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Role of Prothrombin Fragment 1 in the Pathway of Regulatory Exosite I Formation during Conversion of Human Prothrombin to Thrombin

2003; Elsevier BV; Volume: 278; Issue: 45 Linguagem: Inglês

10.1074/jbc.m306916200

1083-351X

Patricia J. Anderson, Paul Bock,

Hemophilia Treatment and Research

Prothrombin (Pro) activation by factor Xa generates the thrombin catalytic site and exosites I and II. The role of fragment 1 (F1) in the pathway of exosite I expression during Pro activation was characterized in equilibrium binding studies using hirudin54–65 labeled with 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoate ([NBD]Hir54–65(SO3-)) or 5-(carboxy)fluorescein ([5F]Hir54–65(SO3-)). [NBD]Hir54–65(SO3-) distinguished exosite I environments on Pro, prethrombin 1 (Pre 1), and prethrombin 2 (Pre 2) but bound with the same affinities as [5F]Hir54–65(SO3-). Conversion of Pro to Pre 1 caused a 7-fold increase in affinity for the peptides. Conversely, fragment 1.2 (F1.2) decreased the affinity of Pre 2 for [5F]Hir54–65(SO3-) by 3-fold. This was correlated with a 16-fold increased affinity of F1.2 for Pre 2 in comparison to thrombin, demonstrating an enhancing effect of F1 on F1.2 binding. The active intermediate, meizothrombin, demonstrated a 50- to 220-fold increase in exosite affinity. Free thrombin and thrombin·F1.2 complex bound [5F]Hir54–65(SO3-) with indistinguishable affinity, indicating that the effect of F1 on peptide binding was eliminated upon expression of catalytic activity and exosite I. The results demonstrate a new zymogen-specific role for F1 in modulating the affinity of ligands for exosite I. This may reflect a direct interaction between the F1 and Pre 2 domains in Pro that is lost upon folding of the zymogen activation domain. The effect of F1 on (pro)exosite I and the role of (pro)exosite I in factor Va-dependent substrate recognition suggest that the Pro activation pathway may be regulated by (pro)exosite I interactions with factor Va. Prothrombin (Pro) activation by factor Xa generates the thrombin catalytic site and exosites I and II. The role of fragment 1 (F1) in the pathway of exosite I expression during Pro activation was characterized in equilibrium binding studies using hirudin54–65 labeled with 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoate ([NBD]Hir54–65(SO3-)) or 5-(carboxy)fluorescein ([5F]Hir54–65(SO3-)). [NBD]Hir54–65(SO3-) distinguished exosite I environments on Pro, prethrombin 1 (Pre 1), and prethrombin 2 (Pre 2) but bound with the same affinities as [5F]Hir54–65(SO3-). Conversion of Pro to Pre 1 caused a 7-fold increase in affinity for the peptides. Conversely, fragment 1.2 (F1.2) decreased the affinity of Pre 2 for [5F]Hir54–65(SO3-) by 3-fold. This was correlated with a 16-fold increased affinity of F1.2 for Pre 2 in comparison to thrombin, demonstrating an enhancing effect of F1 on F1.2 binding. The active intermediate, meizothrombin, demonstrated a 50- to 220-fold increase in exosite affinity. Free thrombin and thrombin·F1.2 complex bound [5F]Hir54–65(SO3-) with indistinguishable affinity, indicating that the effect of F1 on peptide binding was eliminated upon expression of catalytic activity and exosite I. The results demonstrate a new zymogen-specific role for F1 in modulating the affinity of ligands for exosite I. This may reflect a direct interaction between the F1 and Pre 2 domains in Pro that is lost upon folding of the zymogen activation domain. The effect of F1 on (pro)exosite I and the role of (pro)exosite I in factor Va-dependent substrate recognition suggest that the Pro activation pathway may be regulated by (pro)exosite I interactions with factor Va. In the