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Interaction of the Factor XIII Activation Peptide with α-Thrombin

2000; Elsevier BV; Volume: 275; Issue: 47 Linguagem: Inglês

10.1074/jbc.m006076200

ISSN

1083-351X

Autores

C. Sadasivan, Vivien C. Yee,

Tópico(s)

Erythrocyte Function and Pathophysiology

Resumo

The serine protease thrombin proteolytically activates blood coagulation factor XIII by cleavage at residue Arg37; factor XIII in turn cross-links fibrin molecules and gives mechanical stability to the blood clot. The 2.0-Å resolution x-ray crystal structure of human α-thrombin bound to the factor XIII-(28–37) decapeptide has been determined. This structure reveals the detailed atomic level interactions between the factor XIII activation peptide and thrombin and provides the first high resolution view of this functionally important part of the factor XIII molecule. A comparison of this structure with the crystal structure of fibrinopeptide A complexed with thrombin highlights several important determinants of thrombin substrate interaction. First, the P1 and P2 residues must be compatible with the geometry and chemistry of the S1 and S2 specificity sites in thrombin. Second, a glycine in the P5 position is necessary for the conserved substrate conformation seen in both factor XIII-(28–37) and fibrinopeptide A. Finally, the hydrophobic residues, which occupy the aryl binding site of thrombin determine the substrate conformation further away from the catalytic residues. In the case of factor XIII-(28–37), the aryl binding site is shared by hydrophobic residues P4 (Val34) and P9 (Val29). A bulkier residue in either of these sites may alter the substrate peptide conformation. The serine protease thrombin proteolytically activates blood coagulation factor XIII by cleavage at residue Arg37; factor XIII in turn cross-links fibrin molecules and gives mechanical stability to the blood clot. The 2.0-Å resolution x-ray crystal structure of human α-thrombin bound to the factor XIII-(28–37) decapeptide has been determined. This structure reveals the detailed atomic level interactions between the factor XIII activation peptide and thrombin and provides the first high resolution view of this functionally important part of the factor XIII molecule. A comparison of this structure with the crystal structure of fibrinopeptide A complexed with thrombin highlights several important determinants of thrombin substrate interaction. First, the P1 and P2 residues must be compatible with the geometry and chemistry of the S1 and S2 specificity sites in thrombin. Second, a glycine in the P5 position is necessary for the conserved substrate conformation seen in both factor XIII-(28–37) and fibrinopeptide A. Finally, the hydrophobic residues, which occupy the aryl binding site of thrombin determine the substrate conformation further away from the catalytic residues. In the case of factor XIII-(28–37), the aryl binding site is shared by hydrophobic residues P4 (Val34) and P9 (Val29). A bulkier residue in either of these sites may alter the substrate peptide conformation. factor XIII factor XIII peptide (Thr28-Val29-Glu30-Leu31-Gln32-Gly33-Val34-Val35-Pro36-Arg37) fibrinopeptide A (Asp7-Phe8-Leu9-Ala10-Glu11-Gly12-Gly13-Gly14-Val15-Arg16) D-Phe-Pro-Arg-chloromethylketone root mean square. The serine protease thrombin plays a central role in the blood coagulation process (1Fenton J.W., II Bing D.H. Semin. Thromb. Hemost. 1986; 12: 200-208Crossref PubMed Scopus (61) Google Scholar, 2Stubbs M.T. Bode W. Thromb. Res. 1993; 69: 1-58Abstract Full Text PDF PubMed Scopus (446) Google Scholar). It proteolytically activates blood coagulation and plasma factors such as factor V, factor VIII, factor XIII, and protein C (3Fenton J.W., II Ann. N. Y. Acad. Sci. 1981; 370: 468-495Crossref PubMed Scopus (199) Google Scholar). Its proteolytic activity is also responsible for catalyzing the conversion of fibrinogen to fibrin (4Fenton J.W., II Olson T.A. Zabinski M.P. Wilner G.D. Biochemistry. 1988; 27: 7106-7112Crossref PubMed Scopus (143) Google Scholar) by the cleavage of fibrinopeptides A and B from the N termini of the fibrinogen α and β chains, respectively. In addition, thrombin is the most potent stimulator of platelet aggregation (5Tollefsen D.M. Feagler J.R. Majerus P.W. J. Biol. Chem. 1974; 249: 2646-2651Abstract Full Text PDF PubMed Google Scholar). Thrombin achieves this diverse yet specific recognition of substrates with a deep active site cleft and by exploiting an apolar binding site near the catalytic residues as well as an anion binding exosite distant from the active site cleft (6Bode W. Turk D. Karshikov A. Protein Sci. 1992; 1: 426-471Crossref PubMed Scopus (648) Google Scholar). Crystal structures of α-thrombin complexed to a number of natural and synthetic inhibitors have been studied in detail and characterized to near atomic resolution (for example, see Refs. 7Bode W. Mayr I. Baumann U. Huber R. Stone S.R. Hofsteenge J. EMBO J. 1989; 8: 3467-3475Crossref PubMed Scopus (823) Google Scholar, 8Rydel T.J. Ravichandran K.G. Tulinsky A. Bode W. Huber R. Roitsch C. Fenton J.W., II Science. 