Interaction of bovine coagulation factor X and its glutamic-acid-containing fragments with phospholipid membranes A surface plasmon resonance study Eva-Maria Erb 1 , Johan Stenflo 1 and Torbjo¨ rn Drakenberg 2 1 Department of Clinical Chemistry, University Hospital Malmo ¨ , Lund University, Malmo ¨ , Sweden; 2 Department of Biophysical Chemistry, Lund University, Lund, Sweden The interaction of blood coagulation factor X and its Gla-containing fragments with negatively charged phos- pholipid membranes composed of 25 mol% phosphatidyl- serine (PtdSer) and 75 mol% phosphatidylcholine (PtdCho) was studied by surface plasmon resonance. The binding to 100 mol% PtdCho membranes was negligible. The calcium dependence in the membrane binding was evaluated for intact bovine factor X (factor X) and the fragment con- taining the Gla-domain and the N-terminal EGF (epidermal growth factor)-like domain, Gla–EGF N ,fromfactorX. Both proteins show the same calcium dependence in the membrane binding. Calcium binding is cooperative and half- maximum binding was observed at 1.5 m M and 1.4 m M , with the best fit to the experimental data with three cooperatively bound calcium ions for both the intact protein and the fragment. The dissociation constant (K d ) for binding to membranes containing 25 mol% PtdSer decreased from 4.6 l M for the isolated Gla-domain to 1 l M for the frag- ments Gla–EGF N and Gla–EGF NC (the Gla-domain and both EGF-like domains) fragments and to 40 n M for the entire protein as zymogen, activated enzyme or in the active- site inhibited form. Analysis of the kinetics of adsorption and desorption confirmed the equilibrium binding data. Keywords: blood coagulation; membrane binding; calcium dependence; factor X; Gla-domain. Blood coagulation factor X belongs to the family of vitamin K-dependent proteins. It consists of an NH 2 -terminal c-carboxyglutamic acid (Gla)-containing domain, followed by two epidermal growth factor (EGF)-like domains and a serine protease (SP) domain [1]. The Gla-domain mediates Ca 2+ -dependent binding to biological membranes, for example the platelet membrane [2]. Binding of factor X and other Gla domain-containing coagulation factors is greatly enhanced after platelet activation, due to the exposure of negatively charged phosphatidylserine (PtdSer) on the cell surface. The crystal structure of the Ca 2+ -loaded form of prothrombin fragment 1 showed that six or seven of the Gla residues ligate four to five Ca 2+ in the interior of the protein and that three conserved residues with hydrophobic side-chains, Phe4, Leu5 and Val8 in bovine factor X, form a hydrophobic patch on the surfase of the domain [3–5]. These residues are thought to mediate membrane-binding by inserting their side-chains into the membrane. This hypothesis gained support from site directed mutagenesis studies. In protein C the Leu5 fi Gln mutation reduces membrane affinity and biological activity [5,6]. NMR studies have illustrated how Ca 2+ induces a drastic conformational transition in the Gla domain [7]. The Gla- residues at positions 6, 7, 16, 20, and 29 (bovine factor X numbering), solvent exposed in the absence of Ca 2+ ,turnto the inside of the domain where they coordinate Ca 2+ , whereas the three hydrophobic residues, Phe4, Leu5 and Val8, located in the interior of the domain in the absence of Ca 2+ , become solvent exposed and form the hydrophobic patch [7]. These results, as well as studies utilizing a synthetic Gla domain with Leu6 and Phe9 (factor IX, residues 5 and 8 in factor X) substituted for a hydrophobic photoactivable crosslinking agent, suggested that there is an important hydrophobic component in the interaction of Gla-contain- ing proteins with biological membranes [8]. Although the Gla domain sequence is highly conserved among the various hemostatic Gla-containing proteins, the dissociation constant (K d ) for binding to model membranes varies by as much as three orders of magnitude [9]. Presumably, this is caused by still poorly understood electrostatic interactions between the Ca 