penultimate step of blood coagulation, thrombin is generated by factor Xa cleavage of Pro at Arg271-Thr272 and Arg320-Ile321. Pro activation is regulated by the protein cofactor, factor Va, phospholipids, and calcium, which together elicit a ∼300,000-fold increase in the activation rate through formation of the membrane-bound, factor Xa·factor Va, prothrombinase complex (1Nesheim M.E. Taswell J.B. Mann K.G. J. Biol. Chem. 1979; 254: 10952-10962Abstract Full Text PDF PubMed Google Scholar, 2Rosing J. Tans G. Govers-Riemslag J.W.P. Zwaal R.F.A. Hemker H.C. J. Biol. Chem. 1980; 255: 274-283Abstract Full Text PDF PubMed Google Scholar, 3Mann K.G. Nesheim M.E. Church W.R. Haley P. Krishnaswamy S. Blood. 1990; 76: 1-16Crossref PubMed Google Scholar, 4Butenas S. Mann K.G. Biochem. (Moscow). 2002; 67: 5-15Crossref Scopus (231) Google Scholar). In the absence of factor Va, factor Xa cleavage of Arg271-Thr272 results in accumulation of prethrombin 2 (Pre 2) 1The abbreviations used are: Pre 1, prethrombin 1; Pre 2, prethrombin 2; Pro, prothrombin; F1, fragment 1; F2, fragment 2; F1.2, fragment 1.2; Gla, γ-carboxyglutamic acid; MzT(-F1), meizothrombin des-fragment 1; MzT, meizothrombin; T, thrombin; FPR-CH2Cl, D-Phe-Pro-Arg-CH2Cl; Hir54–65(SO3-), Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr(SO3-)-Leu-Gln; [5F]Hir54–65(SO3-), Hir54–65(SO3-) labeled at the amino terminus with 5-carboxy(fluorescein); [NBD]Hir54–65(SO3-), Hir54–65(SO3-) labeled at the amino terminus with succinimidyl 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoate; Mes, 4-morpholineethanesulfonic acid. and activation fragment 1.2 (F1.2) noncovalent complex as an activation intermediate. Factor Va confers a substrate specificity change such that factor Xa cleavage of Arg320-Ile321 is preferred, and the catalytically active intermediate meizothrombin (MzT) is generated predominately (5Rosing J. Zwaal R.F.A. Tans G. J. Biol. Chem. 1986; 261: 4224-4228Abstract Full Text PDF PubMed Google Scholar, 6Krishnaswamy S. Mann K.G. Nesheim M.E. J. Biol. Chem. 1986; 261: 8977-8984Abstract Full Text PDF PubMed Google Scholar, 7Krishnaswamy S. Church W.R. Nesheim M.E. Mann K.G. J. Biol. Chem. 1987; 262: 3291-3299Abstract Full Text PDF PubMed Google Scholar, 8Carlisle T.L. Bock P.E. Jackson C.M. J. Biol. Chem. 1990; 265: 22044-22055Abstract Full Text PDF PubMed Google Scholar). Cleavage of the alternative sites in the intermediates generates the products thrombin and F1.2. The physiological substrate specificity of thrombin and the localization of thrombin activity are mediated by one or both of two exosites (I and II) distinct from the catalytic site (9Stubbs M.T. Bode W. Thromb. Res. 1993; 69: 1-58Abstract Full Text PDF PubMed Scopus (445) Google Scholar, 10Di Cera E. Dang Q.D. Ayala Y.M. Cell. Mol. Life Sci. 1997; 53: 701-773Crossref PubMed Scopus (158) Google Scholar). Exosite I is in a precursor state on Pro called proexosite I (11Anderson P.J. Nesset A. Dharmawardana K.R. Bock P.E. J. Biol. Chem. 2000; 275: 16428-16434Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 12Monteiro R.Q. Bock P.E. Bianconi M.L. Zingali R.B. Protein Sci. 2001; 10: 1897-1904Crossref PubMed Scopus (27) Google Scholar). Activation of the catalytic site in the formation of thrombin is accompanied by an overall ∼100-fold increase in affinity of exosite I for hirudin54–65 (Hir54–65(SO3-)). The proexosite has been implicated in the mechanism of factor Va rate acceleration of Pro activation and cofactor-mediated Pro substrate recognition (13Anderson P.J. Nesset A. Dharmawardana K.R. Bock P.E. J. Biol. Chem. 2000; 275: 16435-16442Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 14Monteiro R.Q. Zingali R.B. Thromb. Hemost. 