1990; 249: 277-280Crossref PubMed Scopus (641) Google Scholar, 9Grutter M.G. Priestle J.P. Rahuel J. Grossenbacher H. Bode W. Hofsteenge J. Stone S.R. EMBO J. 1990; 9: 2361-2365Crossref PubMed Scopus (315) Google Scholar, 10Chen Z. Li Y. Mulichak A.M. Lewis S.D. Shafer J.A. Arch. Biochem. Biophys. 1995; 322: 198-203Crossref PubMed Scopus (46) Google Scholar, 11Rehse P.H. Steinmetzer T. Li Y. Konishi Y. Cygler M. Biochemistry. 1995; 3: 11537-11544Crossref Scopus (28) Google Scholar, 12Chirgadze N.Y. Sall D.J. Klimkowski V.J. Clawson D.K. Briggs S.L. Hermann R. Smith G.F. Gifford-Moore D.S. Wery J.-P. Protein Sci. 1997; 6: 1412-1417Crossref PubMed Scopus (24) Google Scholar, 13Weir M.P. Bethell S.S. Cleasby A. Campbell C.J. Dennis R.J. Dix C.J. Finch H. Jhoti H. Mooney C.J. Patel S. Tang C.-M. Ward M. Wonacott A.J. Wharton C.W. Biochemistry. 1998; 37: 6645-6657Crossref PubMed Scopus (55) Google Scholar, 14Krishnan R. Zhang E. Hakansson K. Arni R.K. Tulinsky A. Lim-Wilby M.S.L. Levy O.E. Semple J.E. Brunck T.K. Biochemistry. 1998; 37: 12094-12103Crossref PubMed Scopus (53) Google Scholar). These structures have provided a wealth of information about the interactions between inhibitors and enzyme. However, more limited knowledge about the atomic level interactions between substrate and thrombin comes mainly from the crystal structures of thrombin bound to fibrinopeptide A. Structures of human and bovine α-thrombin bound to the N-terminal peptide of the fibrinogen α-chain (15Stubbs M.T. Oschkinat H. Mayer I. Huber R. Angliker H. Stone S.R. Bode W. Eur. J. Biochem. 1992; 206: 187-195Crossref PubMed Scopus (198) Google Scholar, 16Martin P.D. Robertson W. Turk D. Huber R. Bode W. Edwards B.F.P. J. Biol. Chem. 1992; 267: 7911-7920Abstract Full Text PDF PubMed Google Scholar) show that the C-terminal region of the peptide runs anti-parallel to the Ser214-Glu217segment of thrombin and that the arginine at the cleavage site occupies the S1 specificity pocket. The N-terminal region of the peptide adopts a compact α-helical conformation, folding back toward the active site cleft to bring the hydrophobic residues Leu9 and Phe8 to the apolar binding site (15Stubbs M.T. Oschkinat H. Mayer I. Huber R. Angliker H. Stone S.R. Bode W. Eur. J. Biochem. 1992; 206: 187-195Crossref PubMed Scopus (198) Google Scholar, 16Martin P.D. Robertson W. Turk D. Huber R. Bode W. Edwards B.F.P. J. Biol. Chem. 1992; 267: 7911-7920Abstract Full Text PDF PubMed Google Scholar). In the present study a 2.0-Å resolution crystal structure of the factor XIII activation peptide, encompassing residues 28–37, complexed to human α-thrombin, sheds light on further details of thrombin-substrate interactions. This work describes the second unique structure of a thrombin-substrate complex, reveals conserved structural features as well as conformational differences in substrate and enzyme, and extends understanding of thrombin-substrate interactions. Factor XIII is a transglutaminase and the last enzyme to be activated in the blood coagulation cascade. The zymogen circulates in human plasma as a heterotetramer of two catalytic A-subunits and two carrier B-subunits and is found intracellularly in platelets, megakaryocytes, and macrophages as an A2 dimer (17Folk J.E. Chung S.I. Adv. Enzymol. Relat. Areas Mol. Biol. 1973; 38: 109-191PubMed Google Scholar, 18Folk J.E. Adv. Enzymol. Relat. Areas Mol. Biol. 1983; 54: 1-56PubMed Google Scholar, 19Muszbek L. Adany R. Mikkola H. Crit. Rev. Clin. Lab. Sci. 1996; 33: 357-421Crossref PubMed Scopus (186) Google Scholar, 20Muszbek L. Yee V.C. Hevessy Z. Thromb. Res. 1999; 94: 271-305Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar). Factor XIII is activated by thrombin, which cleaves the A-subunit between Arg37 and Gly38, releasing the N-terminal activation peptide in the presence of Ca2+ ions (21Lorand L. Ann. N. Y. Acad. Sci. 1986; 485: 144-158Crossref PubMed Scopus (48) Google Scholar, 22Hornyak T.J. Bishop P.D. Shafer J.A. Biochemistry. 