transforming growth factor-beta (tgf-b) binding to ... · the membrane environment on the...

Transforming Growth Factor-beta (TGF-b) Binding tothe Extracellular Domain of the Type II TGF-bReceptor: Receptor Capture on a Biosensor SurfaceUsing a New Coiled-coil Capture SystemDemonstrates that Avidity Contributes Significantly toHigh Affinity Binding

Gregory De Crescenzo1†, Phuong L. Pham2, Yves Durocher2 andMaureen D. O’Connor-McCourt1*

1Health Sector, TheBiotechnology ResearchInstitute, National ResearchCouncil of Canada, 6100Royalmount Avenue, MontrealQuebec, Canada H4P 2R2

2Bioprocess Platform, TheBiotechnology ResearchInstitute, National ResearchCouncil of Canada, 6100Royalmount Avenue, MontrealQuebec, Canada H4P 2R2

Mature TGF-b isoforms, which are covalent dimers, signal by binding tothree types of cell surface receptors, the type I, II and III TGF-b receptors.A complex composed of the TGF-b ligand and the type I and II receptorsis required for signaling. The type II receptor is responsible for recruitingTGF-b into the heteromeric ligand/type I receptor/type II receptorcomplex. The purpose of this study was to test for the extent that aviditycontributes to receptor affinity. Using a surface plasmon resonance (SPR)-based biosensor (the BIACORE), we captured the extracellular domain ofthe type II receptor (TbRIIED) at the biosensor surface in an oriented andstable manner by using a de novo designed coiled-coil (E/K coil) hetero-dimerizing system. We characterized the kinetics of binding of threeTGF-b isoforms to this immobilized TbRIIED. The results demonstratethat the stoichiometry of TGF-b binding to TbRIIED was one dimericligand to two receptors. All three TGF-b isoforms had rapid and similarassociation rates, but different dissociation rates, which resulted in theequilibrium dissociation constants being approximately 5 pM for theTGF-b1 and -b3 isoforms, and 5 nM for the TGF-b2 isoform. Since theseapparent affinities are at least four orders of magnitude higher thanthose determined when TGF-b was immobilized, and are close to thosedetermined for TbRII at the cell surface, we suggest that aviditycontributes significantly to high affinity receptor binding both at the bio-sensor and cell surfaces. Finally, we demonstrated that the coiled-coilimmobilization approach does not require the purification of the capturedprotein, making it an attractive tool for the rapid study of any protein–protein interaction.

q 2003 Elsevier Science Ltd. All rights reserved

Keywords: TGF-b; biosensor; coiled-coil; kinetics; type II TGF-b receptor*Corresponding author

0022-2836/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved

† Supported by the Protein Engineering Network of Centers of Excellence.

E-mail address of the corresponding author: maureen.o’[email protected]

Abbreviations used: EDC, N-ethyl-N0-(3-diethylaminopropyl) carbodiimide hydrochloride; NHS, N-hydroxysuccinimide; EDTA, disodium ethylenediaminetetraacetate; HBS, Hepes buffered saline; Hepes, N-(2-hydroxyethyl) piperazine-N0-2-ethane-sulfonic acid; PAGE, polyacrylamide gel electrophoresis; PDEA, 2-(2-pyridinyldithio)ethaneamine; RU, resonance unit; SDS, sodium dodecyl sulfate; SD, standard deviation; SPR, surfaceplasmon resonance; TbRII, type II TGF-b receptor; TbRIIED, TbRII ectodomain; TbRIIED-E5, TbRIIED fused to the E5coil; TGF-b, transforming growth factor-b.