2002; 87: 288-293Crossref PubMed Scopus (27) Google Scholar). The role of proexosite I in substrate recognition is supported further by recent site-directed mutagenesis studies demonstrating that mutation of proexosite I residues in prethrombin 1 (Pre 1) results in loss of factor Va cofactor activity and is correlated with loss of affinity of the proexosite for Hir54–65(SO3-) (15Chen L. Yang L. Rezaie A. J. Biol. Chem. 2003; 278: 27564-27569Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). A natural mutation of Arg67 to His in proexosite I of Pro isolated from a patient with a severe procoagulant defect and mild bleeding phenotype also showed reduced factor Va acceleration of its activation (16Akhavan S. De Cristofaro R. Peyvandi F. Lavoretano S. Landolfi R. Mannucci P.M. Blood. 2002; 100: 1347-1353Crossref PubMed Scopus (28) Google Scholar). In the preceding paper, proexosite I on Pre 1, a Pro analog that lacks the fragment 1 (F1) domain, and Pre 2 was shown to be activated by cleavage of Arg320-Ile321, generating the active intermediate, meizothrombin des-fragment 1 (MzT(-F1)), and the product, thrombin, respectively. Removal of F1 from Pro by thrombin cleavage to yield Pre 1 resulted in a 6-fold increase in affinity for hirudin peptides. This result suggested a new role for F1 in modulating activation of exosite I. Previous studies also suggested a role for F1 in expression of exosite I in the observation that macromolecular exosite I ligands bound to MzT(-F1) but not meizothrombin (MzT) (17Wu Q. Picard V. Aiach M. Sadler J.E. J. Biol. Chem. 1994; 269: 3725-3730Abstract Full Text PDF PubMed Google Scholar). The roles of F1, F2, and the catalytic domain (Pre 2) in factor Va regulation of Pro activation are not fully understood. Early studies demonstrated a rate-enhancing effect of F2 in factor Va-accelerated Pre 2 activation (18Esmon C.T. Jackson C.M. J. Biol. Chem. 1974; 249: 7791-7797Abstract Full Text PDF PubMed Google Scholar), suggesting that F2 mediated Pro binding to factor Va. Subsequent studies demonstrated similarly significant rate-enhancing effects of F2 and F1.2 on Pre 2 activation in solution (19Krishnaswamy S. Walker R.K. Biochemistry. 1997; 36: 3319-3330Crossref PubMed Scopus (38) Google Scholar). A kinetic analysis of Pre 2 activation by the membrane-bound factor Xa·factor Va complex, however, does not support a significant role for F2 (19Krishnaswamy S. Walker R.K. Biochemistry. 1997; 36: 3319-3330Crossref PubMed Scopus (38) Google Scholar) and indicates that Pre 2 substrate recognition is mediated by exosites expressed on factor Xa in the factor Va-assembled complex (20Krishnaswamy S. Betz A. Biochemistry. 1997; 36: 12080-12086Crossref PubMed Scopus (86) Google Scholar, 21Betz A. Krishnaswamy S. J. Biol. Chem. 1998; 273: 10709-10718Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 22Orcutt S.J. Pietropaolo C. Krishnaswamy S. J. Biol. Chem. 2002; 278: 46191-46196Abstract Full Text Full Text PDF Scopus (40) Google Scholar). Productive binding of Pro to factor Va in the prothrombinase complex has also been linked to the Gla domain (23Blostein M.D. Rigby A.C. Jacobs M. Furie B. Furie B.C. J. Biol. Chem. 2000; 275: 38120-38126Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) and kringle domains of both F1 and F2 (24Kotkow K.J. Deitcher S.R. Furie B. Furie B.C. J. Biol. Chem. 1995; 270: 4551-4557Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 25Deguchi H. Takeya H. Gabazza E.C. Nishioka J. Suzuki K. Biochem. J. 1997; 321: 729-735Crossref PubMed Scopus (44) Google Scholar). Our studies of thrombin- and Pro-factor Va interactions support a direct role for proexosite I on the Pro catalytic domain in mediating productive binding to the heavy chain subunit of factor Va within the factor Xa·factor Va complex (13Anderson P.J. Nesset A. Dharmawardana K.R. Bock P.E. J. Biol. Chem. 2000; 275: 16435-16442Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 26Dharmawardana K.R. Bock P.E. Biochemistry. 