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• W2041188984 abstract "Thrombin-activatable fibrinolysis inhibitor (TAFI) is a metallocarboxypeptidase (MCP) that links blood coagulation and fibrinolysis. TAFI hampers fibrin-clot lysis and is a pharmacological target for the treatment of thrombotic conditions. TAFI is transformed through removal of its prodomain by thrombin-thrombomodulin into TAFIa, which is intrinsically unstable and has a short half-life in vivo. Here we show that purified bovine TAFI activated in the presence of a proteinaceous inhibitor renders a stable enzyme-inhibitor complex. Its crystal structure reveals that TAFIa conforms to the α/β-hydrolase fold of MCPs and displays two unique flexible loops on the molecular surface, accounting for structural instability and susceptibility to proteolysis. In addition, point mutations reported to enhance protein stability in vivo are mainly located in the first loop and in another surface region, which is a potential heparin-binding site. The protein inhibitor contacts both the TAFIa active site and an exosite, thus contributing to high inhibitory efficiency. Thrombin-activatable fibrinolysis inhibitor (TAFI) is a metallocarboxypeptidase (MCP) that links blood coagulation and fibrinolysis. TAFI hampers fibrin-clot lysis and is a pharmacological target for the treatment of thrombotic conditions. TAFI is transformed through removal of its prodomain by thrombin-thrombomodulin into TAFIa, which is intrinsically unstable and has a short half-life in vivo. Here we show that purified bovine TAFI activated in the presence of a proteinaceous inhibitor renders a stable enzyme-inhibitor complex. Its crystal structure reveals that TAFIa conforms to the α/β-hydrolase fold of MCPs and displays two unique flexible loops on the molecular surface, accounting for structural instability and susceptibility to proteolysis. In addition, point mutations reported to enhance protein stability in vivo are mainly located in the first loop and in another surface region, which is a potential heparin-binding site. The protein inhibitor contacts both the TAFIa active site and an exosite, thus contributing to high inhibitory efficiency. After blood-vessel injury, hemostasis induces fibrin clot formation to prevent blood loss and trigger vessel repair (Mann et al., 1988Mann K.G. Jenny R.J. Krishnaswamy S. Cofactor proteins in the assembly and expression of blood clotting enzyme complexes.Annu. Rev. Biochem. 1988; 57: 915-956Crossref PubMed Scopus (449) Google Scholar). This clot must be removed after tissue repair to restore blood flow. These processes are tightly regulated by the coagulation and fibrinolytic cascades because imbalance may lead to thrombosis, heart attack, and stroke, or to bleeding as in hemophilia (Boffa et al., 2001Boffa M.B. Nesheim M.E. Koschinsky M.L. Thrombin activable fibrinolysis inhibitor (TAFI): molecular genetics of an emerging potential risk factor for thrombotic disorders.Curr. Drug Targets Cardiovasc. Haematol. Disord. 2001; 1: 59-74Crossref PubMed Scopus (23) Google Scholar). Hemostasis is modulated by thrombin-activatable fibrinolysis inhibitor (TAFI), also known as procarboxypeptidase (PCP) B from plasma, procarboxypeptidase B2, U, and R. It attenuates fibrinolysis by removing surface-exposed C-terminal lysine residues from the fibrin clot (Arolas et al., 2007Arolas J.L. Vendrell J. Avilés F.X. Fricker L.D. Metallocarboxypeptidases: emerging drug targets in biomedicine.Curr. Pharm. Des. 2007; 13: 349-366Crossref PubMed Scopus (65) Google Scholar, Bajzar et al., 1995Bajzar L. Manuel R. Nesheim M.E. Purification and characterization of TAFI, a thrombin-activable fibrinolysis inhibitor.J. Biol. Chem. 1995; 270: 14477-14484Abstract Full Text Full Text PDF PubMed Scopus (655) Google Scholar, Boffa et al., 2001Boffa M.B. Nesheim M.E. Koschinsky M.L. Thrombin activable fibrinolysis inhibitor (TAFI): molecular genetics of an emerging potential risk factor for thrombotic disorders.Curr. Drug Targets Cardiovasc. Haematol. Disord. 