2+ -bound Gla domain and phosphate head groups in the phospholipid membrane. This notion also gains support from numerous studies where site-directed mutagenesis was employed to establish the functional role of individual amino acids in Gla domains [9–11]. Membrane binding of vitamin K-dependent coagulation factors has previously been studied by ellipsometry [12,13], light scattering [9,14–16] and fluorescence polarization [17]. The K d values determined for the same coagulation factor Correspondence to T. Drakenberg, Department of Biophysical Chemistry, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden. Fax: + 46 46 222 45 43, Tel.: + 46 46 222 44 70, E-mail: Torbjorn.Drakenberg@bpc.lu.se Abbreviations: PtdSer, phosphatidylserine; PtdCho, phosphatidtylcholine; Gla, c-carboxy glutamic acid; EGF-like, epidermal growth factor-like; Gla–EGF N , a fragment comprising the Gla domain and the first EGF domain of factor X; Gla-EGF NC ,a fragment comprising the Gla domain, the first and the second EGF domain of factor X; RU, response units. Note: this work was funded in part by the EU Biotechnology program (contract no BIO4-CT96-0662). (Received 20 December 2001, revised 23 April 2002, accepted 7 May 2002) Eur. J. Biochem. 269, 3041–3046 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02981.x under similar conditions by different methods varied by as much as two orders of magnitude [12,13,17]. We therefore decided to investigate membrane binding by surface plasmon resonance. With this method the kinetics of membrane interaction is measured in real time. Also, the proteins do not have to be labeled with fluorescent compounds as in, for instance, fluorescence energy transfer studies. We have previously characterized the surfaces generated by liposome binding to the Biacore L1 sensor chip [18]. This sensor chip consists of a dextran matrix to which hydrophobic residues are covalently bound. Our results indicate that the liposomes were captured on the modified dextran matrix and subsequently fuse to generate a homogeneous lipid membrane. Moreover, a flat mem- brane is favorable as compared to the curvature of the liposomes [19–21]. To elucidate the impact of domains other than the Gla domain on membrane binding, we have now investigated the membrane-binding properties of coagulation factor X and Gla domain-containing frag- ments of this protein. MATERIALS AND METHODS Materials The lipids 1-palmitoyl 2-oleoyl-sn-glycero-3-phosphocho- line and 1,2-dioleoyl-sn-glycero-3-[phospho- L -serine] were obtained from Avanti Polar Lipids (Alabaster, AL, USA), polycarbonate filters were from SPI suppllies (West Chester, PA, USA). All other reagents were obtained from Merck (Darmstadt, Germany) or Sigma (St Louis, MO, USA). The peptide corresponding to the Gla domain (residues 1–46) of factor X, was chemically synthesized using standard Fmoc chemistry. The fragments Gla–EGF N (residues 1–86) Gla– EGF NC (residues 1–140, 154–183) were generated by digestion of bovine factor X with trypsin [22]. Bovine factor X, factor Xa and DEGR-factor Xa were purchased from Haematologic Technologies Inc. (Burlington, VT, USA). All surface plasmon resonance experiments were performed on either a BIAcore X or a BIA2000 together with L1 pioneer sensor chips (Biacore AB, Uppsala, Sweden). Membrane generation Liposomes were prepared by the extruder technique and bound to the L1 sensor chip as described previously [18]. In brief, liposomes containing either 100 mol% PtdCho, 10 mol% PtdSer/90 mol% PtdCho, 25% mol% PtdSer/ 75 mol% PtdCho or 40 mol% PtdSer/60 mol% PtdCho were injected into a Biacore instrument equipped with a L1 sensor chip. The flow rate was 10 lLÆmin )1 . Liposomes were captured on the sensor chip and spontaneously fused to generate a flat lipid membrane surface. Excess liposomes were removed by two 60 s pulses with 5 m M EDTA, pH 8.0 at a flow rate of 5 lLÆmin )1 . The running buffer was then changed to 10 m M Tris/HCl, 150 m M NaCl, pH 7.4 (Tris buffer) containing 0.1% (w/v) bovine serum albumin (BSA). For titration