1989; 28: 7326-7332Crossref PubMed Scopus (53) Google Scholar). The activated enzyme catalyzes the formation of γ-glutamyl-ε-lysyl amide cross-links between polypeptide chains in adjacent fibrin molecules, rendering the blood clot mechanically stable and resistant to fibrinolysis. The residues surrounding the thrombin cleavage site in factor XIII are of particular interest. Val34 has drawn much recent attention, because a common Leu34 polymorphism has been associated with a lower incidence of myocardial infarction in patients with coronary artery disease (23Kohler H.P. Stickland M.H. Ossei-Gerning N. Carter A. Mikkola H. Grant P.J. Thromb. Haemost. 1998; 79: 8-13Crossref PubMed Scopus (252) Google Scholar, 24Wartiovaara U. Perola M. Mikkola H. Totterman K. Savolainen V. Penntila A. Grant P.J. Tikkanen M.J. Vartiainen E. Karhunen P.J. Peltonen L. Palotie A. Atherosclerosis. 1999; 142: 295-300Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 25Franco R.F. Pazin-Filho A. Tavella M.H. Simoes M.V. Marin-Neto J.A. Zago M.A. Haematologica. 2000; 85: 67-71PubMed Google Scholar) and of deep venous thrombosis (26Catto A.J. Kohler H.P. Coore J. Mansfield M.W. Stickland M.H. Grant P.J. Blood. 1999; 93: 906-908Crossref PubMed Google Scholar, 27Franco R.F. Reitsma P.H. Lourenco D. Maffei F.H. Morelli V. Tavella M.H. Araujo A.G. Piccinato C.E. Zago M.A. Thromb. Haemost. 1999; 81: 676-679Crossref PubMed Scopus (133) Google Scholar). In addition, the Val34Leu polymorphism was found to be more common in patients with primary intracerebral hemorrhage in one study (28Catto A.J. Kohler H.P. Bannan S. Stickland M. Carter A. Grant P.J. Stroke. 1998; 29: 813-816Crossref PubMed Scopus (142) Google Scholar) and to be less common in patients with brain infarction in another (29Elbaz A. Poirier O. Canaple S. Chedru F. Cambien F. Amarenco P. Blood. 2000; 95: 586-591Crossref PubMed Google Scholar). Recombinant and purified factor XIII Leu34variant has a significantly higher specific activity than the Val form (30Kangsadalampai S. Board P.G. Blood. 1998; 92: 2766-2770Crossref PubMed Google Scholar, 31Kohler H.P. Ariens R.A.S. Whitaker P. Grant P.J. Thromb. Haemost. 1998; 80: 704Crossref PubMed Scopus (19) Google Scholar) and is activated more quickly (32Balogh I. Szoke G. Karpati L. Wartiovaara U. Katona E. Komaromi I. Haramura G. Pfliegler G. Mikkola H. Muszbek L. Blood. 2000; 96: 2479-2486Crossref PubMed Google Scholar, 33Ariens R.A.S. Philippou H. Nagaswami C. Weisel J.W. Lane D.A. Grant P.J. Blood. 2000; 96: 988-995Crossref PubMed Google Scholar). The proximity of Val34 to the thrombin cleavage site at Arg37suggests that the Leu34 substitution may affect thrombin cleavage and be responsible thus for the difference in activation and specific activity (33Ariens R.A.S. Philippou H. Nagaswami C. Weisel J.W. Lane D.A. Grant P.J. Blood. 2000; 96: 988-995Crossref PubMed Google Scholar, 34Trumbo T.A. Maurer M.C. J. Biol. Chem. 2000; 275: 20627-20631Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Crystal structures of factor XIII A2 in zymogen, thrombin-cleaved, and Ca2+-bound forms have been determined (35Yee V.C. Pedersen L.C. Le Trong I. Bishop P.D. Stenkamp R.E. Teller D.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7296-7300Crossref PubMed Scopus (323) Google Scholar, 36Yee V.C. Pedersen L.C. Bishop P.D. Stenkamp R.E. Teller D.C. Thromb. Res. 1995; 78: 389-397Abstract Full Text PDF PubMed Scopus (83) Google Scholar, 37Fox B.A. Yee V.C. Pedersen L.C. Le Trong I. Bishop P.D. Stenkamp R.E. Teller D.C. J. Biol. Chem. 1999; 274: 4917-4923Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). In all structures, the 37-residue N-terminal activation peptide remains in the same position and conformation with respect to the rest of the molecule, even after thrombin cleavage or cation binding. As well, in all structures the activation peptide near the Arg37 cleavage site is highly disordered and poorly defined in the electron density maps. The present crystal structure of the factor XIII-(28–37) decapeptide bound to human α-thrombin reveals the detailed atomic level interactions between the activation peptide and thrombin and provides the first high resolution view of this functionally important part of the factor XIII molecule. Human α-thrombin (Enzyme Research Laboratories, Inc., South Bend, IN) and the factor XIII (fXIII1) peptide as a chloromethylketone derivative (Thr28-Val29-Glu30-Leu31-Gln32-Gly33-Val34-Val35-Pro36-Arg37-CMK, AnaSpec, Inc., San Jose, CA) were obtained from commercial sources. The enzyme was concentrated to 8 mg/ml and mixed with a 10-fold molar excess of peptide dissolved in water. Crystals of the thrombin-peptide complex were grown by hanging drop vapor diffusion at room temperature, from 24% polyethylene glycol 8000 and 200 mm sodium chloride