2001; 1: 59-74Crossref PubMed Scopus (23) Google Scholar, Bouma and Mosnier, 2003Bouma B.N. Mosnier L.O. Thrombin activatable fibrinolysis inhibitor (TAFI) at the interface between coagulation and fibrinolysis.Pathophysiol. Haemost. Thromb. 2003; 33: 375-381Crossref PubMed Scopus (81) Google Scholar, Eaton et al., 1991Eaton D.L. Malloy B.E. Tsai S.P. Henzel W. Drayna D. Isolation, molecular cloning, and partial characterization of a novel carboxypeptidase B from human plasma.J. Biol. Chem. 1991; 266: 21833-21838Abstract Full Text PDF PubMed Google Scholar, Hendriks et al., 1989Hendriks D. Scharpé S. van Sande M. Lommaert M.P. Characterisation of a carboxypeptidase in human serum distinct from carboxypeptidase N.J. Clin. Chem. Clin. Biochem. 1989; 27: 277-285PubMed Google Scholar, Willemse and Hendriks, 2007Willemse J.L. Hendriks D.F. A role for procarboxypepidase U (TAFI) in thrombosis.Front. Biosci. 2007; 12: 1973-1987Crossref PubMed Scopus (32) Google Scholar). TAFI is the zymogen of a B-type zinc-dependent metallocarboxypeptidase (MCP), which is produced and secreted by the liver, and apart from carboxypeptidase N, it is the only known MCP found in human plasma. TAFI is similar in sequence to human pancreatic PCPs (see Figure 1 and Arolas et al., 2007Arolas J.L. Vendrell J. Avilés F.X. Fricker L.D. Metallocarboxypeptidases: emerging drug targets in biomedicine.Curr. Pharm. Des. 2007; 13: 349-366Crossref PubMed Scopus (65) Google Scholar, Pereira et al., 2002Pereira P.J.B. Segura-Martín S. Oliva B. Ferrer-Orta C. Avilés F.X. Coll M. Gomis-Rüth F.X. Vendrell J. Human procarboxypeptidase B: three-dimensional structure and implications for thrombin-activatable fibrinolysis inhibitor (TAFI).J. Mol. Biol. 2002; 321: 537-547Crossref PubMed Scopus (62) Google Scholar). However, it differs from these proenzymes in that the prodomain is highly glycosylated at four sites, the glycosylation accounting for ∼20% of the total molecular mass (Valnickova et al., 2006Valnickova Z. Christensen T. Skottrup P. Thogersen I.B. Hojrup P. Enghild J.J. Post-translational modifications of human thrombin-activatable fibrinolysis inhibitor (TAFI): evidence for a large shift in the isoelectric point and reduced solubility upon activation.Biochemistry. 2006; 45: 1525-1535Crossref PubMed Scopus (30) Google Scholar). During coagulation, TAFI is processed by thrombin/thrombomodulin to TAFIa through removal of its 92 residue prodomain. In contrast to pancreatic MCPs, both TAFI and TAFIa uniquely display carboxypeptidase activity against larger substrates. However, while TAFIa has a half-life of less than 10 min, in contrast to its robust pancreatic counterparts, TAFI is stable in circulation (Boffa et al., 1998Boffa M.B. Wang W. Bajzar L. Nesheim M.E. Plasma and recombinant thrombin-activable fibrinolysis inhibitor (TAFI) and activated TAFI compared with respect to glycosylation, thrombin/thrombomodulin-dependent activation, thermal stability, and enzymatic properties.J. Biol. Chem. 1998; 273: 2127-2135Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, Valnickova et al., 2007Valnickova Z. Thogersen I.B. Potempa J. Enghild J.J. Thrombin-activable fibrinolysis inhibitor (TAFI) zymogen is an active carboxypeptidase.J. Biol. Chem. 2007; 282: 3066-3076Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Protein inhibitors account for a control mechanism