Binding of the COOH-terminal Lysine Residue of Streptokinase to Plasmin(ogen) Kringles Enhances Formation of the Streptokinase·Plasmin(ogen) Catalytic Complexes
2006; Elsevier BV; Volume: 281; Issue: 37 Linguagem: Inglês
10.1074/jbc.c600171200
ISSN1083-351X
AutoresPeter Panizzi, Paul D. Boxrud, Ingrid M. Verhamme, Paul Bock,
Tópico(s)Calpain Protease Function and Regulation
ResumoStreptokinase (SK) activates human fibrinolysis by inducing non-proteolytic activation of the serine proteinase zymogen, plasminogen (Pg), in the SK·Pg* catalytic complex. SK·Pg* proteolytically activates Pg to plasmin (Pm). SK-induced Pg activation is enhanced by lysine-binding site (LBS) interactions with kringles on Pg and Pm, as evidenced by inhibition of the reactions by the lysine analogue, 6-aminohexanoic acid. Equilibrium binding analysis and [Lys]Pg activation kinetics with wild-type SK, carboxypeptidase B-treated SK, and a COOH-terminal Lys414 deletion mutant (SKΔK414) demonstrated a critical role for Lys414 in the enhancement of [Lys]Pg and [Lys]Pm binding and conformational [Lys]Pg activation. The LBS-independent affinity of SK for [Glu]Pg was unaffected by deletion of Lys414. By contrast, removal of SK Lys414 caused 19- and 14-fold decreases in SK affinity for [Lys]Pg and [Lys]Pm binding in the catalytic mode, respectively. In kinetic studies of the coupled conformational and proteolytic activation of [Lys]Pg, SKΔK414 exhibited a corresponding 17-fold affinity decrease for formation of the SKΔK414·[Lys]Pg* complex. SKΔK414 binding to [Lys]Pg and [Lys]Pm and conformational [Lys]Pg activation were LBS-independent, whereas [Lys]Pg substrate binding and proteolytic [Lys]Pm generation remained LBS-dependent. We conclude that binding of SK Lys414 to [Lys]Pg and [Lys]Pm kringles enhances SK·[Lys]Pg* and SK·[Lys]Pm catalytic complex formation. This interaction is distinct structurally and functionally from LBS-dependent Pg substrate recognition by these complexes. Streptokinase (SK) activates human fibrinolysis by inducing non-proteolytic activation of the serine proteinase zymogen, plasminogen (Pg), in the SK·Pg* catalytic complex. SK·Pg* proteolytically activates Pg to plasmin (Pm). SK-induced Pg activation is enhanced by lysine-binding site (LBS) interactions with kringles on Pg and Pm, as evidenced by inhibition of the reactions by the lysine analogue, 6-aminohexanoic acid. Equilibrium binding analysis and [Lys]Pg activation kinetics with wild-type SK, carboxypeptidase B-treated SK, and a COOH-terminal Lys414 deletion mutant (SKΔK414) demonstrated a critical role for Lys414 in the enhancement of [Lys]Pg and [Lys]Pm binding and conformational [Lys]Pg activation. The LBS-independent affinity of SK for [Glu]Pg was unaffected by deletion of Lys414. By contrast, removal of SK Lys414 caused 19- and 14-fold decreases in SK affinity for [Lys]Pg and [Lys]Pm binding in the catalytic mode, respectively. In kinetic studies of the coupled conformational and proteolytic activation of [Lys]Pg, SKΔK414 exhibited a corresponding 17-fold affinity decrease for formation of the SKΔK414·[Lys]Pg* complex. SKΔK414 binding to [Lys]Pg and [Lys]Pm and conformational [Lys]Pg activation were LBS-independent, whereas [Lys]Pg substrate binding and proteolytic [Lys]Pm generation remained LBS-dependent. We conclude that binding of SK Lys414 to [Lys]Pg and [Lys]Pm kringles enhances SK·[Lys]Pg* and SK·[Lys]Pm catalytic complex formation. This interaction is distinct structurally and functionally from LBS-dependent Pg substrate recognition by these complexes. Streptokinase (SK) 2The abbreviations used are: SK, streptokinase; wtSK, recombinant wild-type SK; SKΔK414, streptokinase mutant lacking the