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Characterization of the cysteine protease domain of Semliki Forest virus replicase protein nsP2 by in vitro mutagenesis
Author(s) -
Golubtsov Andrey,
Kääriäinen Leevi,
Caldentey Javier
Publication year - 2006
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2006.01.071
Subject(s) - semliki forest virus , proteases , protease , site directed mutagenesis , cysteine , cysteine protease , biochemistry , biology , mutagenesis , enzyme , thermostability , escherichia coli , rna dependent rna polymerase , ns2 3 protease , cleavage (geology) , in vitro , ns3 , mutation , rna , mutant , polymerase , gene , paleontology , fracture (geology)
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.