1998; 37: 13143-13152Crossref PubMed Scopus (49) Google Scholar, 27Dharmawardana K.R. Olson S.T. Bock P.E. J. Biol. Chem. 1999; 274: 18635-18643Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The proexosite I-factor Va interactions are thought to be influenced by changes in exosite I expression during Pro activation. To resolve the role of F1 in the activation of exosites I and II during Pro activation, binding of ligands specific for either exosite were characterized quantitatively for the Pro activation intermediates. Fluorescent derivatives of Hir54–65(SO3-) labeled at the amino termini with 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoate ([NBD]Hir54–65(SO3-)) or 5-carboxy(fluorescein) ([5F]Hir54–65(SO3-)) were used as specific probes of exosite I formation. [NBD]Hir54–65(SO3-) displayed fluorescence spectral changes upon binding to the Pro activation intermediates that reported different proexosite I environments on Pro, Pre 1, and Pre 2. Expression of exosite I on the Pro activation intermediates, as measured by the increase in affinity for the peptides, displayed a similar pattern as for the Pre 1 activation intermediates, where initial cleavage at Arg320-Ile321 caused simultaneous activation of the active site and full activation of exosite I (28Anderson P.J. Nesset A. Bock P.E. J. Biol. Chem. 2003; 278: 44482-44488Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Binding of F1.2 to Pre 2 decreased the affinity of the fluorescein-labeled peptide for exosite I by 3-fold, whereas no effect of F1.2 was observed for thrombin. The attenuating effect of F1 was correlated with binding of F1.2 to Pre 2 with a 16-fold higher affinity compared with thrombin. The results indicate that F1.2 interacts with the zymogen proteinase domain with greater affinity than the active proteinase and decreases the affinity of exosite I for hirudin peptides only in the zymogen forms. The results characterize a new role of F1 in the expression of exosites I and II, which is disengaged on folding of the proteinase “activation domain” (29Huber R. Bode W. Accts. Chem. Res. 1978; 11: 114-122Crossref Scopus (610) Google Scholar) into the catalytically active form, simultaneous with activation of exosite I. These observations suggest that changes in (pro)exosite I interactions mediated by the F1 domain and differential expression of exosite I on the Pro activation intermediates may regulate factor Va-Pro interactions that control the activation pathway. Protein Purification and Characterization—Human Pro, Pre 2, and thrombin were prepared as described in the preceding paper (28Anderson P.J. Nesset A. Bock P.E. J. Biol. Chem. 2003; 278: 44482-44488Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Hir54–65(SO3-) was labeled with 5-carboxy(fluorescein) ([5F]Hir54–65(SO3-)) or succinimidyl 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-hexanoate ([NBD]Hir54–65(SO3-)) and characterized as previously described (30Bock P.E. Olson S.T. Björk I. J. Biol. Chem. 1997; 272: 19837-19845Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Traces of thrombin were removed from Pro by chromatography on sulfopropyl-Sephadex. Thrombin was active-site labeled with 6-(iodoacetamido)fluorescein and characterized as previously described (31Bock P.E. J. Biol. Chem. 1992; 267: 14963-14973Abstract Full Text PDF PubMed Google Scholar). Active-site blocked MzT was prepared by activation of 20 μm Pro with 5 units/ml of ecarin (Sigma) in the presence of 400 μm FPR-CH2Cl at 