doi:10.1016/S0022-2836(03)00360-7 J. Mol. Biol. (2003) 328, 1173–1183

Introduction

Transforming growth factor-b (TGF-b) is the pro-totypic member of a family of dimeric growth fac-tors that includes activins and bone morphogenicproteins (BMPs). These factors are involved in theregulation of growth, tissue repair, development,host defense and tumorigenesis.1 – 6 Three TGF-bisoforms (TGF-b1, -b2 and -b3) are present inmammalian cells. They signal by complexing threetypes of receptors, namely the type I, the type IIand the type III (also known as betaglycan) TGF-breceptors. The minimal complex required for sig-naling is composed of the TGF-b ligand, and thetype I and type II receptors.7 Upon ligand binding,the type II receptor phosphorylates the type Ireceptor in a unidirectional manner. The activatedtype I receptor then phosphorylates members ofthe Smad family of intracellular signaling mol-ecules. The type III receptor modulates TGF-bresponses by interacting with the signaling recep-tors through both its extracellular and cytoplasmicdomains.8 These type III receptor interactionsresult in the presentation of ligand to the type Iand type II receptors9 and the enhancement ofsignaling.8 Ligand presentation by the type IIIreceptor is particularly obvious in the case of theTGF-b2 ligand since the affinity of this isoform forthe type I and II receptors is low as compared tothe affinities of the other isoforms.10

We have previously shown using a surface plas-mon resonance (SPR)-based biosensor that allowsthe real-time monitoring of the interaction betweena surface immobilized protein and its bindingpartner, that, when TGF-b is immobilized on thebiosensor surface, the extracellular domain of thetype II receptor (TbRIIED) binds with a two recep-tor-to-one ligand stoichiometry, and that both bind-ing sites in the TGF-b molecule are independent.11

The equilibrium dissociation constants for thebinding of TGF-b to TbRIIED were found to beapproximately 100 nM for both TGF-b1 and TGF-b3 isoforms, whereas the TGF-b2/TbRIIED affinitywas below the level of detection when using thisapproach with the biosensor. This 100 nM affinityis significantly lower than that determined forbinding of TGF-bs to the full-length type II recep-tor at the cell surface, which is observed to be inthe picomolar range for the TGF-b1 and -b3isoforms.12 This discrepancy may be due to thepresence of the type I and III receptors at the cellsurface, or may occur because of an influence ofthe membrane environment on the ectodomain ofTbRII. Alternatively, this difference may resultfrom the one ligand to two receptor stoichiometryof the TGF-b/TbRIIED complex. That is, if TGF-bis able to bind simultaneously to two cell surfacetype II receptors, then an avidity situation, whichwill stabilize the ternary complex, may be created.

The purpose of the present study was to test forthe extent that avidity contributes to receptoraffinity. To accomplish this, we used a de novodesigned coiled-coil system (the E5/K5 hetero-

dimerizing coiled-coil) to capture TbRIIED on thebiosensor surface. We have demonstrated that thiscoiled-coil motif has kinetic properties whichshould make it ideal for immobilizing proteins inan oriented manner at the biosensor surface. Thatis, the E/K coiled-coil pair exhibits a very slow dis-sociation rate.13 In this report, we demonstrate theutility of this approach by capturing E5 coil-taggedTbRIIED on a K5 coil derivatized biosensor sur-face. The results of TGF-b binding to this capturedTbRIIED confirmed that the stoichiometry of bind-ing was one ligand to two TbRIIED. Furthermore,the apparent affinities of captured TbRIIED for theTGF-b isoforms were high, and agreed well withthose reported for the cell surface type II receptor,suggesting that an avidity effect occurs at both thebiosensor and cell surface. Additionally, weshowed that our immobilization methodologydoes not require purification of the fusion proteinprior to its capture, hence making this approachattractive for rapid protein–protein interactionstudies.

Results

Production, purification and characterization ofthe TbRIIED-E5 fusion protein

In a classical BIACORE experiment, one of thebinding partners is covalently immobilized ornon-covalently captured on the sensor chip surfaceand the other interactant is injected over that sur-face. As the injection is proceeding, the massaccumulation of the analyte (the injected speciesin BIACORE terminology), as it binds to the ligand(the immobilized species), is recorded in arbitraryresonance units (RU). This corresponds to thewash-on phase of the experiment. The analyte sol-ution is then replaced by buffer and dissociationof the surface complexes is recorded (the wash-offphase). If complexes still remain in the matrix atthe end of the wash-off phase, their elution can beachieved by injecting a regeneration solution thatpromotes dissociation of the complexes. This seriesof steps constitutes a sensorgram. By repeating theexperiment with a series of different analyte con-centrations, a set of sensorgrams is generated.Since the data are recorded in real-time, it ispossible to derive kinetic parameters from theanalysis of the set of curves. This experimentalapproach, in combination with numerical inte-gration methods that globally fit all the sensor-grams in a set at the same time, has been shownto be reliable for discriminating between differentmechanisms of binding and for determining thevalues of the related kinetic constants.14 – 16

