Role of the streptokinase alpha-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 </=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.
Bean
Ronald R
RR
Department of Pathology, School of Medicine, Vanderbilt University, Nashville, Tennessee 37232, USA.
Verhamme
Ingrid M
IM
Bock
Paul E
PE
D015119:Aminocaproic Acid / Q000737:chemistry; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D002713:Chlorine / Q000737:chemistry; D005341:Fibrinolysin / Q000737:chemistry*; D017353:Gene Deletion; D006801:Humans; D008070:Lipopolysaccharides / Q000737:chemistry; D008239:Lysine / Q000737:chemistry; D008856:Microscopy, Fluorescence; D009154:Mutation; D010958:Plasminogen / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D013300:Streptokinase / Q000737:chemistry*; D013816:Thermodynamics
D008070:Lipopolysaccharides; D011994:Recombinant Proteins; D002713:Chlorine; D010958:Plasminogen; D013300:Streptokinase; D005341:Fibrinolysin; D008239:Lysine; D015119:Aminocaproic Acid