COOH-terminal lysine; CpB, carboxypeptidase B; 6-AHA; 6-aminohexanoic acid; pNA, p-nitroaniline; Pg, plasminogen; [Glu]Pg, compact form of Pg; [Lys]Pg, [Glu]Pg lacking the 77-residue NH2-terminal peptide; [Lys]Pm, Pm, plasmin; fluorescein-labeled analogues of Pg or Pm prepared with Nα-[(acetylthio)acetyl]-(d-Phe)-Phe-Arg-CH2Cl and 5-(iodoacetamido)fluorescein are represented by [5F]FFR-Pg or –Pm; LBS, lysine-binding site; TEV, tobacco etch virus.2The abbreviations used are: SK, streptokinase; wtSK, recombinant wild-type SK; SKΔK414, streptokinase mutant lacking the COOH-terminal lysine; CpB, carboxypeptidase B; 6-AHA; 6-aminohexanoic acid; pNA, p-nitroaniline; Pg, plasminogen; [Glu]Pg, compact form of Pg; [Lys]Pg, [Glu]Pg lacking the 77-residue NH2-terminal peptide; [Lys]Pm, Pm, plasmin; fluorescein-labeled analogues of Pg or Pm prepared with Nα-[(acetylthio)acetyl]-(d-Phe)-Phe-Arg-CH2Cl and 5-(iodoacetamido)fluorescein are represented by [5F]FFR-Pg or –Pm; LBS, lysine-binding site; TEV, tobacco etch virus. activates the human fibrinolytic system by activating the zymogen, plasminogen (Pg) to form the fibrin-degrading proteinase, plasmin (Pm) (1Collen D. Lijnen H.R. Blood. 1991; 78: 3114-3124Crossref PubMed Google Scholar). The mechanism of SK-activated Pm formation is unique in that it is initiated by formation of an SK·Pg* complex in which the zymogen catalytic site is activated non-proteolytically (2McClintock D.K. Bell P.H. Biochem. Biophys. Res. Commun. 1971; 43: 694-702Crossref PubMed Scopus (153) Google Scholar, 3Reddy K.N. Markus G. J. Biol. Chem. 1972; 247: 1683-1691Abstract Full Text PDF PubMed Google Scholar, 4Schick L.A. Castellino F.J. Biochem. Biophys. Res. Commun. 1974; 57: 47-54Crossref PubMed Scopus (66) Google Scholar, 5Boxrud P.D. Verhamme I.M. Bock P.E. J. Biol. Chem. 2004; 279: 36633-36641Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). SK·Pg* binds free Pg and converts it into Pm by intermolecular proteolytic cleavage (6Boxrud P.D. Bock P.E. J. Biol. Chem. 2004; 279: 36642-36649Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Pm binds tightly to SK in the catalytic mode (7Boxrud P.D. Fay W.P. Bock P.E. J. Biol. Chem. 2000; 275: 14579-14589Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 8Boxrud P.D. Bock P.E. Biochemistry. 2000; 39: 13974-13981Crossref PubMed Scopus (42) Google Scholar) and SK·Pm propagates proteolytic Pg activation (6Boxrud P.D. Bock P.E. J. Biol. Chem. 2004; 279: 36642-36649Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 9Gonzalez-Gronow M. Siefring Jr., G.E. Castellino F.J. J. Biol. Chem. 1978; 253: 1090-1094Abstract Full Text PDF PubMed Google Scholar, 10Wohl R.C. Summaria L. Robbins K.C. J. Biol. Chem. 1980; 255: 2005-2013Abstract Full Text PDF PubMed Google Scholar) through expression of a Pg substrate binding exosite (7Boxrud P.D. Fay W.P. Bock P.E. J. Biol. Chem. 2000; 275: 14579-14589Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). The mechanism is regulated by intrinsic differences in affinity of SK for [Glu]Pg, [Lys]Pg, and [Lys]Pm (5Boxrud P.D. Verhamme I.M. Bock P.E. J. Biol. Chem. 2004; 279: 36633-36641Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 6Boxrud P.D. Bock P.E. J. Biol. Chem. 2004; 279: 36642-36649Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 7Boxrud P.D. Fay W.P. Bock P.E. J. Biol. Chem. 2000; 275: 14579-14589Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 8Boxrud P.D. Bock P.E. Biochemistry. 2000; 39: 13974-13981Crossref PubMed Scopus (42) Google Scholar, 11Bock P.E. Day D.E. Verhamme I.M. Bernardo M.M. Olson S.T. Shore J.D. J. Biol. Chem. 1996; 271: 1072-1080Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). [Glu]Pg consists of an NH2-terminal 77-residue peptide, five kringle domains, and a serine proteinase catalytic domain (12Ponting C.P. Marshall J.M. Cederholm-Williams S.A. Blood Coagul. Fibrinolysis. 