In order to simulate the cell surface behavior ofthe TGF-b type II receptor, we took the approachof capturing the extracellular domain of the typeII receptor (TbRIIED) on a biosensor surface. Toaccomplish that, we designed and expressed afusion protein (TbRIIED-E5), which consists of the

1174 Kinetics of TGF-b Binding to Captured TbRIIED

receptor extracellular domain with a N-terminalmyc-tag for detection, and C-terminal (His)6 andE5 coil tags for purification and capture on the bio-sensor, respectively (see Figure 1).

HEK 293SF-EBNA1 cells grown in a shaker-flaskwere transiently transfected (500 ml scale) with thepTT2-TbRIIED-E5 expression vector, using poly-ethylenimine (PEI) as a transfection vehicle.17

The recombinant protein was purified from themedium by standard Ni-NTA affinity chromato-graphy. Coomassie blue staining of SDS-PAGEshowed that the purified TbRIIED-E5 proteinmigrated as a broad band of approximately37 kDa, with no detectable contaminating bands(Figure 2). Western blotting with an anti-myc anti-body confirmed that the Coomassie blue stainedband was myc tagged TbRIIED-E5 (data not

shown). The broadness of the band is consistentwith the presence of three putative glycosylationsites on TbRIIED.18

In order to compare the affinity of the taggedTbRIIED-E5 fusion protein to that of untaggedTbRIIED, we first analyzed the interaction ofTbRIIED-E5 with TGF-b1 by covalently couplingTGF-b1 on the sensor chip surface and injectingdifferent concentrations (0–225 nM) of TbRIIED-E5. As shown in Figure 3(a), these TbRIIED-E5experiments resulted in sensorgrams similar to theones that we had obtained with untaggedTbRIIED.11 The sensorgrams were globally fit (allcurves at the same time) using the SPRevolutionsoftware package11,19 with a kinetic model depict-ing the presence of two independent binding siteson the TGF-b1 molecule (two-to-one stoichiometry,continuous lines in Figure 3(a)). This model waschosen since we had previously shown that itadequately represented the untagged TbRIIED/TGF-b isoform interactions.11 Figure 3(a) and (b)illustrate that, for the TbRIIED-E5/TGF-b1 data

Figure 2. TbRIIED-E5 purification. TbRIIED-E5protein was purified by standard Ni-NTA affinitychromatography. Coomassie blue staining of variousfractions collected during purification after resolving theproteins (20 ml loaded) on a 4–12% gradient gel(reducing conditions). Lanes W1 and W2 correspond tothe two first buffer A wash steps. Lane FT correspondsto the flow through after the last column loading. Lane1 corresponds to the first elution.

Figure 1. Construction of the expression vectorencoding the TbRIIED-E5 fusion protein. (a) Map of thepTT2 vector. This vector was derived from the pTTvector17 by inserting a multiple cloning site cassette. Thesequence of the multiple cloning site cassette (bp 1277to 1357) is shown in (b). (c) Amino acid sequence of theTbRIIED-E5 fusion protein. Residues 1–26 correspondto residues 1–26 of the TbRII sequence according to thenumbering used in the Swiss-Protein database (accessionnumber: P37173); TbRIIED-E5 residues 27–36 (under-lined) correspond to myc tag; TbRIIED-E5 residues37–170 correspond to residues 27–160 of the TbRIIsequence in the Swiss-Protein database; residues 171–181(underlined) correspond to the 11 amino acid linker;residues 182–216 correspond to the E5 coil and residues217–224 correspond to the His tag (underlined) separatedfrom the E5 coil sequence by two glycine residues.

