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.
Highlights
Streptokinase (SK)1 is a widely used bacterial thrombolytic protein that is secreted by several species of -hemolytic streptococci [1, 2]
This remarkable alteration of the macromolecular substrate specificity of HPN by SK as a result of the latter’s “protein co-factor” property, which has been a subject of intense investigations, is currently thought to be due to exosites generated on the SK1⁄7HPN activator complex, as demonstrated recently by the elegant use of active sitelabeled fluorescent HPN derivatives [10]
A pertinent question that can be posed in the above context is whether any of the isolated domains of SK possess, like the single domain of SAK, the ability to bind with human plasminogen (HPG) in both substrate and partner modes and, if the answer is affirmative, whether this binding is functionally translated into a capability, even if highly compromised compared with native SK, to switch the nonspecific substrate preference of partner plasmin to that of a HPG activator enzyme
Summary
HPG was either purchased from Roche Applied Science or purified from human plasma by affinity chromatography [25]. DEAE-Sepharose (Fast-flow) and Chelating Sepharose were procured from Amersham Biosciences, and phenyl-agarose for hydrophobic interaction chromatography was purchased from Affinity Chromatography Ltd. The T7 RNA polymerase promoter-based expression vector, pET-23d and Bug buster® (a commercial reagent for rapid bacterial cell lysis) were procured from Novagen Inc. The inclusion bodies of the ␣ domain dissolved in 8 M urea and the soluble ␥ domain obtained after cell lysis were further diluted 15-fold in 50 mM sodium phosphate buffer (pH 7.5) containing 10 mM imidazole and 250 mM NaCl before loading on the immobilized metal affinity chromatography matrices. The purification involved lyzing the cells by sonication followed by ammonium sulfate precipitation (60% saturation) and two tandem chromatographic steps, namely hydrophobic ion chromatography on phenyl-agarose and ion exchange chromatography on DEAE-Sepharose (Fast-flow) essentially as described for SK above. Secondary structure analysis to compute the content of ␣ helix,  sheet, random coil, and other secondary structure(s) were carried out using the algorithm described by Yang et al [31]
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