1992; 3: 605-614Crossref PubMed Scopus (199) Google Scholar). [Glu]Pg is maintained in a compact conformation through intramolecular interaction of the NH2-terminal peptide with kringles 4 and 5 (13Ponting C.P. Holland S.K. Cederholm-Williams S.A. Marshall J.M. Brown A.J. Spraggon G. Blake C.C. Biochim. Biophys. Acta. 1992; 1159: 155-161Crossref PubMed Scopus (42) Google Scholar, 14Marshall J.M. Brown A.J. Ponting C.P. Biochemistry. 1994; 33: 3599-3606Crossref PubMed Scopus (105) Google Scholar, 15McCance S.G. Castellino F.J. Biochemistry. 1995; 34: 9581-9586Crossref PubMed Scopus (38) Google Scholar). Pm cleavage of the NH2-terminal peptide of [Glu]Pg generates the more reactive [Lys]Pg, which assumes an extended conformation with expression of enhanced lysine-binding site (LBS) interactions (12Ponting C.P. Marshall J.M. Cederholm-Williams S.A. Blood Coagul. Fibrinolysis. 1992; 3: 605-614Crossref PubMed Scopus (199) Google Scholar, 14Marshall J.M. Brown A.J. Ponting C.P. Biochemistry. 1994; 33: 3599-3606Crossref PubMed Scopus (105) Google Scholar, 15McCance S.G. Castellino F.J. Biochemistry. 1995; 34: 9581-9586Crossref PubMed Scopus (38) Google Scholar, 16Violand B.N. Byrne R. Castellino F.J. J. Biol. Chem. 1978; 253: 5395-5401Abstract Full Text PDF PubMed Google Scholar).Formation of SK·[Lys]Pg* and SK·[Lys]Pm catalytic complexes and subsequent [Lys]Pg substrate recognition are enhanced by interactions of SK with LBS of Pg and Pm kringle domains (5Boxrud P.D. Verhamme I.M. Bock P.E. J. Biol. Chem. 2004; 279: 36633-36641Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 6Boxrud P.D. Bock P.E. J. Biol. Chem. 2004; 279: 36642-36649Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 7Boxrud P.D. Fay W.P. Bock P.E. J. Biol. Chem. 2000; 275: 14579-14589Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 8Boxrud P.D. Bock P.E. Biochemistry. 2000; 39: 13974-13981Crossref PubMed Scopus (42) Google Scholar, 11Bock P.E. Day D.E. Verhamme I.M. Bernardo M.M. Olson S.T. Shore J.D. J. Biol. Chem. 1996; 271: 1072-1080Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 17Lin L.F. Houng A. Reed G.L. Biochemistry. 2000; 39: 4740-4745Crossref PubMed Scopus (33) Google Scholar, 18Conejero-Lara F. Parrado J. Azuaga A.I. Dobson C.M. Ponting C.P. Protein Sci. 1998; 7: 2190-2199Crossref PubMed Scopus (40) Google Scholar, 19Dhar J. Pande A.H. Sundram V. Nanda J.S. Mande S.C. Sahni G. J. Biol. Chem. 2002; 277: 13257-13267Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 20Chaudhary A. Vasudha S. Rajagopal K. Komath S.S. Garg N. Yadav M. Mande S.C. Sahni G. Protein Sci. 1999; 8: 2791-2805Crossref PubMed Scopus (38) Google Scholar). Recent studies of the Pg activation mechanism demonstrate that LBS interactions enhance SK·[Lys]Pg* catalytic complex formation and Pg substrate binding but are not absolutely required for these interactions (5Boxrud P.D. Verhamme I.M. Bock P.E. J. Biol. Chem. 2004; 279: 36633-36641Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 6Boxrud P.D. Bock P.E. J. Biol. Chem. 2004; 279: 36642-36649Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). SK binding to the compact conformation of [Glu]Pg in the catalytic mode is LBS-independent (5Boxrud P.D. Verhamme I.M. Bock P.E. J. Biol. Chem. 2004; 279: 36633-36641Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 6Boxrud P.D. Bock P.E. J. Biol. Chem. 2004; 279: 36642-36649Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 8Boxrud P.D. Bock P.E. Biochemistry. 