Kinetics of TGF-b Binding to Captured TbRIIED 1175

set, the quality of the fit, when using the two-to-one stoichiometry model, was also adequate sincethe residuals (difference between the calculatedand experimental data points) were minimal andrandomly distributed around a zero value. Thestandard deviation of the residuals (SD) confirmedthat the two-to-one stoichiometry model fit betterthan a simple model (SD ¼ 0.489 versusSD ¼ 0.574, respectively). Furthermore, in the caseof the two-to-one stoichiometry model, Table 1summarizes the kinetic and thermodynamic con-stants derived from the global fitting of TbRIIEDand TbRIIED-E5 experiments (Figure 3 and datanot shown). It can be seen that both the kineticand thermodynamic constants were similar fortagged and untagged TbRIIED (Table 1), thusindicating that the interaction of TbRIIEDwith immobilized TGF-b1 was unaffected by thepresence of the myc, (His)6 and E5 coil tags.

Efficiency of the E5 coil tag to capture thefusion protein on a K5 coil surface

We tested the ability of the TbRIIED-E5 fusionprotein to form a stable complex with the K5 coilby injecting different concentrations of TbRIIED-E5 (from 0 nM to 100 nM) over a K5 coil sensorchip surface (less than 50 RUs coupled). A surfacewithout immobilized K5 coil was used as control.Figure 4 shows the resulting set of sensorgramsafter data preparation by the double referencingmethod.20 The sensorgrams were globally fit witha simple one-to-one model, which we had pre-viously found to be adequate when fitting the syn-thetic E5/K5 coiled-coil interaction.21 The qualityof the fit was not as good as that obtained for thesynthetic E5/K5 interaction, as evidenced by thefact that the corresponding residual plot showed anoticeable trend in the dissociation phase (datanot shown). A more complex one-to-one stoichi-ometry model, which includes an additionalrearrangement of the K5/TbRIIED-E5 complex,was then tested. We used this model since we hadfound that it depicted the mechanism of bindingof the streptavidin-bound E5/K5 coiled-coil inter-action well (G.D.C., unpublished results). As canbe seen in Figure 4, this model was also found torepresent the K5/TbRIIED-E5 interaction betterthan the simple model, as judged visually(Figure 4(a) and (b) and data not shown) andby statistical tests (SD ¼ 0.798 for the rearrange-ment model and SD ¼ 1.086 for the simplemodel). The kinetic and thermodynamic valuesderived from global fitting are listed in Table 2.From these kinetic constants, it is possible to deter-mine a global dissociation rate constant ðkdiss app ¼kdiss#1 £ k2r=ðkr þ k2rÞÞ; which takes into accountboth rearranged and unrearranged coiled-coilpopulations. This apparent dissociation rate of theK5/TbRIIED-E5 complex (2.0 £ 1024 s21) is veryslow and equal to that of the synthetic E5/K5interaction ((2.0 ^ 0.1) £ 1024 s21, Table 2). How-ever, the E5/K5 association rate was almost tenfold

Table 1. Kinetic and thermodynamic constants for TGF-b1 (coupled) interacting with TbRIIED-E5 or TbRIIED11

Kinetic model: two sites on TGF-b1

Kinetic and thermo-dynamic parameters TbRIIED11 TbRIIED-E5

kass 1 (M21 s21) (5.4 ^ 0.3) £ 105 (6.9 ^ 0.2) £ 105

kdiss 1 (s21) (6.5 ^ 0.2) £ 1022 (9.4 ^ 0.1) £ 1022

kass 2 (M21 s21) (1.8 ^ 0.1) £ 103 (3.0 ^ 0.2) £ 103

kdiss 2 (s21) (1.5 ^ 0.3) £ 1023 (2.3 ^ 0.2) £ 1023

Kd1 (nM) 158 ^ 37 (n ¼ 6) 136 ^ 10 (n ¼ 4)Kd2 (nM) 981 ^ 123 (n ¼ 6) 657 ^ 50 (n ¼ 4)

All the notations for the constants used in the Table are con-sistent with the ones used in Ref. 11. The values given for Kd1

and Kd2 correspond to the average value ^ the standard devi-ation of n independent experiments.