in 50 mm sodium citrate buffer at pH 5.5. Typically crystals grow to full size in about 2 weeks, to maximum dimensions of 0.1 × 0.2 × 0.4 mm. A crystal stabilized in a cryoprotectant containing 15% glycerol was cooled in a stream of cold nitrogen (−180 °C) for data collection. Diffraction data were collected on an in-house Rigaku R-AXIS IV imaging plate system using CuKα radiation (λ = 1.5418 Å) from a Rigaku H3R rotating anode x-ray generator operated at 50 kV, 100 mA and equipped with Yale focusing mirrors. A total of 127 images was collected with 60-min exposure, 1.5° oscillation step, and a crystal-detector distance of 100 mm. Data to 2.0-Å resolution were processed using the HKL program suite (38Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38617) Google Scholar); data processing statistics are listed in Table I. The unit cell parameters are a = 54.07, b = 81.26, c = 85.46 Å, and β = 101.62° with space group P21. The calculated Matthews number of 2.7 (39Matthews B.W. J. Mol. Biol. 1968; 33: 491-497Crossref PubMed Scopus (7927) Google Scholar) corresponds to a solvent content of 55% and Z = 4, indicating that there are two independent molecules in the asymmetric unit.Table IData collection and final refinement statisticsData collection Resolution range (Å)20.0–2.0(2.07–2.0) 1-aValues in parentheses are for the highest resolution bin. Number of observations124,210 Number of unique reflections45,245(3873) Data completeness (%)91.8(79.3) R merge0.053(0.157)Refinement Resolution range (Å)10.0–2.0 Number of reflections44,341 Number of non-hydrogen protein atoms4485 Number of peptide atoms154 Number of sodium ions2 Number of solvent molecules479 Rfactor0.194 Free R factor0.257r.m.s. deviation from ideality Bond distance (Å)0.009 Bond angle (°)2.46 Dihedral angle (°)24.75 Improper angle (°)1.64Average B factor (Å2) Protein molecule 122.6 Protein molecule 235.5 Peptide 126.4 Peptide 271.3 Sodium ions19.9 Solvent40.6DDQ parameters DDQW11.22 DDQR52.291-a Values in parentheses are for the highest resolution bin. Open table in a new tab The structure was solved by molecular replacement (40Rossmann M.G. Blow D.M. Acta Crystallogr. 1962; 15: 24-31Crossref Google Scholar) using the program AMoRe (41Navaza J. Acta Crystallogr. A. 1994; 50: 157-163Crossref Scopus (5030) Google Scholar), with human α-thrombin coordinates from the 1.6-Å resolution crystal structure of its complex with hirugen and p-amidinophenylpyruvate, and Protein Data Bank code 1AHT (10Chen Z. Li Y. Mulichak A.M. Lewis S.D. Shafer J.A. Arch. Biochem. Biophys. 1995; 322: 198-203Crossref PubMed Scopus (46) Google Scholar) as the search model. The cross rotation calculation showed two peaks with correlation coefficients 34.5% and 28.3%, respectively, which were significantly higher than the rest. Translation function calculations for these peaks gave two clear solutions and the overall correlation coefficient increased to 61.3% with an R factor of 37.0%. Rigid body refinement gave a correlation coefficient and R factor of 69.9% and 33.4%, respectively, for 10.0- to 3.5-Å resolution data and indicated the solution was correct. A rigid body refinement with the two molecules in the asymmetric unit treated as independent rigid groups was then carried out using X-PLOR (42Brunger A.T. XPLOR version 3.1. Yale University Press, New Haven, CT1992Google Scholar). The model was refined further by the gradual extension of data to 2.0-Å resolution, alternating with manual model fitting to the 2‖F o‖ − ‖F c‖ and ‖F o‖ − ‖F c‖ electron density maps using the program O (43Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar). A molecular dynamics refinement using the simulated annealing technique was also carried out. The fXIII-(28–37) residues initially built into 2‖F o‖ − ‖F c‖ and ‖F o‖ − ‖F c‖ omit electron density contoured at 1.0 and 2.5 ς, respectively (Fig. 1), before being included in the refinement. Water molecules were selected from ‖F o‖ − ‖F c‖ peaks higher than 4ς; the sodium ions were selected from positive ‖F o‖ − ‖F c‖ peaks, which corresponded to the binding site characterized elsewhere (44Di Cera E. Guinto E.R. Vindigni A. Dang Q.D. Ayala Y.M. Wuyi M. Tulinsky A. J. Biol. Chem. 1995; 270: 22089-22092Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 45Zhang E. Tulinsky A. Biophys. Chem. 1997; 63: 185-200Crossref PubMed Scopus (101) Google Scholar). These positions were checked after each cycle of refinement and retained only if the electron density remained clear in the 2‖F o‖ − ‖F c‖ map. The final R and free R values are 19.3% and 25.6%, respectively, for 10- to 2.0-Å resolution data. The model quality was checked using PROCHECK (46Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Cryst. 