©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Na Binding Site of Thrombin (*)

(Received for publication, July 19, 1995)

Enrico Di Cera (1)(§) Enriqueta R. Guinto (1) Alessandro Vindigni (1)(¶) Quoc D. Dang (1) Youhna M. Ayala (1) Meng Wuyi (2) Alexander Tulinsky (2)

From the  (1)Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110 and (2)Department of Chemistry, Michigan State University, East Lansing, Michigan 48824-1322

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Thrombin is an allosteric serine protease existing in two forms, slow and fast, targeted toward anticoagulant and procoagulant activities. The slow fast transition is induced by Na binding to a site contained within a cylindrical cavity formed by three antiparallel beta-strands of the B-chain (Met-Tyr, Lys-Tyr, and Val-Gly) diagonally crossed by the Glu-Glu strand. The site is shaped further by the loop connecting the last two beta-strands and is located more than 15 Å away from the catalytic triad. The cavity traverses through thrombin from the active site to the opposite surface and contains Asp of the primary specificity site near its midpoint. The bound Na is coordinated octahedrally by the carbonyl oxygen atoms of Tyr, Arg, and Lys, and by three highly conserved water molecules in the D-Phe-Pro-Arg chloromethylketone thrombin. The sequence in the Na binding loop is highly conserved in thrombin from 11 different species and is homologous to that found in other serine proteases involved in blood coagulation. Mutation of two Asp residues flanking Arg (D221A/D222K) almost abolishes the allosteric properties of thrombin and shows that the Na binding loop is also involved in direct recognition of protein C and antithrombin.


INTRODUCTION

Thrombin is a serine protease playing a key role in hemostasis(1, 2) . The enzyme is allosteric and exists in two forms, slow and fast (3) . The slow form plays an anticoagulant role because it activates protein C (PC) (^1)more specifically, generating a potent inhibitor of blood coagulation; the fast form has a procoagulant role because it cleaves fibrinogen with higher specificity, promoting the formation of a blood clot(4) . The slow fast transition is triggered by the specific binding of a Na ion(3, 5) . Other monovalent cations are less effective and bind with much lower affinity. Under physiological conditions of Na concentration, pH, and temperature, the slow and fast forms are almost equally populated (3) and help maintain the balance between procoagulant and anticoagulant activities that is crucial for effective hemostasis. Furthermore, the slow fast equilibrium in vivo is under conditions where allosteric regulation is most efficient and can be exploited in much needed therapeutic treatments of thrombotic and bleeding disorders(2, 6) .

Specific monovalent cation effects in proteins appear to be widespread and have been documented for a long time(7, 8) . In the realm of blood coagulation, these effects were first reported for factor Xa (9) and then for thrombin (10) and activated PC(11) . Monovalent cations can act as allosteric effectors, influencing the overall structure of an enzyme, or as cofactors that interact directly with substrates. Detailed structural information on the molecular basis underlying these effects has recently emerged for enzymes with specific requirement for K(12, 13, 14) . In the case of Na specific effects, as seen for thrombin, identification of the bound Na ion in the crystal structure is complicated by the comparable electron density of a water molecule and this monovalent cation. For this reason, the exact position of the Na binding site of thrombin has remained elusive, notwithstanding a number of crystallographic structures of the enzyme complexed with various ligands and inhibitors that have been reported in the presence of NaCl (15) . Given the crucial physiological effect of Na on thrombin(4) , identification of the Na binding site should reveal key aspects of structure-function relationships for this important enzyme.


