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.2001 Sep;10(9):1897-904.
doi: 10.1110/ps.09001.

Characterization of bothrojaracin interaction with human prothrombin

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Characterization of bothrojaracin interaction with human prothrombin

R Q Monteiro et al. Protein Sci.2001 Sep.

Abstract

Bothrojaracin (BJC) is a 27-kD snake venom protein from Bothrops jararaca that has been characterized as a potent thrombin inhibitor. BJC binds to exosites I and II, with a dissociation constant of 0.7 nM, and influences but does not block the proteinase catalytic site. BJC also binds prothrombin through an interaction that has not been characterized. In the present work we characterize the interaction of BJC with prothrombin quantitatively for the first time, and identify the BJC binding site on human prothrombin. Gel filtration chromatography demonstrated calcium-independent, 1:1 complex formation between fluorescein-labeled BJC ([5F]BJC) and prothrombin, whereas no interactions were observed with activation fragments 1 or 2 of prothrombin. Isothermal titration calorimetry showed that binding of BJC to prothrombin is endothermic, with a dissociation constant of 76 +/- 32 nM. The exosite I-specific ligand, hirudin(54-65) (Hir(54-65) (SO(3)(-)), displaced competitively [5F]BJC from prothrombin. Titration of the fluorescent hirudin(54-65) derivative, [5F]Hir(54-65)(SO(3)(-)), with human prothrombin showed a dissociation constant of 7.0 +/- 0.2 microM, indicating a approximately 100-fold lower binding affinity than that exhibited by BJC. Both ligands, however, displayed a similar, approximately 100-fold increase in affinity for exosite I when prothrombin was activated to thrombin. BJC efficiently displaced [5F]Hir(54-65)(SO(3)(-)) from complexes formed with thrombin or prothrombin with dissociation constants of 0.7 +/- 0.9 nM and 11 +/- 80 nM, respectively, indicating that BJC and Hir(54-65)(SO(3)(-)) compete for the same exosite on these molecules. The results indicate that BJC is a potent and specific probe of the partially exposed anion-binding exosite (proexosite I) of human prothrombin.

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Figures

Fig. 1.
Fig. 1.
Formation of [5F]BJC–prothrombin complex and the absence of interaction of [5F]BJC with prothrombin fragments investigated by gel filtration chromatography. Superose 12 was equilibrated in TBS, and elution of [5F]BJC was monitored by the elution volume of the fluorescein fluorescence in the absence and presence of other proteins. The following samples (20-μL loop) were applied onto the column: (A) 0.33 μM [5F]BJC (0.18 μg); (B) 0.33 μM [5F]BJC (0.18 μg) + 1 μM human prothrombin (1.4 μg); (C) 0.3 μM [5F]BJC (0.18 μg) + 30 μM prothrombin fragment 1 (14 μg); (D) 0.3 μM [5F]BJC (0.18 μg) + 30 μM prothrombin fragment 2 (11 μg).
Fig. 2.
Fig. 2.
Calorimetric titration of BJC with human prothrombin. The binding isotherm at 25°C for titration of prothrombin (Pro) in the calorimetric cell containing 1 μM BJC was analyzed according to the independent single-site model as described in Materials and Methods. The solid line represents the least-squares fit of the experimental data with the parameters given in the text.
Fig. 3.
Fig. 3.
Fluorescence polarization titration of [5F]BJC–prothrombin complex with prothrombin fragments. Titration with prothrombin (Pro) fragment 1 (open circles) or fragment 2 (closed circles) was performed in TBS containing 100 nM [5F]BJC and 250 nM prothrombin. Experiments were performed at 25°C and fluorescence polarization measured as described in Materials and Methods. The arrow indicates the polarization value obtained for free [5F]BJC (closed diamonds).Inset: Direct titration of 100 nM [5F]BJC with human prothrombin (Pro) along with the fitted curve calculated with the parameters given in the text.
Fig. 4.
Fig. 4.
Direct titration of [5F]Hir54–65(SO3−) with human prothrombin. The change in fluorescence polarization of a solution containing 100 nM [5F]Hir54–65(SO3−) titrated with prothrombin (Pro) was performed in TBS at 25°C as described in Materials and Methods.Inset: Scatchard plot for the [5F]Hir54–65(SO3−) interaction with prothrombin.
Fig. 5.
Fig. 5.
Competitive titration of the [5F]BJC–prothrombin complex with Hir54–65(SO3−). Titration with Hir54–65(SO3−) was performed in TBS containing 100 nM [5F]BJC and 250 nM prothrombin. Experiments were performed at 25°C, and polarization was measured as described in Materials and Methods. The solid line represents the nonlinear least-squares fit by the model for competitive binding of [5F]BJC and the unlabeled hirudin peptide to prothrombin, with the fitted parameters given in the text.
Fig. 6.
Fig. 6.
Competitive titration of the [5F]Hir54–65(SO3−)–thrombin and [5F]Hir54–65(SO3−)–prothrombin complexes with BJC. Titration with BJC of solutions containing: (A) 160 nM thrombin and 40 nM [5F]Hir54–65 (SO3−). (B) Titration of 2.1 μM prothrombin plus 40 nM [5F]Hir54–65 (SO3−) with BJC. Experiments were performed and analyzed as described in Materials and Methods and the text. The solid lines represent the best fits with the parameters given in the text. For the analysis of the thrombin titration, the dissociation constant for the hirudin peptide binding to thrombin was fixed at the determined value of 25 nM (Anderson et al. 2000a). Similarly, for the prothrombin titration, the dissociation constant for hirudin peptide binding to prothrombin was fixed at the determined value of 7 μM.
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References

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