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Function of the central domain of streptokinase in substrate plasminogen docking and processing revealed by site‐directed mutagenesis
Author(s) -
Chaudhary Anita,
Vasudha S.,
Rajagopal K.,
Komath Sneha Sudha,
Garg Nandita,
Yadav M.,
Mande Shekhar C.,
Sahni Girish
Publication year - 1999
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.8.12.2791
Subject(s) - mutant , streptokinase , plasmin , chemistry , active site , plasminogen activator , mutagenesis , alanine , site directed mutagenesis , linker , peptide , docking (animal) , enzyme kinetics , biochemistry , activator (genetics) , enzyme , stereochemistry , amino acid , biology , receptor , gene , genetics , medicine , nursing , myocardial infarction , computer science , operating system
Abstract The possible role of the central β‐domain (residues 151–287) of streptokinase (SK) was probed by site‐specifically altering two charged residues at a time to alanines in a region (residues 230–290) previously identified by Peptide Walking to play a key role in plasminogen (PG) activation. These mutants were then screened for altered ability to activate equimolar “partner” human PG, or altered interaction with substrate PG resulting in an overall compromised capability for substrate PG processing. Of the eight initial alanine‐linker mutants of SK, one mutant, viz. SK KK256, 257aa (SK‐D1), showed a roughly 20‐fold reduction in PG activator activity in comparison to wild‐type SK expressed in Escherichia coli (nSK). Five other mutants were as active as nSK, with two [SK RE248.249aa and SKE K281.282aa , referred to as SK(C) and SK(H), respectively] showing specific activities approximately one‐half and two‐thirds, respectively, that of nSK. Unlike SK(C) and SK(H), however, SK(D1) showed an extended initial delay in the kinetics of PG activation. These features were drastically accentuated when the charges on the two Lys residues at positions 256 and 257 of nSK were reversed, to obtain SK KK256.257ee [SK(D2)]. This mutant showed a PG activator activity approximately 10‐fold less than that of SK(D1). Remarkably, inclusion of small amounts of human plasmin (PN) in the PG activation reactions of SK(D2) resulted in a dramatic, PN dose‐dependent rejuvenation of its PG activation capability, indicating that it required pre‐existing PN to form a functional activator since it could not effect active site exposure in partner PG on its own, a conclusion further confirmed by its inability to show a “burst” of p‐nitrophenol release in the presence of equimolar human PG and p ‐nitrophenyl guanidino benzoate. The steady‐state kinetic parameters for HPG activation of its 1:1 complex with human PN revealed that although it could form a highly functional activator once “supplied” with a mature active site, the K m for PG was increased nearly eightfold in comparison to that of nSK‐PN. SK mutants carrying simultaneous two‐ and three‐site charge‐cluster alterations, viz., SK RE248.249aa;ek281.282aa [SK(CH)], SK EK272.273AA;EK281.282AA [SK(FH)], and SK RE248.249AA;EK272.273AA;EK281.282AA [SK(CFH)], showed additive/synergistic influence of multiple charge‐cluster mutations on HPG activation when compared to the respective “single‐site” mutants, with the “triple‐site” mutant [SK(CFH)] showing absolutely no detectable HPG activation ability. Nevertheless, like the other constructs, the double‐ and triple‐charge cluster mutants retained a native like affinity for complexation with partner PG. Their overall structure also, as judged by far‐ultraviolet circular dichroism, was closely similar to that of nSK. These results provide the first experimental evidence for a direct assistance by the SK β‐domain in the docking and processing of substrate PG by the activator complex, a facet not readily evident probably because of the flexibility of this domain in the recent X‐ray crystal structure of the SK‐plasmin light chain complex.