Figure 3. Evaluation of the kinetics of binding ofTbRIIED-E5 to coupled TGF-b1 by SPR. (a) Globalanalysis of the sensorgrams using a model describingthe presence of two independent binding sites on theTGF-b1 ligand. Different concentrations of TbRIIED-E5(0, 44.4, 66.7, 100, 150 and 225 nM) were injected induplicate over 200 RUs of immobilized TGF-b1 andover a control surface. The points correspond to theexperimental data and the continuous line to the globalfit using the two-to-one stoichiometry model. The kineticand thermodynamic constants related to the global fit arelisted in Table 1. For this set of sensorgrams, the SDvalue was determined to be equal to 0.489 for the two-to-one stoichiometry model (a SD value of 0.574 wasdetermined by globally fitting the data with a simplemodel). (b) Residuals from (a).


slower when TbRIIED was fused to the E5 coil(compare kass#1 ¼ 4:4 £ 105 M21 s21 and3:2 £ 106 M21 s21, Table 2). Nevertheless, since theapparent off rate remained very slow, the E5/K5

heterodimerizing system should be ideal for cap-turing coil tagged proteins at the biosensor surfacein an oriented manner.

TGF-b binding to TbRIIED-E5 captured on theBIACORE surface

We next analyzed the effect of immobilizingTbRIIED (instead of TGF-b) upon the kinetics ofTGF-b binding to TbRIIED-E5 in order to deter-mine whether one TGF-b molecule is able to simul-taneously bind to two TbRIIED molecules, andwhether this creates an avidity effect which stabil-izes the ternary complex. We covalently coupledthe K5 coil to the sensor chip surface and capturedTbRIIED-E5 on the surface through the coiled-coilinteraction. TGF-b was then injected over theTbRIIED-E5 surface at a flow rate of 100 ml/minuteto reduce mass transport artifacts.20 After completeregeneration of the surface, i.e. removal ofTbRIIED-E5 and TGF-b, a control sensorgram wasgenerated by injecting TGF-b over the same K5coil surface without captured TbRIIED-E5. Inorder to obtain a set of sensorgrams for globalanalysis, this series of injections was then repeatedby reloading the surface with TbRIIED-E5 andflowing varying concentrations of TGF-b. Further-more, the effect of surface loading conditions wasevaluated by repeating the overall experimentwith different amounts of K5 coil coupled to sensorchip surfaces (250–500 RUs) and with differentamounts of TbRIIED-E5 captured (150–350 RUs).

Figure 5(a) shows a representative data set fromexperiments in which varying TGF-b1 concen-trations were injected over the K5 coil surfacewith or without captured TbRIIED-E5. In order todetermine if this type of BIACORE immobilizationapproach could be performed without prior purifi-cation of the E5 coil tagged protein, another set ofexperiments was conducted by replacing purifiedTbRIIED-E5 with conditioned medium from theHEK 293 cells which contained TbRIIED-E5(Figure 5(b)). The data shown in Figure 5(a) and(b), were prepared for analysis by the “doublereferencing” method20 and are shown in Figure5(c1) and (e1), respectively. Representative sets ofsensorgrams for the other TGF-b isoforms areshown in Figure 5(d1) (TGF-b2) and (f1) (TGF-b3).All these sets of sensorgrams were globally fitusing a simple kinetic model coupled to a masstransport limitation step (fit shown as continuouslines in (c1)–(f1), and related residuals plotted in(c2)–(f2) in Figure 5). Mass transport limitationoccurs when the kinetics of binding are fast ascompared to the diffusion of the injected speciesfrom the bulk to the vicinity of the surface. Thediffusion of the injected species then becomesrate limiting and influences the shape of thesensorgram. The SD values shown in Table 3indicate that the addition of a mass transportlimitation step to the simple model improved thefit for all the TGF-b/captured TbRIIED-E5interactions. All the apparent kinetic and

Table 2. Kinetic and thermodynamic constants for K5coil (coupled) interacting with E5 coil21 or TbRIIED-E5

Kinetic andthermodynamicparameters

E5/K5 (simplemodel)21

TbRIIED-E5/K5(rearrangement model)

kass#1 (M21 s21) (3.17 ^ 0.05) £ 106 (4.43 ^ 0.09) £ 105

kdiss#1 (s21) (2.0 ^ 0.1) £ 1024 (5.3 ^ 0.8) £ 1023

kr (s21) N/A (7.9 ^ 0.1) £ 1023

k2r (s21) N/A (3.2 ^ 0.4) £ 1024

Kd (M) (6.3 ^ 0.5) £ 10211 (13.6 ^ 2) £ 1029

Kr (no unit) N/A (4.07 ^ 0.02) £ 1022

Kdapp (M) (6.3 ^ 0.5) £ 10211 (5 ^ 1) £ 10210

N/A: non-applicable.

