Function of the central domain of streptokinase in substrate plasminogen docking and processing revealed by site‐directed mutagenesis

Abstract

The possible role of the central b-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 KK256,257AÃ 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 SK EK281.282AA , 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 additive0synergistic 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 b-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 dom...

Plasminogen Substrate Recognition by the Streptokinase-Plasminogen Catalytic Complex Is Facilitated by Arg253, Lys256, and Lys257 in the Streptokinase β-Domain and Kringle 5 of the Substrate

“…Previous structure-function studies have yielded diverse interpretations and conclusions regarding the structural basis of LBS-dependent Pg substrate recognition (23,(27)(28)(29)(30)(31)(32)(33)(34). Each of the three domains of SK has been implicated in this regard (29,30,35,36), and binding of two Pg molecules to the residue 1-59 sequence of the ␣-domain has been reported (36).…”

“…In particular, segments 16 -36, 41-48, 48 -59, and 88 -97 of the SK ␣-domain have been concluded to play a role in Pg substrate recognition (32,33,37,38). For several SK mutants, a complex mixture of functional effects on their binding to [Glu]Pg and its conformational and proteolytic activation has been reported (28,31,33). Some of these effects may result from the inherent flexibility of SK when bound to Pg or Pm (39), and others may be due to the use of kinetic approaches that do not clearly discriminate between conformational and proteolytic activation.…”

“…) in the SK ␤-domain in Pg substrate recognition (27,28,31,34). This loop is disordered in the structure of the SK⅐Pm complex but is ordered in the structure of the isolated ␤-domain (3,40).…”

“…This loop is disordered in the structure of the SK⅐Pm complex but is ordered in the structure of the isolated ␤-domain (3,40). Deletion of the 250-loop, Ala substitution of Lys 256 and Lys 257 at the apex of the loop, and substitution of multiple residues near and within the loop resulted in disparate effects on K m and k cat for [Glu]Pg activation (27,28,31). The conclusions of these studies were that Lys 256 and Lys 257 are involved in SK binding and conformational activation of [Glu]Pg in addition to proteolytic processing of Pg as a substrate.…”

“…The conclusions of these studies were that Lys 256 and Lys 257 are involved in SK binding and conformational activation of [Glu]Pg in addition to proteolytic processing of Pg as a substrate. Some of these studies are problematic because the natural NH 2 -terminal Ile 1 residue necessary for conformational activation is preceded either by an additional methionine (27,31) or maltose-binding protein (28) in the recombinant SK species used.…”