2000; 39: 13974-13981Crossref PubMed Scopus (42) Google Scholar, 11Bock P.E. Day D.E. Verhamme I.M. Bernardo M.M. Olson S.T. Shore J.D. J. Biol. Chem. 1996; 271: 1072-1080Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar).Several studies have sought to define SK lysine residues that mediate its interactions with Pg and Pm kringles. The crystal structure of SK bound to the isolated Pm catalytic domain (micro-Pm) shows that SK consists of three homologous, independently folded β-grasp domains connected by flexible linking sequences (21Wang X. Lin X. Loy J.A. Tang J. Zhang X.C. Science. 1998; 281: 1662-1665Crossref PubMed Scopus (219) Google Scholar). SK forms a “crater” around the Pm catalytic site which provides a surface for Pg substrate binding (21Wang X. Lin X. Loy J.A. Tang J. Zhang X.C. Science. 1998; 281: 1662-1665Crossref PubMed Scopus (219) Google Scholar). Studies of SK domain truncation and deletion mutants, isolated domains, and point mutants have led to diverse interpretations, indicating that each of the SK domains may participate in kringle interactions (18Conejero-Lara F. Parrado J. Azuaga A.I. Dobson C.M. Ponting C.P. Protein Sci. 1998; 7: 2190-2199Crossref PubMed Scopus (40) Google Scholar, 19Dhar J. Pande A.H. Sundram V. Nanda J.S. Mande S.C. Sahni G. J. Biol. Chem. 2002; 277: 13257-13267Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 20Chaudhary A. Vasudha S. Rajagopal K. Komath S.S. Garg N. Yadav M. Mande S.C. Sahni G. Protein Sci. 1999; 8: 2791-2805Crossref PubMed Scopus (38) Google Scholar, 22Loy J.A. Lin X. Schenone M. Castellino F.J. Zhang X.C. Tang J. Biochemistry. 2001; 40: 14686-14695Crossref PubMed Scopus (45) Google Scholar, 23Sazonova I.Y. Robinson B.R. Gladysheva I.P. Castellino F.J. Reed G.L. J. Biol. Chem. 2004; 279: 24994-25001Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Some studies support a role for the flexible 250-loop of the SK β-domain in LBS-dependent Pg substrate binding (19Dhar J. Pande A.H. Sundram V. Nanda J.S. Mande S.C. Sahni G. J. Biol. Chem. 2002; 277: 13257-13267Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 20Chaudhary A. Vasudha S. Rajagopal K. Komath S.S. Garg N. Yadav M. Mande S.C. Sahni G. Protein Sci. 1999; 8: 2791-2805Crossref PubMed Scopus (38) Google Scholar). By contrast, no studies have demonstrated the structural basis for the LBS dependence of catalytic complex formation.Kringles of Pg and Pm bind zwitterionic COOH-terminal lysine residues and lysine analogues specifically, notably 6-aminohexanoic acid (6-AHA) (13Ponting C.P. Holland S.K. Cederholm-Williams S.A. Marshall J.M. Brown A.J. Spraggon G. Blake C.C. Biochim. Biophys. Acta. 1992; 1159: 155-161Crossref PubMed Scopus (42) Google Scholar, 24Castellino F.J. McCance S.G. Ciba Found. Symp. 1997; 212: 46-60PubMed Google Scholar). This is the basis for Pg binding by several proteins, including fibrin (25Lucas M.A. Fretto L.J. McKee P.A. J. Biol. Chem. 1983; 258: 4249-4256Abstract Full Text PDF PubMed Google Scholar, 26Bok R.A. Mangel W.F. Biochemistry. 1985; 24: 3279-3286Crossref PubMed Scopus (75) Google Scholar), antiplasmin (27Hortin G.L. Gibson B.L. Fok K.F. Biochem. Biophys. Res. Commun. 1988; 155: 591-596Crossref PubMed Scopus (43) Google Scholar), histidine-rich glycoprotein (28Lijnen H.R. Hoylaerts M. Collen D. J. Biol. Chem. 1980; 255: 10214-10222Abstract Full Text PDF PubMed Google Scholar), and tetranectin (29Clemmensen I. Petersen L.C. Kluft C. Eur. J. Biochem. 1986; 156: 327-333Crossref PubMed Scopus (175) Google Scholar). In fibrinolysis, LBS interactions with fibrin mediated by COOH-terminal lysine residues localize and accelerate Pg activation and fibrin degradation by Pm (25Lucas M.A. Fretto L.J. McKee P.A. J. Biol. Chem. 1983; 258: 4249-4256Abstract Full Text PDF PubMed Google Scholar, 26Bok R.A. Mangel W.F. Biochemistry. 