Figure 4. Global analysis of the TbRIIED-E5 inter-action with coupled K5 coil. (a) Global fit of theTbRIIED-E5/K5 coil interaction using a rearrangementmodel. Different concentration of the TbRIIED-E5 fusionprotein (0, 3, 10, 30 and 100 nM) were injected in dupli-cate over approximately 20 RUs of immobilized K5 coiland over a control surface. The points correspond to theexperimental data and the continuous line to the globalfit using a rearrangement model. The kinetic andthermodynamic constants related to the global fit arelisted in Table 2. The SD value was determined to beequal to 0.798 for the rearrangement model (a SD valueof 1.086 was determined by globally fitting the datawith a simple model). (b) Residuals from (a).


Figure 5. TGF-b isoform interactions with coiled-coil captured TbRIIED-E5. (a) and (b) Injections of (1) purifiedTbRIIED-E5 or (2) TbRIIED-E5 in medium (1/10 dilution) over two immobilized K5 coil surfaces (200 RU and450 RU, (a) and (b), respectively) were followed by TGF-b injections (4). Control sensorgrams were generated overthe same surfaces by repeating the same TGF-b injections except that buffer injection (3) replaced TbRIIED-E5 injec-tion. (c1), (d1), (e1) and (f1) correspond to representative sets of sensorgrams, after data preparation, for the differentTGF-b isoforms interacting with captured TbRIIED-E5 using the same experimental procedure as shown in (a) and(b). All the data sets were globally analyzed using a simple binding model combined with a mass transport limitation


thermodynamic constants, determined when usingthe simple model coupled to a mass transport limi-tation step, for the TGF-b isoform interactions withcaptured TbRIIED-E5 are listed in Table 3.

Each TGF-b isoform interaction with the cap-tured TbRIIED-E5 was characterized by a rapidand similar on-rate (kass between 2.3 M21 s21 and7.8 £ 107 M21 s21). In contrast, whereas TGF-b1and TGF-b3 exhibited very slow dissociation rates(kdiss ¼ 1.5 s21 and 3.2 £ 1024 s21 for TGF-b1 andTGF-b3, respectively), the TGF-b2 dissociationrate was more rapid (kdiss ¼ 1.0 £ 1021 s21). Thisresults in an almost three orders of magnitudedifference in the apparent thermodynamic dis-sociation constants ðKdappÞ for TGF-b2 as comparedto TGF-b1 and -b3 (4.5 nM for the TGF-b2 isoformversus 6 pM for the TGF-b1 and TGF-b3 isoforms).These apparent affinities are at least four orders ofmagnitude higher than those observed when TGF-b, instead of TbRIIED-E5, was coupled to thebiosensor surface.11 Moreover, for all the TGF-b iso-forms, the ratio of the amount of capturedTbRIIED-E5 (determined from the RUs of capturedpurified TbRIIED-E5) to the amount of TGF-b-binding sites on the captured TbRIIED-E5 (calcu-lated by global fitting) was found to be close to 2(Table 3). This confirms the one-to-two stoichi-ometry for the TGF-b/TbRII interaction and sup-ports the hypothesis that an avidity effect isresponsible for stabilizing the ligand/receptorternary complex at the surface of the biosensor.Finally, we observed that the kinetic and thermo-dynamic constants for the interaction of TGF-b1with purified and non-purified TbRIIED-E5 arestrikingly similar (Table 3).

Discussion

We have previously characterized the inter-actions of the extracellular domain of the type IITGF-b receptor (TbRIIED) with its cognatemammalian ligands (TGF-b1, -b2, -b3) using anapproach in which TGF-b ligands were immobi-lized to the sensor chip of a surface plasmon reson-ance-based biosensor (the BIACORE).11 In thoseexperiments, we determined that the stoichiometryof TbRIIED binding to the TGF-b1 and -b3isoforms was two-to-one and that the twoTbRIIED-binding sites on TGF-b1 and -b3 wereindependent. This two TbRIIED to one TGF-bstoichiometry is supported by results fromMatsuzaki et al.22 who reported the formation of aTbRII/TGF-b1 complex comprising minimallytwo cell surface receptors and one ligand in insectcells. More recently, Hart and colleagues solvedthe X-ray structure of the TGF-b3/TbRIIEDcomplex23 and showed that each monomer withinthe dimeric TGF-b3 ligand is binding to oneTbRIIED, hence confirming a two-to-one recep-tor–ligand stoichiometry.

