Mapping of the plasminogen binding site of streptokinase with short synthetic peptides

Nihalani, Deepak ; Raghava, G. P. S. ; Sahni, Girish (1997) Mapping of the plasminogen binding site of streptokinase with short synthetic peptides Protein Science, 6 (6). pp. 1284-1292. ISSN 0961-8368

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Although several recent studies employing various truncated fragments of streptokinase (SK) have demonstrated that the high-affinity interactions of this protein with human plasminogen (HPG) to form the activator complex (SK-HPG) are located in the central region of SK, the exact location and nature of such HPG interacting site(s) is still unclear. In order to locate the "core" HPG binding ability in SK, we focused on the primary structure of a tryptic fragment of SK derived from the central region (SK 143-293) that could bind as well as activate HPG, albeit at reduced levels in comparison to the activity of the native, full-length protein. Because this fragment was refractory to further controlled proteolysis, we took recourse to a synthetic peptide approach wherein the HPG interacting properties of 16 overlapping 20-mer peptides derived from this region of SK were examined systematically. Only four peptides from this set, viz., SK234-253, SK254-273, SK274-293, and SK263-282, together representing the contiguous sequence SK234-293, displayed HPG binding ability. This was established by a specific HPG-binding ELISA as well as by dot blot assay using 125I-labeled HPG. These results showed that the minimal sequence with HPG binding function resided between residues 234 and 293. None of the synthetic SK peptides was found to activate HPG, either individually or in combination, but, in competition experiments where each of the peptides was added prior to complex formation between SK and HPG, three of the HPG binding peptides (SK234-253, SK254-273, and SK274-293) inhibited strongly the generation of a functional activator complex by SK and HPG. This indicated that residues 234-293 in SK participate directly in intermolecular contact formation with HPG during the formation of the 1:1 SK-HPG complex. Two of the three peptides (SK234-253 and SK274-293), apart from interfering in SK-HPG complex formation, also showed inhibition of the amidolytic activity of free HPN by increasing the Km by approximately fivefold. A similar increase in Km for amidolysis by HPN as a result of complexation with SK has been interpreted previously to arise from the steric hinderance at or near the active site due to the binding of SK in this region. Thus, our results suggest that SK234-253 and SK274-293 also, like SK, bound close to the active site of HPN, an event that was reflected in the observed alteration in its substrate accessibility. By contrast, whereas the intervening peptide (SK254-273) could not inhibit amidolysis by free HPN, it showed a marked inhibition of the activation of "substrate" PG (human or bovine plasminogen) by activator complex, indicating that this particular region is intimately involved in interaction of the SK-HPG activator complex with substrate plasminogen during the catalytic cycle. This finding provides a rational explanation for one of the most intriguing aspects of SK action, i.e., the ability of the SK-HPG complex to catalyze selectively the activation of substrate molecules of PG to PN, whereas free HPN alone cannot do so. Taken together, the results presented in this paper strongly support a model of SK action in which the segment 234-293 of SK, by virtue of the epitopes present in residues 234-253 and 274-293, binds close to the active center of HPN (or, a cryptic active site, in the case of HPG) during the intermolecular association of the two proteins to form the equimolar activator complex; the segment SK254-273 present in the center of the core region then imparts an ability to the activator complex to interact selectively with substrate PG molecules during each PG activation cycle.

Item Type:Article
Source:Copyright of this article belongs to Cold Spring Harbor Laboratory Press.
Keywords:Active Site; Peptide Walking; Plasminogen; Plasminogen Binding; Streptokinase; Streptokinase-derived Peptides
ID Code:43058
Deposited On:09 Jun 2011 11:31
Last Modified:18 May 2016 00:09

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