experiments the buffer was made 0–10 m M in CaCl 2 . For binding experiments, the Ca 2+ concentration was 10 m M . All solutions used in the Biacore experiments were degassed and filtered through 0.22 lmfilters. Ca 2+ -dependence of membrane binding Factor X and Gla–EGF N were diluted in the Tris buffer containing 0.1% (w/v) BSA, 0–10 m M CaCl 2 to a final concentration of 39 n M and 2 l M CaCl 2 , respectively. The running buffer always had the same Ca 2+ concentration as the protein containing buffer. Association was followed for 180 s at a flow rate of 10 lLÆmin )1 , followed by a 600-s dissociation phase using the same flow rate. The membrane was regenerated by two 60 s pulses with 5 m M EDTA pH 8.0 at a flow rate of 5 lLÆmin )1 . The binding data were fittedtoEqn(1). Y ¼ R ½Ca 2þ  n =ð½Ca 2þ  n þ K n 0:5 Þð1Þ where R is the maximum response signal, n is the number of cooperatively bound Ca 2+ ions needed for membrane binding and K 0.5 is the Ca 2+ concentration at which half- maximum binding occurs. Kinetics of membrane binding Membrane binding experiments on factor X, factor Xa, DEGR-factor Xa and the Gla-containing fragments of factor X were performed with membranes containing either 25 mol% PtdSer and 75 mol% PtdCho or 100 mol% PtdCho in the presence of 10 m M Ca 2+ .The Ca 2+ concentration used here would be expected to almost completely saturate the Ca 2+ binding sites in the Gla domain. The response signal, when using membranes containing 25 mol% PtdSer, was corrected for the back- ground binding to membranes composed of 100% PtdCho. Data were evaluated with the program BIAEVAL- UATION 3.0 using either the simple bimolecular interaction model or a two-step binding model as described by the following equations. The rate equation for the bivalent analyte model: A þ B ) * k on;1 k off;1 AB ð2Þ AB þ B ) * k on;2 k off;2 AB 2 ð3Þ where d½B=dt ¼À2k on;1 ½A½Bþk off;1 ½ABÀk on;2 ½AB[B] þ 2k off;2 ½AB 2 ð4Þ d½AB=dt ¼ 2k on;1 ½A½BÀk off;1 ½AB À k on;2 ½AB[B] þ 2k off;2 ½AB 2 ð5Þ d½AB 2 =dt ¼ k on;2 ½AB½BÀ2k off;2 ½AB 2 ð6Þ The rate equations for the conformational change model: A þ B ) * k on;1 k off;1 AB ð7Þ AB ) * k on;2 k off;2 AB à ð8Þ where d½B=dt ¼Àk on;1 ½A½Bþk off;1 ½ABð9Þ 3042 E M. Erb et al.(Eur. J. Biochem. 269) Ó FEBS 2002 d½AB=dt ¼ k on;1 ½A½BÀk off;1 ½ABÀk on;2 ½AB þ k off;2 ½AB à ð10Þ d½AB à =dt ¼ k on;2 ½ABÀk off;2 ½AB à ð11Þ The concentrations at t ¼ 0are[B] 0 ¼ R max , R max ¼ response at full saturation, [AB] 0 ¼ 0and[AB 2 ] 0 ¼ 0. The total response signal is the sum of the initial response signal R i plus the signals from the complexes AB and AB 2 or AB* for the bivalent model or for the conformational change model, respectively. Equilibrium response signals Equilibrium response signals were plotted vs. the protein concentration. The K d values were determined by fitting the data to Eqn (2) assuming a single class of binding sites: saturation ¼½protein=ð½proteinþK d Þ: ð12Þ The equilibrium response signal is the sum of the signals from the intermediate complex AB and the final complex AB 2 . However, the contribution of the second binding step to the total response is about 15%, and therefore the evaluation of the equilibrium response signals by Eqn (2) gives a good approximation for the K d values of the first binding step. The uncertainties given in Table 1 are therefore set to 15%. RESULTS Ca 2+ -dependence of membrane binding The Ca 2+ concentration dependence of membrane binding was determined by measuring the equilibrium response signal at different Ca 2+ concentrations. Factor X and the fragment Gla–EGF N were bound to membranes containing 25 mol% PtdSer/75 mol% PtdCho at a concentration of 39 n M and 2 l M , respectively. Binding of both species to membranes composed of 100 mol% PtdCho was less then 5% of the binding to membrane containing 25 mol% PtdSer. The Ca 2+ titration curves of factor X and Gla– EGF N binding indicate cooperative binding (Fig. 1). Half- maximal binding occurred at a calcium concentration of 1.5 and 1.4 m M for factor X and Gla–EGF N , respectively, which