1985; 24: 3279-3286Crossref PubMed Scopus (75) Google Scholar, 30Wiman B. Collen D. Nature. 1978; 272: 549-550Crossref PubMed Scopus (296) Google Scholar) and protect fibrin-bound Pm from inactivation by antiplasmin (31Wiman B. Collen D. Eur. J. Biochem. 1978; 84: 573-578Crossref PubMed Scopus (234) Google Scholar). Surprisingly, the fact that the COOH-terminal residue of SK is Lys414 (32Jackson K.W. Tang J. Biochemistry. 1982; 21: 6620-6625Crossref PubMed Scopus (117) Google Scholar) has been overlooked in previous studies. Here, we show that Lys414 is responsible for the LBS-dependent enhancement in affinity of SK·[Lys]Pg* and SK·[Lys]Pm catalytic complex formation. This interaction is shown to be structurally and functionally distinct from the LBS-dependent binding of [Lys]Pg as a substrate of the catalytic complexes.EXPERIMENTAL PROCEDURESWild-type SK and an SK Mutant Lacking the COOH-terminal Lys—Wild-type SK (wtSK) was prepared by methods described previously (33Bean R.R. Verhamme I.M. Bock P.E. J. Biol. Chem. 2005; 280: 7504-7510Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 34Boxrud P.D. Verhamme I.M. Fay W.P. Bock P.E. J. Biol. Chem. 2001; 276: 26084-26089Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) or was expressed as a fusion protein with a tobacco etch virus (TEV) proteinase cleavage site (underlined) encoded before the wtSK protein, Met-His6-Ser-Ala-Gly-Gly-Ser-Pro-Trp-Asn-Glu-Asn-Leu-Try-Phe-Gln-SKIle1-SKAla2-SKGly3... (His6-wtSK). From a pET30a(+) vector backbone, a single nucleotide substitution mutated the P1 3Schechter-Berger (36Schechter I. Berger A. Biochem. Biophys. Res. Commun. 1967; 27: 157-162Crossref PubMed Scopus (4727) Google Scholar) notation referring to the residues of a substrate (from the NH2-terminal end) as... P4-P3-P2-P1-P1′-P2′... with the scissile bond at P1-P1′. residue of a thrombin cleavage site (from Arg to Trp), which eliminated this unnecessary site and generated a NcoI restriction site. Flanking NcoI and XhoI restriction sites (underlined) where incorporated into the 5′- and 3′-PCR primers, respectively, with the sense primer, 5′-TCACTCCGCGGGTGGTAGTCCATGGAACGAGAACCTGTATTTTCAGATTGCTGGACCTGAGTGGCTG-3′, the same for wtSK and SKΔK414 constructs, and the antisense primer, 5′-ATAATGGTGCTCGAGTTATTTGTCGTTAGGGTTATCAGG-3′, only differed by the Lys414 codon (bold). A construct that encoded a His6-tagged TEV proteinase was kindly provided by Dr. Laura Mizoue of the Vanderbilt University Center for Structural Biology and used to remove the His6-tag from the NH2 terminus of wtSK.His6-wtSK was expressed from Rosetta(DE3) pLysS cells induced with 20 g/liter lactose for 12–16 h at 37 °C. Cells were harvested by centrifugation, resuspended in 50 mm Hepes, 125 mm NaCl, 1 mg/ml polyethylene glycol 8000, pH 7.4 (Buffer A) with 1 mm EDTA and 0.2% sodium azide, lysed by three cycles of sonication (∼45 s cycles) on ice, and centrifuged to clarify lysates. The pellet was resuspended in Buffer A containing 3 m NaSCN. The solubilized wtSK was dialyzed into 50 mm Hepes, 400 mm NaCl, 50 mm imidazole, pH 7.4 (Buffer B) and purified by Ni2+-iminodiacetic acid-Sepharose chromatography with a 50–500 mm imidazole gradient in Buffer B. TEV proteinase was added to the eluted protein in a 1 to 5 molar ratio of enzyme to substrate. The reaction mixture was first dialyzed overnight into 50 mm Hepes, 300 mm NaCl, 1 mm dithiothreitol, 5% glycerol, pH 7.8 at 4 °C, and subsequently dialyzed back into Buffer B. Uncleaved fusion protein, cleaved His6-tag, and the TEV proteinase bound to Ni2+-iminodiacetic acid-Sepharose, and wtSK was obtained from the column flow-through. wtSK was dialyzed against Buffer A without polyethylene glycol, quick-frozen, and stored at –80 °C. SKΔ414K was prepared following an identical procedure. The correct NH2-terminal sequence for wtSK and SKΔK414 was confirmed.Native and Carboxypeptidase B (CpB)-treated SK—Native SK purchased from Diapharma and purified as described previously (7Boxrud P.D. Fay W.P. Bock P.E. J. Biol. Chem. 2000; 275: 14579-14589Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 8Boxrud P.D. Bock P.E. Biochemistry. 