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 </=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.