MATERIALS AND METHODS

Crystals of thrombin inhibited at the active site with PPACK were grown from sodium phosphate and PEG 8000 in the presence of NaCl as described previously(16, 17) . The mother liquor solution containing PPACK-thrombin crystals was exchanged with K ion solution of identical composition replacing Na with K ions (0.1 M potassium phosphate buffer, pH 7.3, 32% PEG 8000, 0.187 M KCl), by adding a 2-µl aliquot to a 10-µl hanging drop and withdrawing 2 µl from the other side of the drop. This was done four times over 2 days. To complete the Na-K exchange, the crystals were transferred directly into 1 ml of the potassium solution. The Na-Rb exchange was accomplished using a capillary soaking method (18) with the potassium phosphate solution containing RbCl instead of KCl. The crystal was placed in a capillary tube used to mount crystals for diffraction purposes. Then 20 µl of the potassium phosphate solution was placed over the crystal, upon which was layered 20 µl of a similar solution that contained 1.88 M RbCl, followed by another 20-µl layer of the potassium phosphate solution. Diffusion was allowed to take place for 90 h before removing the solution and mounting the crystal in a usual manner. The final concentration of the Rb ion in the capillary is estimated to be 0.625 M. The x-ray diffraction pattern of the Na ion exchanged crystal was measured with an R-AXIS imaging plate detector and used to determine and refine the Rb ion PPACK-thrombin structure (R = 0.19 without solvent structure).

The double mutation D221A/D222K was obtained by amplification of the BglII/EcoRI sequence of pNUT-hII (19) using appropriate primers for the polymerase chain reaction. The double mutant was expressed in baby hamster kidney cells (BHK21) as prethrombin-1 using the pNUT vector constructed with the bovine factor V signal peptide and a 12-amino acid HPC4 epitope(20) . The prethrombin-1 mutant preceded by a 12-amino acid HPC4 epitope was purified on an HPC4 Affi-Gel 10 column. The zymogen form was activated, and the thrombin form was purified and tested for activity as described (21) .

Human alpha-thrombin was purified and tested for activity as described (3, 21) . Recombinant desulfatohirudin variant 1 (Revasc(TM)) was obtained from Ciba-Geigy Pharmaceuticals (Horsham, United Kingdom). The concentration of hirudin was determined by amino acid analysis. Human fibrinogen, PC, antithrombin (AT), and rabbit lung thrombomodulin (TM) were from Hematologic Technologies (Essex Junction, VT). Heparin was from Sigma. The release of fibrinopeptides A and B (FpA and FpB), binding of hirudin, and cleavage of PC by the slow and fast forms of wild-type thrombin and the ARK mutant were studied and analyzed as described(4, 5) . Inhibition of thrombin activity by AT was studied using the procedure of Olson (22) and was analyzed using KINSIM and FITSIM (23, 24) to obtain the apparent second-order rate constant for formation of the thrombin-antithrombin complex (k in Table 2). Experimental conditions were: 5 mM Tris, 0.1% PEG, pH 8.0, 25 °C, and 0.2 M NaCl for the fast form, or 0.2 M choline (Ch) chloride for the slow form. The concentration of TM was 10 nM, and heparin was used at concentrations of 1 unit/ml.




RESULTS AND DISCUSSION

The Na binding site of thrombin has been unequivocally identified here by exchanging Na with Rb ions in crystals of PPACK-thrombin. The site displays octahedral coordination involving Tyr, Arg, Lys, and three highly occupied water molecules (Fig. 1). It is located more than 15 Å away from the catalytic triad of thrombin and lies near Asp of the primary specificity site within a cylindrical cavity (26) formed by three antiparallel beta-strands of the B-chain (Met-Tyr, Lys-Tyr, and Val-Gly) diagonally crossed by Glu-Glu and shaped by the loop connecting the last two beta-strands (Fig. 2). The cavity traverses through thrombin from the active site to the opposite surface with Asp near its midpoint. Very significantly, the channel is filled with about 20 water molecules, most of which are conserved in different thrombin structures. Furthermore, they are linked among each other by a complicated hydrogen bonding network, which also links up to the peptide chain of the protein and appears to stabilize the cavity. A Na ion and a water molecule are practically equivalent in an electron density map. Examination of different crystal structures of thrombin reveals an octahedrally coordinated water molecule at the Na site in over 20 of them, most with exceptionally small contacts to the central water molecule (2.2-2.4 Å).