of proteolytic activity. Latexin, alias ECI, is the only known endogenous A/B-type MCP protein inhibitor found in mammals (Pallarès et al., 2005Pallarès I. Bonet R. García-Castellanos R. Ventura S. Avilés F.X. Vendrell J. Gomis-Rüth F.X. Structure of human carboxypeptidase A4 with its endogenous protein inhibitor, latexin.Proc. Natl. Acad. Sci. USA. 2005; 102: 3978-3983Crossref PubMed Scopus (81) Google Scholar). Its role as a TAFIa inhibitor in fibrinolysis is questionable, however, as it is not found in blood. In contrast, TCI is a physiologically relevant inhibitor that has been found in the hematophagous ixodid tick, Rhipicephalus bursa, which only feeds on ruminants. Such parasites need to inactivate host inflammation and defense mechanisms and prevent coagulation in the gut during feeding through protein inhibitors. TCI displays the highest affinity for TAFIa (equilibrium dissociation constants of 1.3 and 1.2 nM for bovine and human TAFIa, respectively) and strongly accelerates fibrinolysis similarly to the A/B-type MCP inhibitors from potato (PCI) and leech (LCI), which also target TAFIa. These protein inhibitors have proven potential as therapeutic adjuvants, and they are now under clinical trials in various cardiovascular conditions (Arolas et al., 2007Arolas J.L. Vendrell J. Avilés F.X. Fricker L.D. Metallocarboxypeptidases: emerging drug targets in biomedicine.Curr. Pharm. Des. 2007; 13: 349-366Crossref PubMed Scopus (65) Google Scholar). In addition, stimulation of the TAFI pathway is being examined as an approach to the treatment of hemophilia (Mosnier and Bouma, 2006Mosnier L.O. Bouma B.N. Regulation of fibrinolysis by thrombin activatable fibrinolysis inhibitor, an unstable carboxypeptidase B that unites the pathways of coagulation and fibrinolysis.Arterioscler. Thromb. Vasc. Biol. 2006; 26: 2445-2453Crossref PubMed Scopus (128) Google Scholar, Walsh et al., 1971Walsh P.N. Rizza C.R. Matthews J.M. Eipe J. Kernoff P.B. Coles M.D. Bloom A.L. Kaufman B.M. Beck P. Hanan C.M. Biggs R. ɛ-Aminocaproic acid therapy for dental extractions in haemophilia and Christmas disease: a double blind controlled trial.Br. J. Haematol. 1971; 20: 463-475Crossref PubMed Scopus (99) Google Scholar). Such research attests to the importance of TAFI as a pharmacological target for cardiovascular disease treatment (Do et al., 2005Do Y.H. Gifford-Moore D.S. Beight D.W. Rathnachalam R. Klimkowski V.J. Warshawsky A.M. Lu D. Inhibition of thrombin activatable fibrinolysis inhibitor by cysteine derivatives.Thromb. Res. 2005; 116: 265-271Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar, Hsu et al., 2007Hsu, M.-Y., Matsueda, G.R., and Tamura, J.K. (2007). Baboon TAFI polypeptides. March 13, 2007. U.S. patent 7189829 (United States, Bristol-Myers Squibb Company).Google Scholar, Klement et al., 1999Klement P. Liao P. Bajzar L. A novel approach to arterial thrombolysis.Blood. 1999; 94: 2735-2743Crossref PubMed Google Scholar, Refino et al., 2000Refino C.J. de Guzman L. Schmitt D. Smyth R. Jeet S. Lipari M.T. Eaton D. Bunting S. Consequences of inhibition of plasma carboxypeptidase B on in vivo thrombolysis, thrombosis and hemostasis.Fibrinol Proteol. 2000; 14: 305-314Crossref Scopus (31) Google Scholar). Recent data show that the TAFI plasma concentration in humans varies strongly and that high concentrations are a risk factor for thrombosis and coronary artery disease (Silveira et al., 2000Silveira A. Schatteman K. Goossens F. Moor E. Scharpe S. Stromqvist M. Hendriks D. Hamsten A. Plasma procarboxypeptidase U in men with symptomatic coronary artery disease.Thromb. Haemost. 