is close to the concentration of free calcium in blood of 1.2 m M . The best fit to the data in Fig. 1 was obtained assuming three cooperatively bound Ca 2+ ions. As shown in Fig. 1 the membrane binding of intact factor X and the Gla– EGF N fragment, showed very similar Ca 2+ -dependencies, indicating that neither the second EGF domain nor the serine protease domain alter those Ca 2+ -binding properties of factor X that are relevant to membrane binding. Experi- ments using membranes containing either 10 mol% PtdSer/ 90 mol% PtdCho or 40 mol% PtdSer/60 mol% PtdCho showedthesameCa 2+ -dependence as 25 mol% PtdSer/ 75 mol% PtdCho for binding intact factor X and Gla– EGF N (data not shown). Kinetics of membrane binding The kinetics of binding to PL membranes of the zymogen factor X, activated factor X (factor Xa) and the active site inhibited form DEGR-factor Xa as well as the the factor X peptides were studied with surface plasmon resonance. The Ca 2+ concentration was 10 m M to ascertain that the Ca 2+ binding sites of the Gla domain were completely satur- ated. Figure 2 presents the binding of factor X to the Table 1. Kinetic constants for binding of factor X and its Gla-containing fragments to membranes containing 25 mol% PtdSer in the presence of 10 m M Ca 2+ obtained by evaluation of association and dissociation phases (I) and equilibrium binding data (II) as described in Materials and methods. k on (MÆs) )1 k off (s )1 ) K d ( M ) (I) K d ( M ) (II) Gla (8.0 ± 2.2) · 10 3 (3.7 ± 0.2) · 10 )2 (4.6 ± 1.3) · 10 )6 (9.4 ± 1.4) · 10 )6 Gla–EGF N (4.5 ± 1.1) · 10 4 (3.8 ± 0.2) · 10 )2 (8.4 ± 2.1) · 10 )7 (1.7 ± 0.3) · 10 )6 Gla–EGF N,C (6.7 ± 2.1) · 10 4 (4.3 ± 0.2) · 10 )2 (6.4 ± 2.0) · 10 )7 (2.0 ± 0.3) · 10 )6 Factor X (8.3 ± 1.9) · 10 5 (3.2 ± 0.2) · 10 )2 (3.9 ± 0.9) · 10 )8 (3.7 ± 0.6) · 10 )8 Factor Xa (4.5 ± 0.8) · 10 5 (3.6 ± 0.2) · 10 )2 (8.0 ± 1.5) · 10 )8 (5.2 ± 0.8) · 10 )8 DEGR-factor Xa (5.3 ± 1.3) · 10 5 (3.7 ± 0.2) · 10 )2 (8.0 ± 1.5) · 10 )8 (6.2 ± 0.9) · 10 )8 Fig. 1. Ca 2+ -dependence in the membrane binding of factor X (A) and the fragment Gla–EGF N (B) as determined by surface plasmon reson- ance. Binding experiments were performed on 25 mol% PtdSer-con- taining membranes (solid symbols) and 100 mol% PtdCho-containing membranes (open symbols). The solid curve is the best fit to the experimental data points obtained by Eqn (1), assuming n ¼ 3 (c 2 ¼ 359.2); the dotted line assuming n ¼ 4(c 2 ¼ 595.7); the dashed line assuming n ¼ 2(c 2 ¼ 715.3). Ó FEBS 2002 Membrane binding of coagulation factor X (Eur. J. Biochem. 269) 3043 phospholipid membrane at various protein concentrations. Similar sensorgrams were obtained for the other forms of factor X and fragments, although with different concentra- tions for half maximum binding (data not shown). In a first attempt the association and dissociation processes were treated as simple one step processes. However, with this approach it was not possible to obtain a reasonable agree- ment between observed and calculated sensorgrams. Mod- els with two on-rates and two off-rates improved the fit significantly. Moreover, a model including a conformation- al change and a model including a bivalent analyte both gave good fits to the experimental data. The results obtained with the bivalent analyte model is shown in Fig. 2. In all cases there is a dominating fast process with an almost constant off-rate for all the proteins (3.2–4.8 10 )2 Æs )1 ). The difference in binding affinity is therefore the result of different on-rates (Table 1). The isolated Gla domain (the fragment with the lowest molecular mass, about 5 kDa) shows the lowest on rate, even though from thermodynamic aspects it would be expected to show a higher on rate. This may be explained by assuming that only a small fraction of the fragment has a conformation that is commensurate with membrane-bind- ing. The on-rates for Gla–EGF N and Gla–EGF NC are about a factor of five higher than for the Gla-domain. This can presumably be attributed to a stabilizing effect of the N-terminal EGF domain