1993; 26: 283-291Crossref Google Scholar) and DDQ (47van den Akker F. Hol W.G.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 206-218Crossref PubMed Scopus (49) Google Scholar). All the main-chain torsion angles for non-glycine and non-proline residues are within the allowed regions of the Ramachandran plot (48Ramachandran G.N. Sasisekharan V. Adv. Protein. Chem. 1968; 23: 283-438Crossref PubMed Scopus (2770) Google Scholar). The refinement and geometric parameters are given in Table I. The asymmetric unit consists of two thrombin molecules (MOL1 and MOL2), two peptide molecules (PEP1 and PEP2), 479 waters, and 2 sodium ions. The residue numbering in thrombin is based on that of chymotrypsin (7Bode W. Mayr I. Baumann U. Huber R. Stone S.R. Hofsteenge J. EMBO J. 1989; 8: 3467-3475Crossref PubMed Scopus (823) Google Scholar). Peptide residues are numbered 28 to 37 to conform to the intact factor XIII sequence. Terminal residues 1H–1B and 14L–15 in the thrombin light chain and residues 245–247 and 148–149E in the heavy chain lack continuous and well defined electron density, and were not included in the model. Well defined electron density was observed for PEP1 (Fig. 1) and the C-terminal portion of PEP2. However the electron density for N-terminal residues 28–30 of PEP2 was found be relatively weak, as reflected in high B factors (Table I). The structures of the two thrombin molecules observed in this complex crystal are quite similar to other thrombin crystal structures reported in the literature. The r.m.s. deviation for the Cα atoms of the first and second molecules from those of the initial search model are 0.59 and 0.46 Å, respectively, whereas the r.m.s. deviation between the two molecules in the asymmetric unit is 0.51 Å. The two protein molecules in the asymmetric unit of this crystal structure participate in substantially different crystal packing interactions. MOL1 participates in 323 intermolecular contacts (≤4.0 Å) with its 6 surrounding protein molecules, whereas only 175 such contacts with 4 surrounding protein molecules are observed for MOL2. The tight packing of MOL1 compared with MOL2 in the crystal structure is also reflected in the different average B factors for the two molecules of 22.6 and 35.5 Å2, respectively (Table I). The fXIII-(28–37) peptides PEP1 and PEP2 are bound to thrombin molecules MOL1 and MOL2, respectively. The two peptides have roughly the same backbone conformation: the r.m.s. deviation of the ten Cα atoms after superposition of the two peptides is 0.75 Å. Side-chain atoms of N-terminal residues show considerable conformational differences between the two peptides, as evidenced by a high r.m.s. deviation for all atoms between the two peptides of 1.80 Å. The C-terminal segment Val34-Arg37 of the peptides adopts an extended β-sheet arrangement with main-chain hydrogen bonds with thrombin and runs anti-parallel to the Ser214-Glu217 segment of the enzyme (Fig.2). The peptide N-terminal residues Thr28 to Gln32 form a short stretch of irregular α-helix with main-chain hydrogen bonds between residues Thr28-Leu31, (Thr28-Gln32 in the case of PEP2), Val29-Gln32, and Glu30-Gly33. The N-terminal segment folds back toward the active site cleft and Gly33 facilitates this folding, bringing Val29 and Val34, rather distant along the peptide chain, close enough to form main-chain hydrogen bonds. The hydrophobic side chains of both these valine residues are in close proximity and point in the same direction. The side chains of residues Thr28, Glu30, and Leu31 are clustered together and point in the opposite direction. This arrangement gives a compact structure for the segment Thr28-Val34 (Fig. 1). TableII lists the hydrogen bond interactions between the peptide and thrombin in each complex; MOL1-PEP1 interactions are illustrated in Fig. 2. Arg37 makes a number of hydrogen bonds with the thrombin residues constituting the active site cleft. It forms main-chain hydrogen bonds with thrombin residues Ser214, Gly193, and Ser195; its side chain participates in hydrogen bonds with the carbonyl oxygen of Gly219 and side-chain oxygen atoms of Asp189. Glu192 of MOL2 swings in to interact with both Arg37 and Pro36 in PEP2. The peptide structure is stabilized also by two water hydrogen bonds in the specificity pocket. These hydrogen bonds are made by N-ε and N-H1 of Arg37. Other interactions include Val35in the peptide forming main-chain hydrogen bonds with Gly216 of thrombin, and the terminal nitrogen of Thr28 hydrogen bonding to thrombin's carbonyl oxygen of Arg97. A large number of