2000; 84: 364-368Crossref PubMed Scopus (156) Google Scholar, van Tilburg et al., 2000van Tilburg N.H. Rosendaal F.R. Bertina R.M. Thrombin activatable fibrinolysis inhibitor and the risk for deep vein thrombosis.Blood. 2000; 95: 2855-2859Crossref PubMed Google Scholar), while low levels have been correlated with chronic liver disease (Van Thiel et al., 2001Van Thiel D.H. George M. Fareed J. Low levels of thrombin activatable fibrinolysis inhibitor (TAFI) in patients with chronic liver disease.Thromb. Haemost. 2001; 85: 667-670Crossref PubMed Scopus (73) Google Scholar). Accordingly, TAFI has been proposed as a molecular marker for vascular diseases (Boffa and Koschinsky, 2007Boffa M.B. Koschinsky M.L. Curiouser and curiouser: recent advances in measurement of thrombin-activatable fibrinolysis inhibitor (TAFI) and in understanding its molecular genetics, gene regulation, and biological roles.Clin. Biochem. 2007; 40: 431-442Crossref PubMed Scopus (80) Google Scholar, Boffa et al., 2001Boffa M.B. Nesheim M.E. Koschinsky M.L. Thrombin activable fibrinolysis inhibitor (TAFI): molecular genetics of an emerging potential risk factor for thrombotic disorders.Curr. Drug Targets Cardiovasc. Haematol. Disord. 2001; 1: 59-74Crossref PubMed Scopus (23) Google Scholar, Mann et al., 1988Mann K.G. Jenny R.J. Krishnaswamy S. Cofactor proteins in the assembly and expression of blood clotting enzyme complexes.Annu. Rev. Biochem. 1988; 57: 915-956Crossref PubMed Scopus (449) Google Scholar, Wang et al., 2007Wang X. Smith P.L. Hsu M.Y. Tamasi J.A. Bird E. Schumacher W.A. Deficiency in thrombin-activatable fibrinolysis inhibitor (TAFI) protected mice from ferric chloride-induced vena cava thrombosis.J. Thromb. Thrombolysis. 2007; 23: 41-49Crossref PubMed Scopus (35) Google Scholar). In addition to its involvement in fibrinolysis, TAFI has been implicated in wound healing, blood-pressure regulation, tissue remodeling, and inflammation by inactivating plasminogen activation, bradykinin, and inflammatory mediators (for a review, see Arolas et al., 2007Arolas J.L. Vendrell J. Avilés F.X. Fricker L.D. Metallocarboxypeptidases: emerging drug targets in biomedicine.Curr. Pharm. Des. 2007; 13: 349-366Crossref PubMed Scopus (65) Google Scholar), as well as in sepsis (Taylor and Bajzar, 2005Taylor, F.B., and Bajzar, L. (2005). Treatment of sepsis with TAFI. January 4, 2005. U.S. patent US6,838,432B2 (USA, Oklahoma Medical Research Foundation, Oklahoma City, OK, US; and McMaster University, Hamilton, CA).Google Scholar), endometriosis (Bedaiwy and Casper, 2006Bedaiwy, M., and Casper, R. (2006). Diagnosis and treatment of endometriosis. In World Intellectual Property Organization (International Bureau, Mount Sinai Hospital, Toronto, CA), November 9, 2006. Patent no. WO 2006/116873A1.Google Scholar), and pulmonary fibrosis (Gabazza et al., 2006Gabazza, E.C., Taguchi, O., Fujimoto, H., and Nagashima, M. (2006). TAFI inhibitors and their use to treat pulmonary fibrosis. In World Intellectual Property Organization (International Bureau, Schering AG, Berlin, Germany; Mie University Graduate School of Medicine, Mie, Japan; and Michael John Morser, San Francisco, CA, US), April 20, 2006. Patent no. WO 2006/041808 A2.Google Scholar). TAFI is also expressed in the neuronal endoplasmic reticulum of hippocampal neurons, potentially playing a role in the processing of native β-amyloid precursor protein in the brain (Matsumoto et al., 2000Matsumoto A. Itoh K. Matsumoto R. A novel carboxypeptidase B that processes native β-amyloid precursor protein is present in human hippocampus.Eur. J. Neurosci. 2000; 12: 227-238Crossref PubMed Scopus (30) Google Scholar, Matsumoto et al., 2001Matsumoto A. Itoh K. Seki T. Motozaki K. Matsuyama S. Human brain carboxypeptidase B, which cleaves β-amyloid peptides in vitro, is expressed in the endoplasmic reticulum of neurons.Eur. J. Neurosci. 