25 °C in 50 mm Hepes, 110 mm NaCl, 5 mm CaCl2, pH 7.4. After 1 h, FPR-MzT was concentrated by YM10 ultrafiltration and dialyzed against buffer containing 0.1 μm FPR-CH2Cl. Preparation of Fragment 1.2—F1.2 was prepared by activation of 20 μm FPR-MzT with 30 nm factor Xa in the presence of 3 μm Thromstop (American Diagnostica) for 2 h at 25 °C. The reaction was chromatographed on sulfopropyl-Sephadex (2.5 × 21 cm) equilibrated with 20 mm Mes, 0.1 m NaCl, 0.1 mm EDTA, 1 mm benzamidine, pH 6.5. F1, F2, and F1.2 co-eluted from the resin in the void volume. FPR-thrombin was eluted from the column with a 1-liter gradient of buffer up to 0.8 m NaCl. The fragments were dialyzed against 20 mm Hepes, 0.25 m NaCl, 1 mm benzamidine, 2 mm EDTA, 0.02% NaN3, pH 7.4, and F1.2 was separated from F1 and F2 by chromatography on fast-flow Q-Sepharose (2.5 × 13 cm) (Amersham Biosciences). The column was washed with 20 mm Hepes, 0.25 m NaCl, 1 mm benzamidine, 0.02% NaN3, pH 7.4, eluting F2. F1 and F1.2 were separated by a 360-ml gradient of buffer up to 20 mm CaCl2. The purified activation fragments were concentrated by YM3 (F2) or YM10 (F1 and F1.2) ultrafiltration and dialyzed against 50 mm Hepes, 110 mm NaCl, 5 mm CaCl2, 1 mg/ml polyethylene glycol 8000, pH 7.4, containing 0.1 μm FPR-CH2Cl. Protein concentrations were determined by absorbance at 280 nm with the same absorption coefficients ((mg/ml–1)/cm–1) and molecular weights as in the previous paper (28Anderson P.J. Nesset A. Bock P.E. J. Biol. Chem. 2003; 278: 44482-44488Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar) or with the following constants (7Krishnaswamy S. Church W.R. Nesheim M.E. Mann K.G. J. Biol. Chem. 1987; 262: 3291-3299Abstract Full Text PDF PubMed Google Scholar, 32Fenton II, J.W. Fasco M.J. Stackrow A.B. Aronson D.L. Young A.M. Finlayson J.S. J. Biol. Chem. 1977; 252: 3587-3598Abstract Full Text PDF PubMed Google Scholar, 33Mann K.G. Elion J. Butkowski R.J. Downing M. Nesheim M.E. Methods Enzymol. 1981; 80: 286-302Crossref PubMed Scopus (97) Google Scholar): MzT, 1.47, 71,600; F1.2, 1.12, 34,500. Fluorescence Studies—Fluorescence was measured with an SLM 8100 fluorometer using cuvettes coated with polyethylene glycol 20,000. All experiments were performed in 50 mm Hepes, 110 mm NaCl, 5 mm CaCl2, 1 mg/ml polyethylene glycol 8000, pH 7.4, containing 0.1 μm FPR-CH2Cl at 25 °C. Emission spectra (4-nm band pass) of 0.4 μm [NBD]Hir54–65(SO3-) with near-saturating concentrations of Pro, Pre 1, and the activation intermediates were recorded with excitation at 480 nm (16-nm band pass) and normalized to the initial fluorescence intensity. Corrections for background (≤1%) were made from parallel measurements on blanks lacking the probe, and corrections for dilution were ≤ 5%. Direct binding of the labeled peptides to Pro, the Pro activation intermediates, and thrombin was measured by titrating the labeled peptide with each protein. Changes in fluorescence (ΔF/F 0 = F obs – F 0/F 0) were monitored as described for [5F]Hir54–65(SO3-) (28Anderson P.J. Nesset A. Bock P.E. J. Biol. Chem. 2003; 278: 44482-44488Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). For [NBD]Hir54–65(SO3-), fluorescence was measured with excitation at 480 nm (16-nm band pass) and emission at 540 nm (8-nm band pass). The data were fit by the quadratic binding equation to obtain the maximal fluorescence change (ΔF max/F 0) and the dissociation constant (KD) for peptide binding. Competitive binding of labeled and unlabeled Hir54–65(SO3-) was measured by titrations of fixed concentrations of labeled peptide and protein as a function of competing ligand concentration. The direct and competitive binding data were analyzed simultaneously with the cubic binding equation (28Anderson P.J. Nesset A. Bock P.E. J. Biol. Chem. 2003; 278: 44482-44488Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 34Lindahl P. Raub-Segall E. Olson S.T. Björk I. Biochem. J. 1991; 276: 387-394Crossref PubMed Scopus (29) Google Scholar, 35Olson S.T. Bock P.E. Sheffer R. Arch. Biochem. Biophys. 