van der Waals interactions (≤4.0 Å) also are observed between the peptide and the enzyme molecule, as schematically represented in Fig. 3.Table IIPeptide-thrombin hydrogen bondsPeptide atomThrombin atomDistanceMolecule 1Molecule 2ÅThr28NArg97O3.113.01Thr28NGln38O-ε23.26 2-aHydrogen bonds between PEP1 and MOL2, resulting from the crystal packing. No hydrogen bonds are observed between PEP2 and MOL1.Gln32N-ε2Lys36O2.84 2-aHydrogen bonds between PEP1 and MOL2, resulting from the crystal packing. No hydrogen bonds are observed between PEP2 and MOL1.Gln32N-ε2Arg173O2.84Gln32N-ε2Gln38O-ε23.14 2-aHydrogen bonds between PEP1 and MOL2, resulting from the crystal packing. No hydrogen bonds are observed between PEP2 and MOL1.Val35NGly216O3.113.00Val35OGly216N3.423.29Pro36OGlu192O-ε22.71Arg37NSer214O2.782.89Arg37NH1Asp189O-δ12.932.89Arg37NH2Asp189O-δ22.973.02Arg37NH2Gly219O2.843.11Arg37OGly193N3.443.35Arg37OSer195N3.113.12Arg37OGlu192O-ε23.04Intermolecular hydrogen bonds between the peptide and thrombin. Both the peptides make hydrogen bonds with the enzyme to which it is attached.2-a Hydrogen bonds between PEP1 and MOL2, resulting from the crystal packing. No hydrogen bonds are observed between PEP2 and MOL1. Open table in a new tab Intermolecular hydrogen bonds between the peptide and thrombin. Both the peptides make hydrogen bonds with the enzyme to which it is attached. Significant differences in crystal contacts involving the N-terminal regions of the two fXIII-(28–37) peptides are observed. In PEP1, the peptide nitrogen of Thr28 and terminal nitrogen of Gln32 hydrogen bond to O-ε1 of Gln38 in MOL2; Gln32 also hydrogen bonds to the carbonyl oxygen of Lys36 in MOL2. In addition, a number of van der Waals interactions are observed between PEP1 and MOL2. Thr28, Glu30, Leu31, and Gln32 in PEP1 make contacts with Lys36, Gln38, Leu65, Arg67, Tyr76, and Ile82 in MOL2. In contrast, no intermolecular interactions are observed between PEP2 and MOL1. The lack of these stabilizing crystal contacts partially explains the high B factors for PEP2. It should be noted that MOL2 also has a higher average B factor than MOL1. The carbonyl carbon atom of fXIII-(28–37)'s P1 residue Arg37 makes a hemiketal bond with the active site residue Ser195 O-γ, and is also linked to His57N-ε2 through a methylene, because the original peptide was derivatized with a reactive chloromethylketone group. The Arg37 side chain occupies the S12 specificity pocket, whereas Pro36 fills the S2 subsite. The side chain of the P3 residue Val35 points toward the bulk solvent, away from the apolar binding site. Instead, the aryl binding site of thrombin is occupied by fXIII-(28–37)'s hydrophobic residues Val34 and Val29. The final structure contains 489 water molecules and two sodium ions. Each sodium ion coordinates with carbonyl oxygen atoms of Arg221A and Lys224 in a thrombin molecule. The waters which surround each sodium ion are part of a solvent cluster that occupies an internal cavity enclosed by residues Tyr184A-Gly188, Asp221-Tyr224, and Asp189, as reported earlier (44Di Cera E. Guinto E.R. Vindigni A. Dang Q.D. Ayala Y.M. Wuyi M. Tulinsky A. J. Biol. Chem. 1995; 270: 22089-22092Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 45Zhang E. Tulinsky A. Biophys. Chem. 1997; 63: 185-200Crossref PubMed Scopus (101) Google Scholar). This cluster and its linkage to the bulk water has previously been suggested as a route to expel water molecules through the back of the specificity pocket upon insertion of a P1 side chain during substrate/inhibitor binding. Among the many other water clusters also observed in the current structure is a large clustering of solvent molecules between the two thrombin molecules in the asymmetric unit. Many of these water molecules are hydrogen bound to exosite II residues Arg126, Asp178, and Arg233 of MOL1 and Lys135, Glu164, Asp186A, Glu186B, and Lys186D of MOL2. The structure of this thrombin-fXIII-(28–37) complex can be compared with that of the only other substrate complexes studied, those of fibrinopeptide A-(7–16) (FPA) bound to α-thrombin (15Stubbs M.T. Oschkinat H. Mayer I. Huber R. Angliker H. Stone S.R. Bode W. Eur. J. Biochem. 