Interaction of bovine coagulation factor X and itsglutamic-acid-containing fragments with phospholipid membranes A surface plasmon resonance study Eva-Maria Erb1, Johan Stenflo1and Torbj
Trang 1Interaction of bovine coagulation factor X and its
glutamic-acid-containing fragments with phospholipid membranes
A surface plasmon resonance study
Eva-Maria Erb1, Johan Stenflo1and Torbjo¨rn Drakenberg2
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–EGFN, from factor X
Both proteins show the same calcium dependence in the
membrane binding Calcium binding is cooperative and
half-maximum binding was observed at 1.5 mM and 1.4 mM,
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 (Kd) for binding
to membranes containing 25 mol% PtdSer decreased from 4.6 lM for the isolated Gla-domain to 1 lM for the frag-ments Gla–EGFNand Gla–EGFNC(the Gla-domain and both EGF-like domains) fragments and to 40 nMfor 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 NH2-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
Ca2+-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 Ca2+-loaded
form of prothrombin fragment 1 showed that six or seven of
the Gla residues ligate four to five Ca2+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 Leu5fi Gln mutation reduces membrane affinity and biological activity [5,6] NMR studies have illustrated how Ca2+ 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 Ca2+, turn to the inside of the domain where they coordinate Ca2+, whereas the three hydrophobic residues, Phe4, Leu5 and Val8, located in the interior of the domain in the absence of
Ca2+, 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 (Kd) 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 Ca2+-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 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)
Trang 2under 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 [19Ờ21] 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
M A T E R I A L S A N D M E T H O D S
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 1Ờ46) of
factor X, was chemically synthesized using standard Fmoc
chemistry The fragments GlaỜEGFN(residues 1Ờ86) GlaỜ
EGFNC (residues 1Ờ140, 154Ờ183) 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 lLẳmin)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 mM EDTA,
pH 8.0 at a flow rate of 5 lLẳmin)1 The running buffer
was then changed to 10 mM Tris/HCl, 150 mM NaCl,
pH 7.4 (Tris buffer) containing 0.1% (w/v) bovine serum
albumin (BSA) For titration experiments the buffer was
made 0Ờ10 mM in CaCl2 For binding experiments, the
Ca2+concentration was 10 mM All solutions used in the
Biacore experiments were degassed and filtered through
0.22 lm filters
Ca2+-dependence of membrane binding Factor X and GlaỜEGFNwere diluted in the Tris buffer containing 0.1% (w/v) BSA, 0Ờ10 mM CaCl2 to a final concentration of 39 nMand 2 lMCaCl2, respectively The running buffer always had the same Ca2+concentration as the protein containing buffer Association was followed for
180 s at a flow rate of 10 lLẳmin)1, followed by a 600-s dissociation phase using the same flow rate The membrane was regenerated by two 60 s pulses with 5 mM EDTA
pH 8.0 at a flow rate of 5 lLẳmin)1 The binding data were fitted to Eqn (1)
YỬ R ơCa2ợn=đơCa2ợnợ K0:5n ỡ đ1ỡ where R is the maximum response signal, n is the number of cooperatively bound Ca2+ ions needed for membrane binding and K0.5is the Ca2+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 mM Ca2+ The
Ca2+ concentration used here would be expected to almost completely saturate the Ca2+ 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-UATION3.0 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:
Aợ B )*
kon;1
koff;1 AB đ2ỡ
ABợ B )*
k on;2
k off;2
where dơB=dt Ử 2kon;1ơAơB ợ koff;1ơAB kon;2ơAB[B]
dơAB=dt Ử 2kon;1ơAơB koff;1ơAB
kon;2ơAB[B] ợ 2koff;2ơAB2 đ5ỡ dơAB2=dt Ử kon;2ơABơB 2koff;2ơAB2 đ6ỡ The rate equations for the conformational change model:
Aợ B )*
kon;1
k off;1
AB )*
k on;2
koff;2 AB đ8ỡ where
dơB=dt Ử kon;1ơAơB ợ koff;1ơAB đ9ỡ
Trang 3dơAB=dt Ử kon;1ơAơB koff;1ơAB kon;2ơAB
dơAB =dt Ử kon;2ơAB koff;2ơAB đ11ỡ
The concentrations at tỬ 0 are [B]0Ử Rmax, RmaxỬ
response at full saturation, [AB]0Ử 0 and [AB2]0Ử 0
The total response signal is the sum of the initial response
signal Riplus the signals from the complexes AB and AB2or
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 Kdvalues were determined by fitting the
data to Eqn (2) assuming a single class of binding sites:
saturationỬ ơprotein=đơprotein ợ Kdỡ: đ12ỡ
The equilibrium response signal is the sum of the signals
from the intermediate complex AB and the final complex
AB2 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 Kdvalues of the first
binding step The uncertainties given in Table 1 are
therefore set to 15%
R E S U L T S
Ca2+-dependence of membrane binding
The Ca2+concentration dependence of membrane binding
was determined by measuring the equilibrium response
signal at different Ca2+concentrations Factor X and the
fragment GlaỜEGFNwere bound to membranes containing
25 mol% PtdSer/75 mol% PtdCho at a concentration of
39 nM and 2 lM, 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 Ca2+ titration curves of factor X and GlaỜ
EGFNbinding indicate cooperative binding (Fig 1)
Half-maximal binding occurred at a calcium concentration of 1.5
and 1.4 mM for factor X and GlaỜEGFN, respectively,
which is close to the concentration of free calcium in blood of
1.2 mM The best fit to the data in Fig 1 was obtained
assuming three cooperatively bound Ca2+ions As shown in
Fig 1 the membrane binding of intact factor X and the GlaỜ
EGFNfragment, showed very similar Ca2+-dependencies,
indicating that neither the second EGF domain nor the
serine protease domain alter those Ca2+-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 showed the same Ca2+-dependence as 25 mol% PtdSer/
75 mol% PtdCho for binding intact factor X and GlaỜ EGFN(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
Ca2+concentration was 10 mMto ascertain that the Ca2+ 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 Ca2+obtained by evaluation of association and dissociation phases (I) and equilibrium binding data (II) as described in Materials and methods.