2000; 39: 13974-13981Crossref PubMed Scopus (42) Google Scholar, 11Bock P.E. Day D.E. Verhamme I.M. Bernardo M.M. Olson S.T. Shore J.D. J. Biol. Chem. 1996; 271: 1072-1080Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar) was treated with porcine pancreatic CpB (Sigma Type-1, diisopropylfluorophosphate-treated, 4.7 mg/ml in 0.1 m NaCl) in 50 mm Hepes, 125 mm NaCl, pH 7.4. SK (2.5 mg/ml) was incubated with CpB (35 μg/ml) for 30 min at 25 °C, and the reaction was stopped by addition of 10 mm EDTA. Titrations of [5F]FFR-[Lys]Pg were performed in 50 mm Hepes, 125 mm NaCl, 1 mm EDTA, 10 μm Val-Phe-Arg-CH2Cl, 1 mg/ml bovine serum albumin, ±100 mm 6-AHA, pH 7.4.Fluorescence Equilibrium Binding—[Glu]Pg, [Lys]Pg, [Lys]Pm, and the active site-labeled fluorescein analogues were prepared as described (5Boxrud P.D. Verhamme I.M. Bock P.E. J. Biol. Chem. 2004; 279: 36633-36641Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 6Boxrud P.D. Bock P.E. J. Biol. Chem. 2004; 279: 36642-36649Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 11Bock P.E. Day D.E. Verhamme I.M. Bernardo M.M. Olson S.T. Shore J.D. J. Biol. Chem. 1996; 271: 1072-1080Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Fluorescence titrations were performed in Buffer A containing 1 mm EDTA and 1 μm d-Phe-Phe-Arg-CH2Cl ± 10 mm 6-AHA as described previously (5Boxrud P.D. Verhamme I.M. Bock P.E. J. Biol. Chem. 2004; 279: 36633-36641Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 6Boxrud P.D. Bock P.E. J. Biol. Chem. 2004; 279: 36642-36649Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 33Bean R.R. Verhamme I.M. Bock P.E. J. Biol. Chem. 2005; 280: 7504-7510Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Fluorescence changes ((Fobs – Fo)/Fo = ΔF/Fo) as a function of total SK concentration, were fit by the quadratic binding equation to determine the maximum fluorescence change (ΔFmax/Fo) and dissociation constant (KD), with the stoichiometric factor (n) fixed at 1. Competitive binding titrations of native [Lys]Pg were performed by addition of wtSK or SKΔ414 to mixtures of [5F]FFR-[Lys]Pg as a function of native [Lys]Pg concentration, as described previously (5Boxrud P.D. Verhamme I.M. Bock P.E. J. Biol. Chem. 2004; 279: 36633-36641Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Results were analyzed by fitting of the cubic binding equation to determine the KD of wtSK and SKΔ414 for native [Lys]Pg.Plasminogen Activation Kinetics—Coupled conformational and proteolytic activation of [Lys]Pg by wtSK and SKΔK414 were quantitated as described previously (5Boxrud P.D. Verhamme I.M. Bock P.E. J. Biol. Chem. 2004; 279: 36633-36641Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 6Boxrud P.D. Bock P.E. J. Biol. Chem. 2004; 279: 36642-36649Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Fitting of parabolic progress curves of d-Val-Leu-Lys-pNA (VLK-pNA) hydrolysis at 200 μm, in the presence of 15 nm [Lys]Pg and increasing wtSK and SKΔK414 concentrations gave the initial rates (v1) of VLK-pNA hydrolysis, reflecting conformational activation of the SK·Pg* complex, and the rates of activity increase (v2), reflecting Pm generation. The SK dependences of v1 and v2 were