1992; 206: 187-195Crossref PubMed Scopus (198) Google Scholar) (Fig. 3). The fXIII-(28–37) and FPA follow roughly the same backbone conformation (Fig. 4) despite the fact that the sequence identity between the two peptides is only 20%. The r.m.s deviations of the Cα atoms between FPA and PEP1, and between FPA and PEP2, are 1.00 and 0.57 Å, respectively. Although PEP1 and PEP2 show similar r.m.s deviations from FPA for P1–P5 residues after superposition of this segment (0.47 and 0.52 Å), the PEP1 shows a larger deviation (0.69 Å) than PEP2 (0.33 Å) in the case of N-terminal residues. The difference in similarity between the two fXIII-(28–37) peptides and FPA may not be significant given the higher B-factors and likely greater positional uncertainty for the atoms in PEP2. A comparison of the fXIII-(28–37) with PPACK bound to thrombin (7Bode W. Mayr I. Baumann U. Huber R. Stone S.R. Hofsteenge J. EMBO J. 1989; 8: 3467-3475Crossref PubMed Scopus (823) Google Scholar) shows that residues P1–P3 superimpose well. This indicates that the C-terminal residues, which are bound to the active site cleft and also involved in the hydrogen bonding with the enzyme, have essentially the identical conformation in fXIII-(28–37), FPA, and PPACK. The arginine side chain of PPACK fills the specificity pocket, and the proline is encapsulated in a hydrophobic cage. The same mode of interaction is observed between the P1 Arg37 residue and thrombin in the FPA and fXIII-(28–37) complexes. The P2 residues, Pro36 in fXIII-(28–37) and Val15 in FPA, occupy the same position as proline in PPACK. Thus the conformations of the C-terminal residues in fXIII-(28–37) and in FPA are determined mainly by the active site geometry of thrombin, because this region has direct, specific interactions with the enzyme. The N-terminal residues, however, have only weak van der Waals interactions with thrombin and are not engulfed by thrombin molecules to give a tight packing. One probable reason for the observed folding of the substrate peptides is the stability offered by the clustering and burial of hydrophobic residues, rather distant along the peptide chain, in the apolar binding site. In PPACK, the benzyl side chain of D-Phe fills the residual part of the hydrophobic cavity called the aryl binding site (6Bode W. Turk D. Karshikov A. Protein Sci. 1992; 1: 426-471Crossref PubMed Scopus (648) Google Scholar). The side chain of a substrate's l-amino acid at the P3 position would interact with a different thrombin site as compared with that of PPACK. In the fXIII-(28–37), the side chain of P3 residue Val35 points toward the bulk solvent, and the side chain of P4 residue Val34 occupies the aryl binding site instead. Based on modeling studies, it has been suggested that the side chain of anl-stereoisomer at P3 would extend into bulk solvent due to steric hindrance by the P2 side chain and the Trp60D indole group of thrombin (7Bode W. Mayr I. Baumann U. Huber R. Stone S.R. Hofsteenge J. EMBO J. 1989; 8: 3467-3475Crossref PubMed Scopus (823) Google Scholar), and probably the side chain of the P4 residue in a polypeptide substrate would point toward the apolar binding site (6Bode W. Turk D. Karshikov A. Protein Sci. 1992; 1: 426-471Crossref PubMed Scopus (648) Google Scholar). Because the P3 and P4 residues in FPA are glycines, this fXIII-(28–37) structure provides the first experimental confirmation for this prediction of substrate conformation when bound to thrombin. The aryl binding site in the fXIII-(28–37) complex is occupied by the aliphatic side chains of P9 residue Val29 and P4 residue Val34 and is bordered by the side chains of thrombin residues Tyr60A, Ile174, Trp215, and Glu217, and main-chain atoms of residues Arg97-Asn98 and Gly216. In the case of FPA, the P9 residue Phe8 occupies almost the same relative position as Val29, and the P8 residue Leu9 is displaced from both the Val29 and Val34 positions in fXIII-(28–37) (Fig.5). The crystal structure of the thrombin-hirudin complex (8Rydel T.J. Ravichandran K.G. Tulinsky A. Bode W. Huber R. Roitsch C. Fenton J.W., II Science. 1990; 249: 277-280Crossref PubMed Scopus (641) Google Scholar) shows that the phenolic side chain of the inhibitor's Tyr3 is located in the aryl binding site (Fig.5). Finally, the naphthyl and tosyl moieties of benzamidine-derived inhibitors, and other aromatic groups in synthetic inhibitors interact in a similar favorable manner with this apolar site (6Bode W. Turk D. Karshikov A. Protein Sci. 1992; 1: 426-471Crossref PubMed Scopus (648) Google Scholar, 49Matsuzaki T. Sasaki T. Okumura C. Umeyama H. J. Biochem. (Tokyo). 1989; 105: 949-952Crossref PubMed Scopus (29) Google Scholar, 50Bode W. Turk D. Sturzebecher J. Eur. J. Biochem. 