wild type Pro39. Mutations M781T, A662T and G577R, found in temperature-sensitive virus strains, rendered the enzyme temperature-sensitive in vitro as well. Five conserved residues were required for the proteolytic activity of Pro39. Changes affecting Cys(478), HiS(548), and Trp(549) resulted in complete inactivation of the enzyme, whereas the replacements N600D and N605D significantly impaired its activity. The importance of Trp(549) for the proteolytic cleavage specificity is discussed and a new structural motif involved in substrate recognition by cysteine proteases is proposed. (c) 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
引用
收藏
页码:1502 / 1508
页数:7
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