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Specificity and reactive loop length requirements for crmA inhibition of serine proteases
Author(s) -
Tesch Lisa D.,
Raghavendra Manikanahally P.,
BedstedFaarvang Tina,
Gettins Peter G.W.,
Olson Steven T.
Publication year - 2005
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.041104905
Subject(s) - proteases , serpin , trypsin , serine , proteolysis , biochemistry , chemistry , chymotrypsin , pancreatic elastase , cysteine , serine protease , serine proteinase inhibitors , protease , elastase , neutrophil elastase , enzyme , microbiology and biotechnology , biology , inflammation , gene , immunology
The viral serpin, crmA, is distinguished by its small size and ability to inhibit both serine and cysteine proteases utilizing a reactive loop shorter than most other serpins. Here, we characterize the mechanism of crmA inhibition of serine proteases and probe the reactive loop length requirements for inhibition with two crmA reactive loop variants. P1 Arg crmA inhibited the trypsin‐like proteases, thrombin, and factor Xa, with moderate efficiencies (∼10 2 –10 4 M −1 sec −1 ), near equimolar inhibition stoichiometries, and formation of SDS‐stable complexes which were resistant to dissociation ( k diss ∼10 −7 sec −1 ), consistent with a serpin‐type inhibition mechanism. Trypsin was not inhibited, but efficiently cleaved the variant crmA as a substrate ( k cat / K M of ∼10 6 M −1 sec −1 ). N‐terminal sequencing confirmed that the P1 Arg–P1′Cys bond was the site of cleavage. Altering the placement of the Arg in a double mutant P1 Gly‐P1′Arg crmA resulted in minimal ability to inhibit any of the trypsin family proteases. This variant was cleaved by the proteases ∼10‐fold less efficiently than P1 Arg crmA. Surprisingly, pancreatic elastase was rapidly inhibited by wild‐type and P1 Arg crmAs (10 5 –10 6 M −1 sec −1 ), although with elevated inhibition stoichiometries and higher rates of complex dissociation. N‐terminal sequencing showed that elastase attacked the P1′Cys–P2′Ala bond, indicating that crmA can inhibit proteases using a reactive loop length similar to that used by other serpins, but with variations in this inhibition arising from different effective P2 residues. These results indicate that crmA inhibits serine proteases by the established serpin conformational trapping mechanism, but is unusual in inhibiting through either of two adjacent reactive sites.