2001; 13: 1653-1657Crossref PubMed Scopus (14) Google Scholar). Here, latexin (alias ECI), which has been detected in rodent and human brain (Arimatsu, 1994Arimatsu Y. Latexin: a molecular marker for regional specification in the neocortex.Neurosci. Res. 1994; 20: 131-135Crossref PubMed Scopus (48) Google Scholar, Normant et al., 1995Normant E. Martres M.P. Schwartz J.C. Gros C. Purification, cDNA cloning, functional expression, and characterization of a 26-kDa endogenous mammalian carboxypeptidase inhibitor.Proc. Natl. Acad. Sci. USA. 1995; 92: 12225-12229Crossref PubMed Scopus (59) Google Scholar), could play a role as an endogenous TAFI inhibitor. Given the importance and key role of the TAFI/TAFIa axis in thrombotic conditions, the detailed structure analysis of TAFIa should contribute to an explanation for its intrinsic instability and provide a mold for the design of small-molecule inhibitors, thus contributing to alternative therapeutic approaches. Glycan-induced heterogeneity, the minute amounts retrievable from single-patient samples, the impossibility of establishing efficient recombinant overexpression systems, and the instability of human TAFIa have hampered structural studies on this protein since its discovery in 1988. We sought to overcome these problems by choosing a highly homologous ortholog from a nonhuman mammalian species, for which large amounts of blood could be obtained from one individual. We purified TAFI from a single cow to homogeneity, essentially in three large-scale liquid chromatography steps, and showed it to be active against a chromogenic substrate (see Experimental Procedures). The kinetic properties of bovine TAFI and human TAFI were compared in parallel experiments, and the data suggested that the two proteins are very similar in terms of activity (Table 1), in agreement with their 78% sequence identity (see Figure 1). Freshly purified TAFI was subsequently incubated in vitro with the physiological processor thrombin in the presence of its modulator thrombomodulin and TCI. The TAFIa/TCI complex was stable for several days/weeks and suitable for structural studies. In addition, our experiments with heparin-Sepharose showed that both human and bovine TAFI bind heparin, since most of the protein eluted only in the presence of 200–500 mM NaCl (data not shown). This suggests that glycosaminoglycans may modulate TAFI as described for antithrombin III and thrombin (Bjork and Lindahl, 1982Bjork I. Lindahl U. Mechanism of the anticoagulant action of heparin.Mol. Cell. Biochem. 1982; 48: 161-182Crossref PubMed Scopus (362) Google Scholar).Table 1Comparison of the Activity of Human and Bovine TAFIEquationHuman TAFIBovine TAFIVmax (μM/min)Km (mM)Vmax (μM/min)Km (mM)Hanes44.842.3647.403.58Eadie-Hofstee44.652.3544.333.14Eisenthal-Cornish-Bowden44.962.4444.613.16Hyperbolic Regression44.99 ± 1.192.35 ± 0.2047.84 ± 3.673.88 ± 0.85Average values44.862.3846.053.44Kcat (min−1)160.21164.46Kcat/Km (min−1/mM)67.3247.81The values for Km and Vmax were determined using the direct fit of the Michaelis-Menten equation employing four graphical methods. The data represent the enzyme-catalyzed reaction for 0.28 μM TAFI. Open table in a new tab The values for Km and Vmax were determined using the direct fit of the Michaelis-Menten equation employing four graphical methods. The data represent the enzyme-catalyzed reaction for 0.28 μM TAFI. TAFIa has a compact globular shape and shows the classic α/β-hydrolase fold of A/B- and N/E-type MCPs (Arolas et al., 2007Arolas J.L. Vendrell J. Avilés F.X. Fricker L.D. Metallocarboxypeptidases: emerging drug targets in