In addition to determining the stoichiometry ofbinding, our biosensor study11 yielded kinetic andthermodynamic constants of binding for TGF-b1and -b3. These were in the range of 100 nM for theKd with relatively rapid dissociation rates (approxi-mately 5 £ 1022 s21). In contrast, the affinity of theTbRIIED/TGF-b2 isoform interaction was too lowto be determined using that approach. This100 nM affinity is, a priori, in disagreement withthat observed for binding of TGF-bs to the type IIreceptor at the cell surface; Kds were estimated to

step. In each panel, the dots correspond to the experimental data points and the continuous line to the global fit. (c1)Global analysis of TGF-b1 (0.3, 1, 3 and 10 nM injections) interacting with purified captured TbRIIED-E5. (c2)Residuals from (c1). (d1) Global analysis of TGF-b2 (2, 6, 20 and 60 nM injections) interacting with purified capturedTbRIIED-E5. (d2) Residuals from (d1). (e1) Global analysis of TGF-b1 (0.1, 0.5, 1, 5 and 10 nM injections) interactingwith non-purified captured TbRIIED-E5. (e2) Residuals from (e1). (f1) Global analysis of TGF-b3 (0.33, 0.5, 1, 2 and3.3 nM injections) interacting with purified captured TbRIIED-E5. (f2) Residuals from (f1).

Table 3. Kinetic and thermodynamic constants for the three TGF-b isoform interactions with captured TbRIIED-E5using a simple binding model coupled to a mass transport limitation step

Kinetic and thermodynamicparameters

TGF-b1 (with puri-fied TbRIIED-E5)

TGF-b1 (with TbRIIED-E5from crude medium)



kass (M21 s21) (2.3 ^ 0.2) £ 107 (2.7 ^ 0.4) £ 107 (3.9 ^ 0.09) £ 107 (7.8 ^ 0.5) £ 107

kdiss (s21) (1.5 ^ 0.2) £ 1024 (1.5 ^ 0.2) £ 1024 (1.05 ^ 0.05) £ 1021 (3.1 ^ 0.2) £ 1024

Captured TbRIIED-E5 (RU) 164 340 242 220Calculated active TbRIIED-E5(RU)

80.5 ^ 1.4 165.2 ^ 6.5 125.4 ^ 2 108.7 ^ 0.5

Ratio (captured TbRIIED-E5/calculated TbRIIED-E5)

2.03 2.06 1.92 2.02

Kdapp (M) (6.0 ^ 2) £ 10212

(n ¼ 2)(4.5 ^ 2) £ 10212 (n ¼ 3) (4.5 ^ 2.0) £ 1029

(n ¼ 3)(4.0 ^ 1.5) £ 10212

(n ¼ 3)SDa 2.083 (2.432) 3.786 (5.686) 2.430 (3.630) 0.813 (1.562)

The values given for Kdapp correspond to the average value ^ the standard deviation of n independent experiments.a SD values corresponding to the global analysis of the data sets shown in Figure 5 with a simple model coupled to a mass trans-

port limitation step (the values in brackets correspond to the SD values from the global fit of the same data sets using a simplemodel without a mass transport limitation step).


be in the range of 5–30 pM for TGF-b1 and TGF-b3and in the nanomolar range for TGF-b2 usingradioligand competition assays.12

In this report, we investigate whether an avidityeffect may provide the simplest explanation forthis observed affinity difference by using an alter-nate experimental approach with the biosensor.We simulated the cell surface presentation ofTbRIIED by capturing it in an oriented and stablemanner on the biosensor chip surface using theE5/K5 heterodimerizing coiled-coil system.13,24,25

Specifically, our approach was to express an E5-tagged TbRIIED fusion protein (TbRIIED-E5, theE5 coil being positioned where the transmembranedomain normally occurs) and capture it through itsinteraction with covalently immobilized K5 coil.We first demonstrated that the interaction ofTbRIIED-E5 with immobilized TGF-b1 was thesame as TbRIIED, i.e. that the TGF-b1-binding siteof TbRIIED was not influenced by the presence ofthe E5 coil tag (Figure 3, Table 1).

