Role of the streptokinase α-domain in the interactions of streptokinase with plasminogen and plasmin

The role of the streptokinase ( SK) alpha-domain in plasminogen ( Pg) and plasmin ( Pm) interactions was investigated in quantitative binding studies employing active site fluorescein-labeled [ Glu] Pg, [ Lys] Pg, and [ Lys] Pm, and the SK truncation mutants, SK-( 55 - 414), SK-( 70 414), and SK-( 152 - 414). Lysine binding site ( LBS)-dependent and - independent binding were resolved from the effects of the lysine analog, 6-aminohexanoic acid. The mutants bound indistinguishably, consistent with unfolding of the alpha-domain on deletion of SK-( 1 - 54). The affinity of SK for [ Glu] Pg was LBS-independent, and although [ Lys] Pg affinity was enhanced 13-fold by LBS interactions, the LBS-independent free energy contributions were indistinguishable. alpha-Domain truncation reduced the affinity of SK for [ Glu] Pg 2-7-fold and [ Lys] Pg less than or equal to 2-fold, but surprisingly, rendered both interactions near totally LBS-dependent. The LBS-independent affinity of SK for [ Lys] Pm, 3000-fold higher compared with [ Lys] Pg, was reduced dramatically by alpha-domain truncation. Thermodynamic analysis demonstrates that the SK alpha-domain contributes substantially to affinity for all Pg/Pm species solely through LBS-independent interactions, and that the higher affinity of SK for [ Lys] Pm compared with [ Lys] Pg involves all three SK domains. The residual affinity of the SK betagamma-fragment for all Pg/Pm species was increased by an enhanced contribution to complex stability from LBS-dependent interactions or free energy coupling between LBS-dependent and - independent interactions. Redistribution of the free energy contributions accompanying alpha-domain truncation demonstrates the interdependence of SK domains in stabilizing the SK- Pg/Pm complexes. The flexible segments connecting the SK alpha, beta, and gamma domains allow their rearrangement into a distinctly different bound conformation accompanying loss of the constraint imposed by interactions of the alpha-domain.