Figure 1: The Na binding site of PPACK-thrombin. Electron density of PPACK-thrombin at 2.5 Å resolution in blue (dotted) contoured at 1.20 (A. Tulinsky, unpublished results); Rb ion difference electron density at same resolution in red at 4, calculated using Fourier coefficients of the form: (F - F(T))exp(-ialpha(T)), where F is the observed structure amplitude of Rb ion soaked PPACK-thrombin, F(T) that of PPACK-thrombin, and alpha(T) the phase angle of PPACK-thrombin without water molecules included; distal water is designated WAT in correct sixth octahedral position, but about 3.4 Å from Na position. The average Na-O distance is 2.9 Å. Numbering is based on topological similarities with chymotrypsin (25) .




Figure 2: MOLSCRIPT plot (27) of the B-chain of human alpha-thrombin, derived from the coordinates of the thrombin-hirudin complex(26) , with the inhibitor removed. The residues of the catalytic triad are shown at the interface between the two six-stranded beta-barrels. The N terminus of the chain is at the bottom, and the C terminus is at the end of the alpha-helix at the top. Important structural domains of the enzyme are noted as follows: 1, Na binding loop; 2, autolysis loop; 3, fibrinogen binding loop; 4, heparin binding site. The Na ion is shown as a redball in the loop connecting the last two beta-strands of the B-chain terminating into the heparin binding site. Binding of Na to this loop induces the slow fast transition and controls allosterically both the activity (3) and specificity (4) of the enzyme. The highly conserved fragment Cys-Gly of this loop (see Table 1) is in yellow.





The sequence Cys-Gly involving the Na binding loop and part of the last beta-strand of the B-chain is highly conserved in thrombin from 11 different species (Table 1), thereby supporting the fundamental role of Na binding in thrombin function. While Asp is the only variable residue, Arg is replaced by a Lys in the hagfish(28) . Comparison with analogous sequences in other serine proteases (Table 1) indicates a high degree of homology, especially with factors IX and X and to a lesser extent with plasmin, PC, and factor VII. Homology is weaker between thrombin and pancreatic serine proteases like trypsin, chymotrypsin, and elastase. Hence, the architecture of the Na binding site of thrombin and the allosteric regulation based on the slow fast equilibrium may be paradigmatic of a molecular strategy shared by other members of the blood coagulation cascade, like factors VII, IX, X, and PC, but not by pancreatic serine proteases. Proteases active in the blood might have evolved to exploit Na-dependent allosteric transitions to accomplish their multifunctional roles, taking advantage of the availability of this monovalent cation in the extracellular environment.

The allosteric nature of thrombin makes it possible to control its function by interfering with Na binding. A mutation involving the two Asp residues flanking Arg (Table 1) in the Na binding loop was made to mimic the ARK sequence found in factor Xa, which has a reduced Na affinity (K = 110 mM at 25 °C) compared to thrombin (K = 22 mM at 25 °C, see (3) ). The difference may be due to a reduction of the negative electrostatic potential at the Na site due to the presence of Ala and Lys, as opposed to Asp and Asp in thrombin. In the crystal structure(25, 29) , Asp and Asp are ion-paired to Arg and make no contacts with synthetic substrates(25) , hirudin(26) , and most likely fibrinogen(30) . This double mutant D221A/D222K (ARK) has lost the ability to bind Na selectively (Fig. 3) and cleaves a synthetic substrate with nearly the same specificity in the presence of Li, Na, K, or the bulky cation Ch. The ARK mutant interacts with fibrinogen and hirudin with increased specificity in the slow form and reduced specificity in the fast form (Table 2). The perturbed energetics originate from an indirect effect, because the mutation impairs Na binding and stabilizes a conformation that is functionally and structurally intermediate between the slow and fast forms of the wild type. Circular dichroic spectra (Fig. 4) support this conclusion. The spectra for the slow and fast forms are significantly different for the wild type, but not for the ARK mutant that behaves as an intermediate between the slow and fast forms of the wild type.