analyzed using the simplified equations described previously (5Boxrud P.D. Verhamme I.M. Bock P.E. J. Biol. Chem. 2004; 279: 36633-36641Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 6Boxrud P.D. Bock P.E. J. Biol. Chem. 2004; 279: 36642-36649Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar).SK+Pg⇄KASK·Pg*+Pg→kPgSK·Pg+Pm(Scheme 1) SK+Pm⇄KA'SK·Pm+Pg→kPmSK·Pm+PmThe data were also analyzed by fitting of the family of progress curves as a function of SK concentration with a more complete mechanism including both SK·Pg*- and SK·Pm-catalyzed Pg activation pathways under bimolecular reaction conditions (Scheme 1). For this analysis, the Km values for chromogenic substrate hydrolysis by Pm, SK·Pg*, and SK·Pm were fixed at the previously determined values, whereas the corresponding kcat values were allowed to vary within the experimental error of their determination to optimize the fit. K′A for SK·Pm binding was fixed at 12 pm (7Boxrud P.D. Fay W.P. Bock P.E. J. Biol. Chem. 2000; 275: 14579-14589Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). The fitted parameters were KA for SK·Pg* formation and the bimolecular rate constants for Pm generation by SK·Pg* (kPg) and SK·Pm (kPm) (Scheme 1) (5Boxrud P.D. Verhamme I.M. Bock P.E. J. Biol. Chem. 2004; 279: 36633-36641Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 6Boxrud P.D. Bock P.E. J. Biol. Chem. 2004; 279: 36642-36649Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Nonlinear least squares fitting was performed with SCIENTIST (MicroMath) or DYNAFIT (35Kuzmic P. Anal. Biochem. 1996; 237: 260-273Crossref PubMed Scopus (1339) Google Scholar). Error estimates represent the 95% confidence interval.RESULTS AND DISCUSSIONBinding of Native SK and CpB-treated SK to [Lys]Pg—Native SK was treated with CpB to remove the COOH-terminal lysine residue, under conditions where there was no detectable degradation of SK observable by SDS-gel electrophoresis (not shown). Titrations of [5F]FFR-[Lys]Pg with native SK and CpB-SK were performed in the absence and presence of saturating 6-AHA to evaluate the effect of CpB treatment on the LBS dependence of SK affinity (Fig. 1A). In the absence and presence of 6-AHA, native SK bound fluorescein-labeled [Lys]Pg with KD 20 ± 5nm and 380 ± 40 nm, respectively, and with ΔFmax/Fo of –15 ± 1% and –19 ± 1%. By contrast, CpB-SK bound labeled [Lys]Pg with KD 130 ± 20 nm and ΔFmax/Fo –17 ± 1% in the absence of 6-AHA, and with KD 300 ± 50 nm and ΔFmax/Fo –19 ± 1% in the presence of 6-AHA. The affinities for native and CpB-treated SK in the presence of 6-AHA were indistinguishable. CpB treatment of SK decreased the effect of 6-AHA on [Lys]Pg affinity from 19-fold to 2.3-fold. This demonstrated that CpB treatment of SK resulted in a selective loss of LBS-dependent affinity for labeled [Lys]Pg.Binding of wtSK and SKΔK414 to [Lys]Pg—Titrations of [5F]FFR-[Lys]Pg with wtSK and SKΔK414 were performed in the absence and presence of 10 mm 6-AHA (Fig. 1B). In the absence and presence of 6-AHA, wtSK bound labeled [Lys]Pg with KD 28 ± 6 nm and 520 ± 70 nm, respectively, and with ΔFmax/Fo of –23 ± 1% and –21 ± 1%. Like CpB-treated SK, SKΔK414 bound to labeled [Lys]Pg with weaker affinity than wtSK, with KD 610 ± 200 nm and 750 ± 350 nm in the absence and presence of 6-AHA, respectively, and ΔFmax/Fo of –24 ± 2% and –24 ± 3%. Native and wtSK bound with indistinguishable affinity to labeled Pg, and their LBS-dependent losses of affinity in the presence of 6-AHA were the same (Fig. 1, A and B). Deletion of the COOH-terminal lysine decreased the weakening effect of 6-AHA on affinity from 19- to 1.2-fold. This demonstrated that the SK mutant lacking the COOH-terminal lysine exhibited a