1991; 286: 533-545Crossref PubMed Scopus (42) Google Scholar). Binding of F1.2 to Thrombin and Pre 2—Binding of F1.2 to thrombin was measured from the change in fluorescence of 190 nm [6F]FPR-thrombin as a function of F1.2 concentration. Competitive binding of Pre 2 was measured by titration of mixtures of 190 nm [6F]FPR-thrombin and 20 μm Pre 2 with F1.2. The fluorescence changes as a function of total F1.2 concentration were fit by the competitive binding model. Effect of F1.2 on Binding of Hirudin Peptides to Pre 2 and Thrombin—The effect of F1.2 on binding of [5F]Hir54–65(SO3-) to Pre 2 was determined by titration of 50 nm [5F]Hir54–65(SO3-) with Pre 2 in the absence and presence of 20 μm F1.2. The change in fluorescence as a function of total Pre 2 concentration was fit by the random addition ternary complex binding model to determine the dissociation constants for [5F]Hir54–65(SO3-) (H) binding to free Pre 2 (P2) and the Pre 2·F1.2 (P2F1.2) complex (K P2(H) and K P2F1.2(H)), and the maximum fluorescence changes for each of the fluorescent species (ΔF max P2(H)/F 0 and ΔF max P2F1.2(H)/F 0) as described previously (28Anderson P.J. Nesset A. Bock P.E. J. Biol. Chem. 2003; 278: 44482-44488Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 36Verhamme I.M. Olson S.T. Tollefsen D.M. Bock P.E. J. Biol. Chem. 2002; 277: 6788-6798Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). The dissociation constant for F1.2 binding to Pre 2 (K P2(F1.2)) was fixed at the value determined as described above. The effect of F1.2 on binding of [5F]Hir54–65(SO3-) to thrombin was determined similarly and analyzed to determine the binding constants for [5F]Hir54–65(SO3-) binding to free thrombin (T) and thrombin·F1.2 complex (K T(H) and K TF1.2(H)), and the maximum fluorescence changes for each of the fluorescent species (ΔF max T(H)/F 0 and ΔF max TF1.2(H)/F 0). The dissociation constant for F1.2 binding to thrombin (K T(F1.2)) was fixed at the determined value. Free Energy Calculations for [5F]Hir54–65(SO3-) Binding to Pro and Pre 1 Activation Intermediates—The change in free energy of association upon [5F]Hir54–65(SO3-) binding to Pro and the activation intermediates was calculated from ΔG = RT ln KD. The change in free energy of peptide binding to each of the activation intermediates relative to Pro was calculated from: ΔΔG = RT ln(K Int/K Pro), where K Int is the dissociation constant for [5F]Hir54–65(SO3-) binding to one of the intermediates and K Pro is the dissociation constant for [5F]Hir54–65(SO3-) binding to Pro (37Segel I.H. Biochemical Calculations. 2nd. Ed. John Wiley & Sons, New York1976: 150-159Google Scholar, 38Hammes G.G. Thermodynamics and Kinetics for the Biological Sciences. John Wiley & Sons, New York2000: 30-32Google Scholar). Nonlinear least squares analysis was performed with SCIENTIST (MicroMath). Reported errors represent ± 2 S.D. Characterization of Fluorescence Spectral Properties of [NBD]Hir54–65(SO3-) Binding to Pro Activation Intermediates— Fluorescence emission spectral studies of [NBD]Hir54–65(SO3-) peptide binding were carried out to investigate further the small changes in the environment of proexosite I on Pro and the Pre 1 activation intermediates observed with the fluorescein-labeled peptide (28Anderson P.J. Nesset A. Bock P.E. J. Biol. Chem. 2003; 278: 44482-44488Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). With the more