Abstract
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.
Highlights
Mogen, plasminogen (Pg) into the clot-dissolving proteinase, plasmin (Pm) (1)
Protein Purification and Characterization—[Glu]Pg carbohydrate variant 2 was purified from human plasma (35, 36). [Lys]Pg was generated by incubation of 40 M [Glu]Pg with 2 M Pm in 50 mM Tris-Cl, 20 mM L-lysine, 0.1 M NaCl, pH 9.0, for 30 min at 25 °C, and isolated by affinity chromatography on soybean trypsin inhibitor-agarose and aminohexyl-agarose as described previously (3, 11). [Lys]Pm was prepared by activation of 10 M [Glu]Pg with 90 units/ml urokinase (Calbiochem) in 10 mM MES, 10 mM Hepes, 0.15 M NaCl, 20 mM 6-aminohexanoic acid (6-AHA), 1 mg/ml polyethylene glycol 8000 (PEG), pH 7.4, at 25 °C, and purified by chromatography on soybean trypsin inhibitor-agarose (9, 37)
Lysine binding site (LBS)-dependent interactions of SK occur through the kringle domains of Pg (8, 12, 14, 32), whereas interactions of SK with the catalytic domain are LBSindependent as shown by the crystal structure of the SK1⁄7micro-Pm complex (17)
Summary
Protein Purification and Characterization—[Glu]Pg carbohydrate variant 2 was purified from human plasma (35, 36). [Lys]Pg was generated by incubation of 40 M [Glu]Pg with 2 M Pm in 50 mM Tris-Cl, 20 mM L-lysine, 0.1 M NaCl, pH 9.0, for 30 min at 25 °C, and isolated by affinity chromatography on soybean trypsin inhibitor-agarose and aminohexyl-agarose as described previously (3, 11). [Lys]Pm was prepared by activation of 10 M [Glu]Pg with 90 units/ml urokinase (Calbiochem) in 10 mM MES, 10 mM Hepes, 0.15 M NaCl, 20 mM 6-AHA, 1 mg/ml polyethylene glycol 8000 (PEG), pH 7.4, at 25 °C, and purified by chromatography on soybean trypsin inhibitor-agarose (9, 37). ATA-FFR-[Lys]Pm was prepared by incubating 19 M active site-titrated Pm with a 6-fold excess of inhibitor in 0.1 M Hepes, 0.3 M NaCl, 1 mM EDTA, 10 mM 6-AHA, 1 mg/ml PEG, pH 7.0, for 1 h at 25 °C, until activity was Ͻ0.01% of the initial value. Preparation of Catalytic Site-labeled Pg—[Glu]Pg and [Lys]Pg were labeled at the catalytic site, separated from free dye on Sephadex G-25, and purified by affinity chromatography on SK-Affi-Gel and PmSulfolink as described previously, with the addition of 20 mM 6-AHA to all reaction buffers (9, 11). Stoichiometries of probe incorporation were 0.8 –1.0 mol fluorescein/mol of Pg for [5F]FFR-[Glu]Pg, and [5F]FFR-[Lys]Pg. Fluorescence Studies—Fluorescence titrations of [5F]FFR-[Glu]Pg, [5F]FFR-[Lys]Pg, and [5F]FFR-Pm with recombinant SK were done by serial addition of small volumes to labeled Pg or Pm in 50 mM Hepes, 0.125 M NaCl, 1 mM EDTA, 1 mg/ml PEG, 1 mg/ml bovine serum albumin, 1 M FFR-CH2Cl, pH 7.4, with and without 10 mM 6-AHA. The change in free energy of association for SK species binding to Pg or Pm was calculated from ⌬G0 ϭ RTln(KD)
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