selective loss of LBS-dependent affinity for labeled [Lys]Pg.Binding of wtSK and SKΔK414 to [Glu]Pg—Titrations of [5F]FFR-[Glu]Pg with wtSK and SKΔK414 were performed in the absence and presence of 10 mm 6-AHA (Fig. 1C). In the absence and presence of 6-AHA, wtSK bound labeled [Glu]Pg with KD 930 ± 120 nm and 471 ± 140 nm, respectively, and with ΔFmax/Fo –37 ± 1% and –19 ± 1%. SKΔK414 bound labeled [Glu]Pg with KD 634 ± 160 nm and 560 ± 150 nm in the absence and presence of 6-AHA, respectively, and with ΔFmax/Fo –30 ± 2% and –19 ± 1%. Although the amplitudes of the fluorescence changes were decreased by 6-AHA, the affinities of wtSK and SKΔK414 for labeled [Glu]Pg in the presence or absence of 6-AHA were indistinguishable and LBS-independent. This was consistent with the LBS independence of SK binding to [Glu]Pg in the compact conformation (5Boxrud P.D. Verhamme I.M. Bock P.E. J. Biol. Chem. 2004; 279: 36633-36641Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 8Boxrud P.D. Bock P.E. Biochemistry. 2000; 39: 13974-13981Crossref PubMed Scopus (42) Google Scholar, 11Bock P.E. Day D.E. Verhamme I.M. Bernardo M.M. Olson S.T. Shore J.D. J. Biol. Chem. 1996; 271: 1072-1080Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar) and an independence of the affinity on deletion of Lys414.Binding of wtSK and SKΔK414 to [Lys]Pm—To determine whether the COOH-terminal lysine of SK also interacted with [Lys]Pm kringles, titrations of [5F]FFR-Pm with wtSK and SKΔK414 were performed in the absence and presence of 10 mm 6-AHA (Fig. 1D). wtSK bound labeled [Lys]Pm with KD 19 ± 7pm, consistent with the previously reported affinity (7Boxrud P.D. Fay W.P. Bock P.E. J. Biol. Chem. 2000; 275: 14579-14589Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) and ΔFmax/Fo of –54 ± 2% in the absence of 6-AHA, compared with a 14-fold higher KD of 260 ± 70 pm and ΔFmax/Fo of –49 ± 2% in the presence of 6-AHA. SKΔK414 bound labeled [Lys]Pm with impaired affinity represented by KD 190 ± 40 pm and 350 ± 80 pm in the absence and presence of 6-AHA, respectively, and with ΔFmax/Fo of –50 ± 1% and –47 ± 2%. The affinities of wtSK for [5F]FFR-Pm in the presence of 6-AHA and that of SKΔK414 in the absence and presence of 6-AHA were indistinguishable within the experimental error. The results indicated that similar to [Lys]Pg, the affinity for complex formation between wtSK and [Lys]Pm was enhanced by interaction of SK Lys414 with kringles on [Lys]Pm.Binding of SKΔK414 and wtSK to Native Pg—The affinities of active site-labeled [Glu]Pg and [Lys]Pg for SK are consistently ∼5-fold lower than those of the native proteins, as determined in previous studies, but maintain the same magnitude of the effects of 6-AHA on binding (5Boxrud P.D. Verhamme I.M. Bock P.E. J. Biol. Chem. 2004; 279: 36633-36641Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 11Bock P.E. Day D.E. Verhamme I.M. Bernardo M.M. Olson S.T. Shore J.D. J. Biol. Chem. 1996; 271: 1072-1080Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). To characterize binding of wtSK and SKΔK414 to native [Lys]Pg, [5F]FFR-[Lys]Pg was used as a probe of the native and labeled [Lys]Pg competitive binding equilibria in the absence of proteinase inhibitors. To resolve the rapid binding equilibrium from slower proteolytic cleavage, individual measurements were made as a function of time after addition of wtSK or SKΔK414 to mixtures of [5F]FFR-[Lys]Pg and various concentrations of native [Lys]Pg at fixed wtSK and SKΔK414 concentrations as described previously (5Boxrud P.D. Verhamme I.M. Bock P.E. J. Biol. Chem. 2004; 279: 36633-36641Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 6Boxrud P.D. Bock P.E. J. Biol. Chem. 20
Referência(s)