on the Gla domain [7]. The entire protein has an on-rate that is two orders of magnitude faster than for the Gla-domain presumably due to a further stabilization of the structure of the Gla-domain, indicating that less than 1% of the free isolated Gla-domain has a conformation that is appropriate for membrane binding. Equilibrium binding isotherms The concentration dependence of factor X binding is shown in Fig. 2. It is apparent that the adsorption is rapid and that a plateau is reached within 100–200 s. Figure 3 shows the binding isotherms of factor X and its peptides. Their mem- brane binding affinities increase in the order Gla < Gla– EGF N ¼ Gla–EGF NC <factor X ¼ factor Xa ¼ DEGR- factor Xa (Table 1). Although both the first and second binding step contribute to the equilibrium response signal, the first binding step is the dominating process and the influence from the second one, whether a conformational change or a bifunctional ligand, has been neglected. The consistency of the K d values resulting from the evaluation of the equilibrium response signals and those obtained by evaluating the first step in the association phase of the sensorgrams justifies this assumption. DISCUSSION Calcium binding to the Gla domain is known to be crucial for the induction of a conformation in the domain that mediates membrane binding. Early studies employing equilibrium dialysis established the existence of about 10 Ca 2+ -binding sites, at least three of which mediate cooper- ative binding [23–26]. By studies of the binding of divalent cations other than Ca 2+ , for example Mg 2+ ,Mn 2+ and Ba 2+ , it became evident that there is one class of binding sites that is cation nonspecific and binds all four metal ions in a cooperative manner [26–29]. Moreover, metal ion- binding to the cation nonspecific sites induces quenching of the intrinsic protein fluorescence [26,28,30]. The Ca 2+ concentration necessary to induce half-maximal fluores- cence quenching in factor X and in the fragment that consists of the Gla domain linked to the first EGF domain was determined to about 0.5 m M [31]. The conformation induced by cation binding to the nonspecific sites does not support membrane-binding [27,29]. The second class of binding sites is Ca 2+ -specific, and metal ion-binding to these sites induces a membrane binding conformation. From NMR studies of the Mg 2+ form of a Gla-domain it became evident that unlike Ca 2+ -binding, Mg 2+ -binding to the Fig. 3. Equilibrium isotherms of factor X and its Gla-containing frag- ments binding to membranes containing 25 mol% PtdSer in the presence of 10 m M Ca 2+ . The measured equilibrium binding signal is plotted against the solution phase concentration of factor X (d), factor Xa (m), DEGR-factor Xa (n), Gla–EGF NC (e), Gla–EGF N (r)andGla (.). Solid lines indicate the least-square fit of the Langmuir model to this data as described in Materials and methods. The estimated binding parameters are listed in Table 1. Fig. 2. Adsorption and desorption kinetics of factor X to 25 mol% PtdSer containing membranes. Experiments were performed using 10 m M Tris/HCl,pH7.5,150m M NaCl, 10 m M CaCl 2 ,0.1%(w/v) BSA as running buffer at a flow rate of 10 lLÆmin )1 .FactorXwas diluted in the same buffer to the final concentration of 44 n M (h), 22 n M (j), 11 n M (n), 5.5 n M (m), 2.8 n M (s)and1.4n M (d). The protein was injected at t ¼ 0 and binding to the membrane is apparent during the association phase (180 s). The protein-containing buffer was then replaced by running buffer, resulting in dissociation of the protein from the membrane. The solid curves were calculated using equations 4–6. 3044 E M. Erb et al.(Eur. J. Biochem. 