creators_name: Sundram, Vasudha
creators_name: Nanda, Jagpreet S
creators_name: Rajagopal, Kammara
creators_name: Dhar, Jayeeta
creators_name: Chaudhary, Anita
creators_name: Sahni, Girish
type: article
datestamp: 2012-01-06 14:58:44
lastmod: 2012-01-06 14:58:44
metadata_visibility: show
title: Domain truncation studies reveal that the streptokinase-plasmin activator complex utilizes long range protein-protein interactions with macromolecular substrate to maximize catalytic turnover.
ispublished: pub
subjects: QD
full_text_status: restricted
note: Copyright of this article belongs to ASBMB.
abstract: To explore the interdomain co-operativity during human plasminogen (HPG) activation by streptokinase (SK), we expressed the cDNAs corresponding to each SK domain individually (alpha, beta, and gamma), and also their two-domain combinations, viz. alphabeta and betagamma in Escherichia coli. After purification, alpha and beta 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 alphabeta and betagamma 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.
date: 2003-08-15
date_type: published
publication: The Journal of biological chemistry
volume: 278
number: 33
publisher: ASBMB
pagerange: 30569-77
refereed: TRUE
issn: 0021-9258
official_url: http://www.jbc.org/content/278/33/30569.long
related_url_url: http://www.jbc.org/content/278/33/30569.long
related_url_type: pub
citation:   Sundram, Vasudha and Nanda, Jagpreet S and Rajagopal, Kammara and Dhar, Jayeeta and Chaudhary, Anita and Sahni, Girish  (2003) Domain truncation studies reveal that the streptokinase-plasmin activator complex utilizes long range protein-protein interactions with macromolecular substrate to maximize catalytic turnover.  The Journal of biological chemistry, 278 (33).  pp. 30569-77.  ISSN 0021-9258     
document_url: http://crdd.osdd.net/open/242/1/sahni2003.pdf