Figure 3: Value of the specificity constant for the hydrolysis of S2238 (H-D-Phe-pipecolyl-Arg-p-nitroanilide) by wild-type thrombin and the ARK mutant, as indicated. Experimental conditions are: 5 mM Tris, 0.1% PEG, pH 8.0, 25 °C. The salt concentration was kept constant at 200 mM with the chloride salts indicated on the abscissa. The large difference seen between Na (fast form) and Ch (slow form) for the wild type, is not observed in the ARK mutant.




Figure 4: Circular dichroic spectra of wild-type thrombin (circle, bullet) and the ARK mutant (box, ) in the slow (circle, box) or fast (bullet, ) forms. Spectra were recorded on a Jasco J600A spectropolarimeter under solution conditions of: 5 mM Tris, 0.1% PEG, pH 8.0, 25 °C, and 0.2 M NaCl for the fast form, or 0.2 M ChCl for the slow form. The concentration of the wild type and the ARK mutant was 1.5-2.0 µM. The pathlength was 1 cm. The slow and fast forms of the wild-type enzyme show significant differences in the regions around 204 and 218 nm, that are not seen for the ARK mutant.



When PC interacts with the ARK mutant, the functional differences between the slow and fast forms are restored (Table 2). Furthermore, the mutation has the noteworthy effect of slightly enhancing the catalytic properties of the slow form compared to the wild type. This result is also observed in the presence of TM. Protein C presumably makes favorable contacts with Lys of ARK, and this effect overwhelms the indirect effect of the mutation on the slow fast equilibrium. In the absence of a direct effect, activation of PC by the ARK mutant would be decreased in the slow form and increased in the fast form compared to the wild type, consistent with the findings on S2238, fibrinogen, and hirudin. Binding of AT in the absence of heparin is perturbed exclusively as a result of the effect of the mutation on the slow fast equilibrium (Table 2). In the presence of heparin, however, binding of AT is decreased by more than 10 times. The contacts made by AT with thrombin are likely different when heparin is bound, as a result of heparin-induced conformational changes. Favorable contacts made by AT with Asp and Asp of the wild type in the presence of heparin may explain the loss of binding affinity in the ARK mutant. Hence, the Na binding loop of thrombin plays an important role in the direct recognition of PC and AT in the presence of heparin.

The architecture of the Na binding site of thrombin reveals important details on the molecular trigger for the slow fast transition that contributes to the conversion of the enzyme from an anticoagulant to a procoagulant factor(4) . The slow form has increased specificity for substrates like PC carrying an acidic residue at P3. Even in the absence of a structure of the slow form, the side chain of Arg, which is ion-paired to Glu in the fast form(25, 29) , is oriented differently and makes a water mediated contact with the acidic side chain of the residue at P3 of substrates like the thrombin platelet receptor peptide (31, 32) and probably PC. The enhanced anticoagulant activity of the ARK mutant may well be a result of the D222K substitution, with the side chain of Lys mimicking or enhancing synergistically the role of Arg. Further mutagenesis analysis of the Na binding loop will reveal the exact origin of Na specificity and the trigger for the allosteric slow fast transition.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Research Grants HL43229 (to A. T.) and HL49413 (to E. D. C.), and by grants from the American Heart Association (to E. D. C.) and Monsanto-Searle (to E. D. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Established Investigator of the American Heart Association and Genentech. To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Box 8231, St. Louis, MO 63110. Tel.: 314-362-4185; Fax: 314-362-7183; enrico{at}caesar.wustl.edu.

W. M. Keck Fellow.

(^1)
The abbreviations used are: PC, protein C; PEG, polyethylene glycol; PPACK, D-Phe-Pro-Arg-chloromethyl ketone; AT, antithrombin; ARK, double mutant D221A/D222K; TM, thrombomodulin.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.