biomedicine.Curr. Pharm. Des. 2007; 13: 349-366Crossref PubMed Scopus (65) Google Scholar, Vendrell et al., 2000Vendrell J. Querol E. Avilés F.X. Metallocarboxypeptidases and their protein inhibitors. Structure, function and biomedical properties.Biochim. Biophys. Acta. 2000; 1477: 284-298Crossref PubMed Scopus (133) Google Scholar). It has a central eight-stranded β sheet (strands β1–β8) of strand connectivity +1, +2, −1x, −2x, −2, +1x, −2, which vertically spans the molecule top to bottom accumulating a vertical twist of ∼130° (Figures 1 and 2A). This gives rise to a concave face, which accommodates helices α4 and α6, and the active-site cleft. At the rear, the convex side of the sheet harbors α1–α3, α5 and α7 and the surface N and C termini of the molecule. The access to the active site is like a funnel, whose rim is shaped by a series of irregular loop segments required for interactions of the protease moiety with the prodomain and with cognate protein inhibitors in A/B-type MCPs (Pallarès et al., 2005Pallarès I. Bonet R. García-Castellanos R. Ventura S. Avilés F.X. Vendrell J. Gomis-Rüth F.X. Structure of human carboxypeptidase A4 with its endogenous protein inhibitor, latexin.Proc. Natl. Acad. Sci. USA. 2005; 102: 3978-3983Crossref PubMed Scopus (81) Google Scholar). These segments include the loop connecting strand β8 with helix α7 (Lβ8α7), Lβ3α2, Lβ5β6, Lβ6α5, as well as the first part of the 53 residue segment connecting α3 and α4 (Lα3α4). This long segment is stabilized by two disulfide bonds (Cys138–Cys161 and Cys152–Cys166; for numbering conventions, see Figure 1) and closes the front and the bottom of the active site and the specificity pocket, thus contributing to the characteristic cul-de-sac of these exopeptidases (Figure 2C).Figure 2Structure of TAFIa in Its Complex with TCIShow full caption(A) Richardson plot of the complex showing TAFIa in standard orientation with yellow β strands (β1–β8), green α helices (α1–α7) and the catalytic zinc ion as a red sphere. Segment α5-Lα5β7-β7 is shown in orange, and the TAFI exosite is shown in magenta. The region of the proposed fibrinolysis switch is shown over gray background. TCI is shown for its NTD (light blue), linker (red) and CTD (navy blue). The disulfide bonds of TCI are also shown. The N and C termini of both molecules are labeled.(B) Close-up view of (A) after a vertical rotation of ∼90° showing the proposed fibrinolysis switch region of TAFIa (yellow) superimposed with the equivalent region of human CPB1 (green). TAFIa regular secondary structure elements are labeled.(C) Close-up view of (A) showing the TAFIa active site and the residues participating in the interaction with TCI CTD.(D) Close-up view of (A) centered on the interaction area of TAFIa with TCI NTD and the participating residues.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Richardson plot of the complex showing TAFIa in standard orientation with yellow β strands (β1–β8), green α helices (α1–α7) and the catalytic zinc ion as a red sphere. Segment α5-Lα5β7-β7 is shown in orange, and the TAFI exosite is shown in magenta. The region of the proposed fibrinolysis switch is shown over gray background. TCI is shown for its NTD (light blue), linker (red) and CTD (navy blue). The disulfide bonds of TCI are also shown. The N and C termini of both molecules are labeled. (B) Close-up view of (A) after a vertical rotation of ∼90° showing the proposed fibrinolysis switch region of TAFIa (yellow) superimposed with the equivalent region of human CPB1 (green). TAFIa regular secondary structure elements are labeled. (C) Close-up view of (A) showing the TAFIa active site and the residues participating in the interaction with TCI CTD. (D) Close-up