environmentally sensitive NBD probe, the fluorescence emission spectra for [NBD]Hir54–65(SO3-) binding to Pro, Pre 1, and the Pro activation intermediates were distinctly different for each of the proteins (Fig. 1). Pro enhanced the fluorescence of the NBD-labeled peptide by 20 ± 1%, blue-shifting the maximum from 544 to 540 nm, while binding of the probe to Pre 1 produced a similar blue shift of ∼4 nm, but a very small change in fluorescence at 540 nm (0.6%) compared with Pro. This indicated that removal of F1 from Pro had a significant effect on the environment of the bound fluorescent peptide. Peptide binding to Pre 2 quenched the NBD fluorescence by 19 ± 1%, whereas MzT, MzT(-F1), and thrombin displayed indistinguishable spectral properties, with maxima at 539 nm and larger quenching of the fluorescence of 44–50%. The results indicated that NBD-labeled hirudin54–65 reported differences in the environments of proexosite I on the Pro, Pre 1, and Pre 2 zymogen forms and the activated enzymes. The probe-peptide environment of exosite I on the enzymes MzT and MzT(-F1) was indistinguishable from that of thrombin. Binding of [NBD]Hir54–65(SO3-) to Pro, Pre 1, and Thrombin—Titrations of NBD-labeled peptide with Pro yielded a dissociation constant of 3.7 ± 0.8 μm (Fig. 2A), which was in excellent agreement with the previously determined value of 3.2 ± 0.3 μm for [5F]Hir54–65(SO3-) (11Anderson P.J. Nesset A. Dharmawardana K.R. Bock P.E. J. Biol. Chem. 2000; 275: 16428-16434Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Binding of [NBD]Hir54–65(SO3-) to Pre 1 resulted in a 12 ± 1% quench of the fluorescence (measured at 580 nm) instead of the enhancement seen with Pro, indicating a significant effect of the removal of F1 on the change in probe environment. [NBD]Hir54–65(SO3-) bound to Pre 1 with a 7-fold increased affinity in comparison to Pro (data not shown, Table I), confirming the 6-fold increase in affinity observed for [5F]Hir54–65(SO3-) (28Anderson P.J. Nesset A. Bock P.E. J. Biol. Chem. 2003; 278: 44482-44488Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Pre 2 bound the NBD-labeled peptide with a slightly lower (<2-fold) affinity compared with Pre 1, which was not considered significant. In contrast to the enhancement seen with Pro, thrombin quenched the fluorescence of [NBD]Hir54–65(SO3-) by 50 ± 1% and bound with a ∼250-fold tighter dissociation constant of 15 ± 3 nm, indistinguishable from the affinity determined for the fluorescein-labeled peptide (Fig. 2B and Table I). Comparison of the results for binding of the two peptides to Pro and thrombin showed a 250-fold increase in affinity for the NBD-labeled peptide binding to thrombin, similar to the 130-fold increased affinity for [5F]Hir54–65(SO3-) seen previously (11Anderson P.J. Nesset A. Dharmawardana K.R. Bock P.E. J. Biol. Chem. 2000; 275: 16428-16434Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Comparison of Pre 2 and Pre 1 showed that, although the spectral changes were distinct, there was no correlation with a change in exosite affinity. The results suggested that removal of F1 from Pro resulted in either a conformational change in proexosite I on formation of Pre 1 or that F1 interfered directly with the binding of peptides to proexosite I on Pro.Table IParameters determined for binding of hirudin54–65 peptides to Pro, the activation intermediates, and productsInteractionAnalysis methodKDSitesΔF max/F 0μ mmol/mol protein%Prothrombin[NBD]Hir54-65(SO3-)Direct titration3.7 ± 0.820 ± 1Prethrombin 1[NBD]Hir54-65(SO3-)Direct titration0.5 ± 0.2-12 ± 1Prethrombin 2[NBD]Hir54-65(SO3-)Direct titration0.9 ± 0.2-19 ± 1[5F]Hir54-65(SO3-)Ternary complex0.43 ± 0.03-26.5 ± 0.5+F1.2: [5F]Hir54-65(SO3-)Ternary