269) Ó FEBS 2002 Gla-domain did not induce the native conformation in residues 1–11 of the Gla-domain [8]. Moreover, NMR studies of the Ca 2+ -free form of the Gla-domain established that the metal ion binding translocated the residues that constitute the hydrophobic patch from the interior of the domain to the surface, allowing them to interact with the phospholipid membrane [7]. Furthermore, these results support the notion that the nature of this drastic conform- ational transition must be highly cooperative with respect to Ca 2+ due to noncompensated electrostatic repulsion between carboxylate groups with, for instance, only one Ca 2+ bound in this region. We have now found that the Ca 2+ concentration that induces half-maximal membrane binding of factor X and the fragment Gla–EGF N to PtdSer-containing membranes is about 1.5 m M . This is consistent with results from light scattering experiments with other Gla domain-containing proteins. Thus the Ca 2+ -concentration necessary to induce half-maximal binding has been determined to be 0.55 m M ,0.9m M and 1.2 m M for factor IX [32], factor VII [33] and protein C [5], respectively. We have found that the membrane-binding of intact factor X and Gla– EGF N show about the same Ca 2+ dependence, indicating that Ca 2+ -binding to domains other than the Gla domain and the N-terminal EGF-like domain does not influence the membrane-binding properties of factor X. Our results also demonstrate that the membrane binding is cooper- ative with respect to Ca 2+ , presumably reflecting the cooperative Ca 2+ -binding to sites in the Gla domain. Interestingly, the Ca 2+ concentration necessary to induce membrane-binding corresponds rather closely to the concentration of free Ca 2+ in blood (1.2 m M ). It is thus possible that binding of at least some Gla domain- containing proteins to biological membranes will be sensitive to local variations in the Ca 2+ concentration in the immediate vicinity of the membrane. We found that the isolated factor X Gla domain exhibits low affinity binding to PtdSer-containing membranes with a K d of 4.6 l M . This agrees well with the value of 2.4 l M for factor IX (1–47) [8] and 3.7 l M for human protein C (1–48) [34] measured under similar conditions (1 l M Ca 2+ ,40% PtdSer) by resonance energy transfer and circular dichro- ism, respectively. The C-terminal helix of the factor X Gla domain of Gla–EGF N (residues 33–41) interacts with the adjacent EGF N domain [8]. Presumably, this interaction stabilizes the Gla domain and contributes to the five-fold higher affinity of Gla–EGF N (K d ¼ 1 l M ) for phospholipid membranes as compared to the isolated Gla domain. The second EGF domain does not appear to provide any further stabilization. The membrane affinity of the intact protein is about 10-fold higher than the affinity for Gla–EGF N and Gla–EGF NC and about 100-fold higher than the affinity to the isolated Gla domain. No significant difference in membrane affinity could be detected between the zymogen, the activated protein and the active site-inhibited form. It should be pointed out that the results from equilibrium binding studies are consistent with the data resulting form the evaluation of association and dissociation phases. The differences in the K d values resulting from the different evaluations of the experiments are in the same range as observed previously [13,35]. The K d determined for factor X is consistent with the value determined by McDonald et al.[9]. The effect of the serine protease domain upon the membrane affinity of the intact protein is enigmatic. It could be due to a long distance conformational change in the protein mediated through the two EGF-domains. In this context it should be noted that mutation of Ca 2+ ligating amino acids in the N-terminal part of the first EGF-like domain of factor X influences the amidolytic activity of the intact protein [36]. However, direct interactions between the Gla and serine protease domains, intra or intermolecular, might also explain the difference in binding affinities. Another factor contributing to the higher on-rate for the intact protein is the net charge. The Gla–EGF NC fragment is highly negatively charge, especially when not saturated with Ca 2+ ()29 without Ca 2+ and )15 with 7Ca 2+ ). 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Bovine factor X, factor Xa and DEGR -factor Xa were purchased from Haematologic Technologies