k on (Mẳs))1 k off (s)1) K d ( M ) (I) K d ( M ) (II)
(3.7 ổ 0.2) ở 10)2 (4.6 ổ 1.3) ở 10)6 (9.4 ổ 1.4) ở 10)6 GlaỜEGF N (4.5 ổ 1.1) ở 10 4
(3.8 ổ 0.2) ở 10)2 (8.4 ổ 2.1) ở 10)7 (1.7 ổ 0.3) ở 10)6 GlaỜEGF N,C (6.7 ổ 2.1) ở 10 4 (4.3 ổ 0.2) ở 10)2 (6.4 ổ 2.0) ở 10)7 (2.0 ổ 0.3) ở 10)6 Factor X (8.3 ổ 1.9) ở 10 5 (3.2 ổ 0.2) ở 10)2 (3.9 ổ 0.9) ở 10)8 (3.7 ổ 0.6) ở 10)8 Factor Xa (4.5 ổ 0.8) ở 10 5
(3.6 ổ 0.2) ở 10)2 (8.0 ổ 1.5) ở 10)8 (5.2 ổ 0.8) ở 10)8 DEGR-factor Xa (5.3 ổ 1.3) ở 10 5 (3.7 ổ 0.2) ở 10)2 (8.0 ổ 1.5) ở 10)8 (6.2 ổ 0.9) ở 10)8
Fig 1 Ca2+-dependence in the membrane binding of factor X (A) and the fragment GlaỜEGF 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-conPtdSer-con-taining membranes (open symbols) The solid curve is the best fit to the experimental data points obtained by Eqn (1), assuming n Ử 3 (c 2
Ử 359.2); the dotted line assuming n Ử 4 (c 2
Ử 595.7); the dashed line assuming n Ử 2 (c 2
Ử 715.3).
Trang 4phospholipid 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 bivconformation-alent anconformation-alyte 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)2Æs)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–EGFNand Gla–EGFNCare 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– EGFN¼ Gla–EGFNC<factor X¼ 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 Kdvalues 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
D I S C U S S I O N
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
Ca2+-binding sites, at least three of which mediate cooper-ative binding [23–26] By studies of the binding of divalent cations other than Ca2+, for example Mg2+, Mn2+and
Ba2+, 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 Ca2+ 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 mM [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 Ca2+-specific, and metal ion-binding to these sites induces a membrane binding conformation From NMR studies of the Mg2+form of a Gla-domain it became evident that unlike Ca2+-binding, Mg2+-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 Ca2+ 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) and Gla (.) 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, pH 7.5, 150 m M NaCl, 10 m M CaCl 2 , 0.1% (w/v)
BSA as running buffer at a flow rate of 10 lLÆmin)1 Factor X was
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) and 1.4 n 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.
Trang 5Gla-domain did not induce the native conformation in
residues 1–11 of the Gla-domain [8] Moreover, NMR
studies of the Ca2+-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 Ca2+ due to noncompensated electrostatic repulsion
between carboxylate groups with, for instance, only one
Ca2+bound in this region
We have now found that the Ca2+concentration that
induces half-maximal membrane binding of factor X and
the fragment Gla–EGFNto PtdSer-containing membranes
is about 1.5 mM This is consistent with results from light
scattering experiments with other Gla domain-containing
proteins Thus the Ca2+-concentration necessary to
induce half-maximal binding has been determined to be
0.55 mM, 0.9 mM and 1.2 mM 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–
EGFNshow about the same Ca2+dependence, indicating
that Ca2+-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 Ca2+, presumably reflecting the
cooperative Ca2+-binding to sites in the Gla domain
Interestingly, the Ca2+concentration necessary to induce
membrane-binding corresponds rather closely to the
concentration of free Ca2+ in blood (1.2 mM) 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 Ca2+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
Kdof 4.6 lM This agrees well with the value of 2.4 lMfor
factor IX (1–47) [8] and 3.7 lMfor human protein C (1–48)
[34] measured under similar conditions (1 lMCa2+, 40%
PtdSer) by resonance energy transfer and circular
dichro-ism, respectively The C-terminal helix of the factor X Gla
domain of Gla–EGFN(residues 33–41) interacts with the
adjacent EGFN domain [8] Presumably, this interaction
stabilizes the Gla domain and contributes to the five-fold
higher affinity of Gla–EGFN(Kd¼ 1 lM) 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–EGFNand
Gla–EGFNCand 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 Kd values resulting from the different
evaluations of the experiments are in the same range as
observed previously [13,35] The Kddetermined 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 Ca2+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–EGFNCfragment is highly negatively charge, especially when not saturated with Ca2+()29 without Ca2+and)15 with 7Ca2+) The C-terminal serine protease domain, however, has a net charge
of +8, making the whole protein less negatively charged Therefore the equilibrium concentration of the intact protein near the negatively charged surface will be higher than for the fragments resulting in a higher apparent on-rate Using the same argument the on-rate of the Gla–EGFNC fragment should be lower than for the Gla-domain as it is more negatively charged The stabilizing effect of EGFNon the structure of the Gla-domain is therefore even more than what
is reflected by the fivefold increase in the on-rate
A C K N O W L E D G E M E N T S
This work was supported by grants from the Swedish Medical Research Council and EU Project BIO-CT-96-0662.
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