Role of the amino‐terminal region of streptokinase in the generation of a fully functional plasminogen activator complex probed with synthetic peptides

Abstract

The mechanism whereby fragments of streptokinase (SK) derived from its N terminus (e.g., SK1-59 or SK1-63) enhance the low plasminogen (PG)-activating ability of other fragments, namely SK64-386, SK60-414, SK60-387, and SK60-333 (reported previously), has been investigated using a synthetic peptide approach. The addition of either natural SK1-59, or chemically synthesized SK16-59, at saturation (about 500-fold molar excess) generated amidolytic and PG activation capabilities in equimolar mixtures of human plasminogen (HPG) and its complementary fragment (either SK60-414 or SK56-414, prepared by expression of truncated SK gene fragments in Escherichia coli) that were approximately 1.2-and 2.5-fold, respectively, of that generated by equimolar mixtures of native SK and HPG. Although in the absence of SK1-59 equimolar mixtures of SK56-414 and HPG could generate almost 80% of amidolytic activity, albeit slowly, less than 2% level of PG activation could be observed under the same conditions, indicating that the contribution of the N-terminal region lay mainly in imparting in SK56-414 an enhanced ability for PG activation. The ability of various synthetic peptides derived from the amino-terminal region (SK16-51, SK16-45, SK37-59, SK1-36, SK16-36, and SK37-51) to (1) complement equimolar mixtures of SK56-414 and HPG for the generation of amidolytic and PG activation functions, (2) inhibit the potentiation of SK56-414 and HPG by SK16-59, and (3) directly inhibit PG activation by the 1:l SK-HPG activator complex was tested. Apart from SK16-59, SK16-51, and 16-45, the ability to rapidly generate amidolytic potential in HPG in the presence of SK56-414 survived even in the smaller SK-peptides, viz., SK37-59 and SK37-51. However, this ability was abolished upon specifically mutating the sequence -LTSRP-, present at position 42-46 in native SK. Although SK16-5 1 retained virtually complete ability for potentiation of PG activation in comparison to SK16-59 or SK1-59, this ability was reduced by approximately fourfold in the case of SK16-45, and completely abolished upon further truncation of the C-terminal residues to SK16-36 or SK1-36. Remarkably, however, these peptides not only displayed ability to bind PG, but also showed strong inhibition of PG activation by the native activator complex in the micromolar range of concentration; the observed inhibition, however, could be competitively relieved by increasing the concentration of substrate PG in the reaction, suggesting that this region in SK contains a site directed specifically toward interaction with substrate PG. This conclusion was substantiated by the observation that the potentiation of PG activating ability was found to be considerably reduced in a peptide (SK25-59) in which the sequence corresponding to this putative locus (residues 16-36) was truncated at the middle. On the other hand, fragments SK37-51 and SK37-59 did not show any inhibition of the PG activation by native activator complex. Taken together, these findings strongly support a model of SK acti...

Involvement of a Nine-residue Loop of Streptokinase in the Generation of Macromolecular Substrate Specificity by the Activator Complex through Interaction with Substrate Kringle Domains

“…Thus, the remarkable alteration of the macromolecular substrate specificity of HPN by SK is currently thought to be due to "exosites" generated on the SK⅐HPN complex, as shown recently by the elegant use of active sitelabeled fluorescent HPN derivatives (10). Peptide walking studies in our laboratory had also indicated that short peptides based on the primary structure of SK, particularly those derived from selected regions in the ␣ and ␤ domains, displayed competitive inhibition for HPG activation by the preformed SK⅐HPN complex under conditions where the 1:1 complexation of SK and HPN was essentially unaffected (11,12) However, the crystal structure of SK complexed with microplasmin(ogen) (8), while providing a high degree of resolution of the residues involved in the SK⅐PN complexation, yielded few unambiguous insights regarding the interactions engendered between the activator complex and substrate HPG. This is probably due to the binary nature of the complex (i.e.…”

“…Recent biochemical studies suggest that an exosite-mediated substrate HPG binding, independent of the primary covalent specificity of the HPN active site, represents the major mechanism of SK-induced changes in the macromolecular substrate specificity of HPN (10,12). The structural basis whereby such an exosite contributes toward the change in substrate specificity of the HPN active site consequent to SK binding has, however, remained essentially unknown so far.…”

“…Unlike free HPN, however, which is essentially a trypsin-like protease with broad substrate preference, SK⅐HPN displays a very narrow substrate specificity (4). The structural basis of the conversion of the broadly specific serine protease HPN to a highly substrate-specific protease, once complexed with the "cofactor" SK, with exclusive propensity for acting on the target scissile peptide bond in HPG has been the subject of active investigations with both fundamental and applied implications (5)(6)(7)(8)(9)(10)(11)(12).…”

“…Of the three domains of SK, the central ␤ domain displays maximal affinity for HPG (15), the N-terminal ␣ domain displays relatively lesser affinity for HPG with the ␥ domain showing much less affinity of for HPG. 2 Solution and structural studies suggest that both ␣ and ␤ domains are involved in the substrate recognition phenomenon (8,12). Mutagenesis studies have also implicated positively charged residues in the ␤ domain to be important but have failed to show that these residues are directly involved in substrate recognition by the binary complex (17).…”

“…The change in absorbance at 405 nm was monitored to compute the kinetics of amidolytic activation (12,24).…”