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J. Biol. Chem., Vol. 278, Issue 33, 30569-30577, August 15, 2003

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Domain Truncation Studies Reveal That the Streptokinase-Plasmin Activator Complex Utilizes Long Range Protein-Protein Interactions with Macromolecular Substrate to Maximize Catalytic Turnover^*

Vasudha Sundram , Jagpreet S. Nanda , Kammara Rajagopal, Jayeeta Dhar, Anita Chaudhary  and Girish Sahni ¶

From the Institute of Microbial Technology, Chandigarh 160036, India

To explore the interdomain co-operativity during human plasminogen (HPG) activation by streptokinase (SK), we expressed the cDNAs corresponding to each SK domain individually (, , and ), and also their two-domain combinations, viz. and in Escherichia coli. After purification, and showed activator activities of approximately 0.4 and 0.05%, respectively, as compared with that of native SK, measured in the presence of human plasmin, but the bi-domain constructs and showed much higher co-factor activities (3.5 and 0.7% of native SK, respectively). Resonant Mirror-based binding studies showed that the single-domain constructs had significantly lower affinities for "partner" HPG, whereas the affinities of the two-domain constructs were remarkably native-like with regards to both binary-mode as well as ternary mode ("substrate") binding with HPG, suggesting that the vast difference in co-factor activity between the two- and three-domain structures did not arise merely from affinity differences between activator species and HPG. Remarkably, when the co-factor activities of the various constructs were measured with microplasminogen, the nearly 50-fold difference in the co-factor activity between the two- and three-domain SK constructs observed with full-length HPG as substrate was found to be dramatically attenuated, with all three types of constructs now exhibiting a low activity of approximately 1–2% compared to that of SK·HPN and HPG. Thus, the docking of substrate through the catalytic domain at the active site of SK-plasmin(ogen) is capable of engendering, at best, only a minimal level of co-factor activity in SK·HPN. Therefore, apart from conferring additional substrate affinity through kringle-mediated interactions, reported earlier (Dhar et al., 2002; J. Biol. Chem. 277, 13257), selective interactions between all three domains of SK and the kringle domains of substrate vastly accelerate the plasminogen activation reaction to near native levels.

Received for publication, April 11, 2003 , and in revised form, May 20, 2003.

^* This work was supported by generous grants from the Department of Biotechnology and the Council of Scientific and Industrial Research, Government of India. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Both authors contributed equally to this work.

Present address: Dept. Environmental Sciences, Indian Agricultural Research Institute, Pusa, Dr. K. S. Krishnan Marg, New Delhi 110012, India.

^¶ To whom correspondence should be addressed: Inst. of Microbial Technology, Sector 39-A, Chandigarh 160036, India. Fax: 91-172-690585 or 91-172-690632; E-Mail: sahni{at}imtech.res.in.

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