view of (A) centered on the interaction area of TAFIa with TCI NTD and the participating residues. The catalytic zinc ion resides at the bottom of the funnel-like cleft and is coordinated by His69 Nδ1 (2.09 Å apart) and by Glu72, in an asymmetric bidentate manner, through its Oɛ2 (2.01 Å) and Oɛ2 (2.76 Å) atoms (Figure 2C). These two residues are embedded in a consensus sequence, HXXE (amino-acid one-letter code; X for any residue), characteristic of A/B- and N/E-type MCPs (Hooper, 1994Hooper N.M. Families of zinc metalloproteases.FEBS Lett. 1994; 354: 1-6Crossref PubMed Scopus (667) Google Scholar). The third TAFIa zinc ligand is His196 Nδ1 (2.08 Å). An acetate ion is found next to the zinc, partially occupying the specificity pocket and hydrogen bonded through one of its carboxylate oxygen atoms to Arg127 Nη2 (3.02 Å), Arg145 Nη2 (2.89 Å), and Tyr248 Oη (2.58 Å). The latter is in the “down” orientation usually found in MCPs with occupied pockets (Reverter et al., 2000Reverter D. Fernandez-Catalan C. Baumgartner R. Pfander R. Huber R. Bode W. Vendrell J. Holak T.A. Aviles F.X. Structure of a novel leech carboxypeptidase inhibitor determined free in solution and in complex with human carboxypeptidase A2.Nat. Struct. Biol. 2000; 7: 322-328Crossref PubMed Scopus (66) Google Scholar). The other acetate carboxylate oxygen atom is bound to Asn144 Nδ2 (2.90 Å) and Arg145 Nη1 (2.80 Å). As shown for other MCPs, the protein residues engaged in substrate binding and catalysis in A/B-type MCPs (Auld, 2004Auld D.S. 240. Carboxypeptidase A.in: Barrett A.J. Rawlings N.D. Woessner Jr., J.F. Handbook of proteolytic enzymes. Elsevier Academic Press, London2004: 812-821Crossref Scopus (15) Google Scholar, Vendrell et al., 2000Vendrell J. Querol E. Avilés F.X. Metallocarboxypeptidases and their protein inhibitors. Structure, function and biomedical properties.Biochim. Biophys. Acta. 2000; 1477: 284-298Crossref PubMed Scopus (133) Google Scholar) are Arg127 and Glu270 forming S1; Arg71, Ser197, Tyr198, and Ser199 delimiting S2; and Phe279 contributing to S3. Typical B-type MCP specificity toward basic side chains in substrates is due to an S1′ pocket that is hydrophobic at its entrance and acidic at its bottom. The pocket is formed by the side chains of Asn144, Arg145, Val203, Gly243, Leu247, Ala250, Thr268, Tyr248, and Asp255. The terminal carboxylate group of a substrate, when it is trapped for scission, is fixed by Asn144, Arg145, and Tyr248, while the scissile carbonyl group is near Glu270, Arg127, and the catalytic zinc (Figure 2C). Heparin has been shown to stabilize TAFIa against spontaneous inactivation (Mao et al., 1999Mao S.S. Cooper C.M. Wood T. Shafer J.A. Gardell S.J. Characterization of plasmin-mediated activation of plasma procarboxypeptidase B. Modulation by glycosaminoglycans.J. Biol. Chem. 1999; 274: 35046-35052Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). In the absence of structural data of a complex with heparin, surface anions may point to potential binding sites. In the present complex, two pairs of sulfate ions found in the structure may represent two potential independent binding sites for heparin sulfate groups. In one case, the two sulfate ions are 10 Å apart, one is bound by Ser158 N and Oγ, and Arg52I Nη1 and Nη2 from the TCI moiety, and the other is bound by Ser160 N and Oγ, and Ala137 N. The distance between sulfate ions is similar to that found in the thrombin/heparin complex structure, in which two sets of sulfate groups from sulfoiduronate or disulfoglucosamine moieties are separated by two (11 Å apart) and three (10 Å apart) monosaccharides, respectively (Protein Data Bank [PDB] access code 1XMN; Carter et al., 2005Carter W.J. Cama