complex1.3 ± 0.2-23 ± 1Meizothrombin des-fragment 1[NBD]Hir54-65(SO3-)Direct titration0.011 ± 0.0041.4 ± 0.1-48 ± 1Meizothrombin[NBD]Hir54-65(SO3-)Direct titration0.017 ± 0.003-44 ± 1[5F]Hir54-65(SO3-)Direct titration0.022 ± 0.0021.1 ± 0.1-32.2 ± 0.3Hir54-65(SO3-)Competitive titration0.058 ± 0.006-32.5 ± 0.5Thrombin[NBD]Hir54-65(SO3-)Direct titration0.015 ± 0.0031.3 ± 0.1-50 ± 1[5F]Hir54-65(SO3-)Ternary complex0.031 ± 0.004-30 ± 1+F1.2: [5F]Hir54-65(SO3-)Ternary complex0.038 ± 0.009-34 ± 2 Open table in a new tab Binding of Fragment 1.2 to [6F]FPR-thrombin and Pre 2— Interactions of F1.2 with exosite II on thrombin and Pre 2 were characterized in competitive binding experiments with fluorescein-labeled thrombin ([6F]FPR-thrombin). The fluorescence of [6F]FPR-thrombin was quenched by 28 ± 3% upon binding of F1.2 with a dissociation constant of 8 ± 2 μm (Fig. 3). Competitive titrations of [6F]FPR-thrombin and unlabeled Pre 2 with F1.2 gave a dissociation constant of 0.5 ± 0.3 μm for F1.2 binding to Pre 2, displaying a 16-fold increased affinity of F1.2 for Pre 2 in comparison to thrombin. By contrast, the affinities of F2 binding to exosite II on Pre 2 and thrombin are the same (28Anderson P.J. Nesset A. Bock P.E. J. Biol. Chem. 2003; 278: 44482-44488Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). The results indicated that F1 increased the affinity of F1.2 for Pre 2 by 16-fold, whereas no effect of F1 on F1.2 binding to thrombin was seen. Binding of Hirudin Peptides to Pre 2 in the Absence and Presence of F1.2—The effect of F1.2 binding on proexosite I of Pre 2 was characterized by direct titration of [5F]Hir54–65(SO3-) (H) with Pre 2 (P2) in the absence and presence of 20 μm F1.2. The results were analyzed by the random addition ternary complex model (28Anderson P.J. Nesset A. Bock P.E. J. Biol. Chem. 2003; 278: 44482-44488Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 36Verhamme I.M. Olson S.T. Tollefsen D.M. Bock P.E. J. Biol. Chem. 2002; 277: 6788-6798Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), which yielded a fitted dissociation constant for the P2·H binary complex (K P2(H)) of 0.43 ± 0.03 μm (Fig. 4 and Table I), the same as the dissociation constant determined by direct titration of Pre 2 alone (0.44 ± 0.04 μm) (28Anderson P.J. Nesset A. Bock P.E. J. Biol. Chem. 2003; 278: 44482-44488Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). The dissociation constant for [5F]Hir54–65(SO3-) binding to P2·F1.2 complex to form the P2·F1.2·H ternary complex corresponded to a 3-fold weaker affinity (K P2F1.2(H) 1.3 ± 0.2 μm) and slightly smaller fluorescence change compared with free Pre 2 (Fig. 4 and Table I). In view of the previous results demonstrating no effect of F2 on binding of peptides to Pre 2, the decrease in affinity of the peptide for the Pre 2·F1.2 complex indicated an effect of the presence of F1 in F1.2 on peptide binding to (pro)exosite I, which approached that seen in comparison of Pro and Pre 1. Binding of Hirudin Peptides to Thrombin in the Absence and Presence of F1.2—The effect of F1.2 on binding of [5F]Hir54–65(SO3-) to exosite I on thrombin was investigated in titrations of thrombin (T) in the absence and presence of near-saturating F1.2 (20 μm) (Fig. 5 and Table I). The data were analyzed with the ternary complex model to obtain the dissociation constants for T·H (K T(H) 31 ± 4 nm) and T·F1.2·H (K TF1.2(H) 38 ± 9 nm), which were indistinguishable from the previous results for [5F]Hir54–65(SO3-) binding (28Anderson P.J. Nesset A. Bock P.E. J. Biol. Chem. 2003; 278: 44482-44488Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar) (Table I). The results demonstrated that binding of F1.2 to exosite II on thrombin did not affect b