1990; 193: 175-182Crossref PubMed Scopus (127) Google Scholar, 51Turk D. Sturzebecher J. Bode W. FEBS Lett. 1991; 287: 33-138Crossref Scopus (74) Google Scholar, 52Malkowski M.G. Martin P.D. Lord S.T. Edwards B.F.P. Biochem. J. 1997; 326: 815-822Crossref PubMed Scopus (17) Google Scholar). In the present crystal structure, Ile174 shows side-chain conformational differences and a shift of about 1 Å compared with thrombin in complexes with FPA or PPACK (Fig.6). This may be due to the steric bulk of the Val34 side chain in the aryl binding site. In addition, Trp215 shows a slight shift in its position. Therefore, a bulkier side chain at P4 position may not be a favorable condition for the observed conformation of the substrate peptides. We have modeled a leucine residue in place of Val34 and have calculated distances to surrounding atoms. This calculation showed that the Leu34 side chain in various orientations makes unfavorable short contacts with either thrombin or N-terminal segments of the peptide or both. Thrombin residues that are involved in these short contacts are Glu217, Ile174, Trp215, and Gly216, and peptide residues involved are Val29 and Gln32. The short contacts between the modeled Leu34 and Val29 of the peptide indicate that the Val34Leu mutation likely leads to a different peptide conformation. Eventually this may affect properties of factor XIII as a substrate. For example, in the crystal structure of thrombin bound to fibrinopeptide A with phenylalanine replaced by the larger tyrosine at P9, a disordered peptide conformation results (52Malkowski M.G. Martin P.D. Lord S.T. Edwards B.F.P. Biochem. J. 1997; 326: 815-822Crossref PubMed Scopus (17) Google Scholar). The observed peptide conformation is incompatible with nucleophilic attack by the catalytic Ser195 and explains how the bulkier tyrosine mutation renders the peptide a less susceptible substrate. Finally, the side-chain arrangement of Glu30 in fXIII-(28–37) is different from that of the corresponding Leu9 in FPA. Although the hydrophobic side chain of leucine in FPA folds toward the apolar binding site, the charged Glu30 side chain in fXIII-(28–37) points toward the bulk solvent (Fig. 4). In FPA Glu11 makes salt bridges with Arg173 of thrombin. This interaction has been thought of as an important factor for the binding of fibrinogen to thrombin (15Stubbs M.T. Oschkinat H. Mayer I. Huber R. Angliker H. Stone S.R. Bode W. Eur. J. Biochem. 1992; 206: 187-195Crossref PubMed Scopus (198) Google Scholar, 16Martin P.D. Robertson W. Turk D. Huber R. Bode W. Edwards B.F.P. J. Biol. Chem. 1992; 267: 7911-7920Abstract Full Text PDF PubMed Google Scholar). In fXIII-(28–37), the corresponding residue Gln32 adopts a different side-chain conformation and makes only a few van der Waals interactions with thrombin. The loss of binding energy due to the absence of this peptide-thrombin electrostatic interaction may be at least partially compensated by the dual hydrophobic insertions into the aryl binding site in the fXIII-(28–37) complex. It has been observed that glycine at P5 position is conserved in fibrinogen, and a non-glycine residue would be unfavorable to the observed peptide folding (15Stubbs M.T. Oschkinat H. Mayer I. Huber R. Angliker H. Stone S.R. Bode W. Eur. J. Biochem. 1992; 206: 187-195Crossref PubMed Scopus (198) Google Scholar, 16Martin P.D. Robertson W. Turk D. Huber R. Bode W. Edwards B.F.P. J. Biol. Chem. 1992; 267: 7911-7920Abstract Full Text PDF PubMed Google Scholar). In fXIII-(28–37) also, the P5 position is occupied by a glycine and adopts a similar main-chain conformation. This confirms an important role for glycine at P5 for substrate conformation and thus for substrate-thrombin interaction. The fXIII-(28–37)- and FPA-bound thrombin crystal structures, along with those for inhibitor complexes, highlight the several important determinants of thrombin substrate structure. First, the P1 and P2 residues must be compatible with the geometry and chemistry of the S1 and S2 specificity sites in thrombin. Second, a glycine in the P5 position is necessary for the conserved substrate conformation seen in both fXIII-(28–37) and FPA. Finally, the hydrophobic residues, which occupy the aryl binding site determine the substrate conformation further away from the catalytic residues. In FPA, the P8 residue Leu9 occupies the aryl binding site and interacts with thrombin's apolar binding site, but a similar interaction was not observed for the corresponding Glu30 in fXIII-(28–37). In the case of fibrinopeptide A, a hydrophobic P9 residue is crucial for thrombin cleavage; a bulkier residue in this position disrupts the geometry necessary for nucleophilic attack by Ser195 and makes the peptide a less favorable substrate (52Malkowski M.G. Martin P.D. Lord S.T. Edwards B.F.P. Biochem. J. 1997; 326: 815-822Crossref PubMed Scopus (17) Google Scholar). In the case of fXIII-(28–37), the aryl binding site is shared by hydrophobic residues P4 (Val34) and P9 (Val29).

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