E. Huntington J.A. Crystal structure of thrombin bound to heparin.J. Biol. Chem. 2005; 280: 2745-2749Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). The sulfur atoms of the second potential TAFIa site are 20 Å apart, which is compatible with the spacing of three or four monosaccharides within a heparin chain (Carter et al., 2005Carter W.J. Cama E. Huntington J.A. Crystal structure of thrombin bound to heparin.J. Biol. Chem. 2005; 280: 2745-2749Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Here, the sulfate groups are bound by His1501 Nɛ2, Ser209 Oγ, and Ser211 Oγ and by His216 Nɛ2, Arg224 Nη2 and Nɛ, and Ser 220 Oγ, respectively, and they affect segment α5-Lα5β7-β7 on top of the molecule (Figure 2A). It is interesting to note that this region contains a high number of residues whose substitution by random mutagenesis has been shown to influence TAFIa stability (Knecht et al., 2006Knecht W. Willemse J. Stenhamre H. Andersson M. Berntsson P. Furebring C. Harrysson A. Hager A.C. Wissing B.M. Hendriks D. Cronet P. Limited mutagenesis increases the stability of human carboxypeptidase U (TAFIa) and demonstrates the importance of CPU stability over proCPU concentration in down-regulating fibrinolysis.FEBS J. 2006; 273: 778-792Crossref PubMed Scopus (38) Google Scholar, Schneider et al., 2002Schneider M. Boffa M. Stewart R. Rahman M. Koschinsky M. Nesheim M. Two naturally occurring variants of TAFI (Thr-325 and Ile-325) differ substantially with respect to thermal stability and antifibrinolytic activity of the enzyme.J. Biol. Chem. 2002; 277: 1021-1030Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar; see Figure 1 and below). Therefore, heparin binding at this site could thus become a regulatory mechanism affecting the half-life of TAFIa. For the last 6 years, the structure of human pancreatic PCPB1, solved by two of our groups, has been a model for TAFI (Marx et al., 2000Marx P.F. Hackeng T.M. Dawson P.E. Griffin J.H. Meijers J.C. Bouma B.N. Inactivation of active thrombin-activable fibrinolysis inhibitor takes place by a process that involves conformational instability rather than proteolytic cleavage.J. Biol. Chem. 2000; 275: 12410-12415Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, Pereira et al., 2002Pereira P.J.B. Segura-Martín S. Oliva B. Ferrer-Orta C. Avilés F.X. Coll M. Gomis-Rüth F.X. Vendrell J. Human procarboxypeptidase B: three-dimensional structure and implications for thrombin-activatable fibrinolysis inhibitor (TAFI).J. Mol. Biol. 2002; 321: 537-547Crossref PubMed Scopus (62) Google Scholar). Sequence and structure comparison of the homology model obtained from the former with bovine TAFIa (Figures 1 and 2B) enables to assess that the gross of the model proposed was valid. In particular, TAFI very likely possesses an equivalent prodomain to PCPB1 with a globular part folded as an α/β open sandwich. This globular part would be linked to the mature enzyme moiety through a connecting segment, which would include an α helix. The prodomain would be likewise placed on top of the active site, which would be preformed in the TAFI zymogen. With respect to the mature enzyme moiety, the overall structure is also very similar, and this similarity comprises the identity and arrangement of the active-site residues (see also below). There are two major points that were anticipated by the PCPB1-based model. (1) An essential salt bridge made up between Asp41 of the prodomain and by an active-site residue, Arg145, is not present in TAFI as inferred from TAFIa. This interaction is thought to account for the lack of activity of B-type MCP zymogens, and therefore it was po" @default.
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