We then evaluated the effect of fusing TbRIIEDto E5 on the E5/K5 interaction (Figure 4). Thisanalysis showed that the binding of TbRIIED-E5to K5 was best described by a one-to-one stoichi-ometry model including an additional rearrange-ment kinetic step. This is in contrast to ourprevious results that showed that a simple modeladequately represents the synthetic E5/K5interaction.21 However, it is in agreement withresults showing that this more complex modeldepicts the mechanism of binding of thestreptavidin-bound E5/K5 coiled-coil interactionwell (G.D.C., unpublished results). In both caseswhere the molecular weight of E5 (or K5) wasartificially increased (by either binding it to strep-tavidin (data not shown) or fusing it to TbRIIED(Table 2)) we observed a change in both on- andoff-rates of the first binding step, relative to thesynthetic E5/K5 interaction. This changeapparently “reveals” the rearrangement step byrendering it kinetically limiting. From the kineticconstants for the TbRIIED-E5/K5 interactiondetermined using the model that includes arearrangement step (Table 2), an apparentdissociation rate constant (defined askdiss app ¼ kdiss#1 £ k2r=ðkr þ k2r)) was calculated asbeing 2.0 £ 1024 s21. This value is the same as theapparent dissociation rate constants determinedfor the synthetic E5/K5 interaction and the K5/streptavidin-bound E5 interaction (Table 2 anddata not shown). These results demonstrate thatthe E5 coil is a suitable tag to capture fusion pro-teins on the surface of the BIACORE since all themeasured apparent dissociation rates were slow(2.0 £ 1024 s21) and were independent of whetherthe E5 coil strand was free, or bound/fused to alarger protein.

Using this immobilization approach, we thenstudied the interactions of the three TGF-b iso-forms with the coiled-coil captured TbRIIED. Thesets of curves corresponding to the three TGF-bisoforms interacting with TbRIIED-E5 were

globally fit using a simple model coupled to amass transport limitation step (Figure 5). Allthe interactions were characterized by fastassociation rates (kass between 2.3 M21 s21 and7.8 £ 107 M21 s21, Table 3). These on-rates are inthe range for which the transport of the species insolution from the bulk to the vicinity of the surfacehas been reported to become rate limiting,26 hencejustifying the use of the mass transport step duringthe fitting process. The SD values shown in Table 3from the use of a simple model with, versus with-out, a mass transport limitation step demonstratethat there was an improvement of the fit whenmass transport limitation was taken into account.The quality of the fit varied between the differentdata sets. For example, the quality of the fit waslower when purified TbRIIED-E5 was replaced byconditioned medium containing TbRIIED-E5(Figure 5(c) and (e)). This may result from non-specific binding of other molecules in the con-ditioned medium. The fact that the quality of thefits for the interactions of TGF-b with captured/purified TbRIIED was relatively good for thesimple model combined with mass transportlimitations could be regarded as being somewhatsurprising, considering that we are suggesting thatan avidity effect is occurring. However, this fit to asimple kinetic model may indicate that the twomonomers within the TGF-b molecule bind to thetwo captured TbRIIED-E5 molecules almostsimultaneously.

Both TGF-b1 and -b3 isoform interactions werecharacterized by apparent slow dissociation rates(1.5–3 £ 1024 s21). In contrast, the TGF-b2 isoformdissociation rate was rapid (kdiss approximately0.1 s21, see Table 3). This resulted in the thermo-dynamic dissociation constants being approxi-mately 5 pM for the TGF-b1 and -b3 isoforms, andapproximately 6 nM for the TGF-b2 isoform. Thefact that we are able to detect TbRIIED-E5 bindingto the TGF-b2 isoform is of interest since it waspreviously thought that TGF-b2 does not interactwith any significant affinity to TbRIIED alone.

The kinetic and thermodynamic constants thatwere determined in this study are strikinglydifferent from those determined when the TGF-bisoforms, instead of TbRIIED, were immobilized(compare the constants in Table 1 to those inTable 3). Specifically, there is an almost two ordersof magnitude increase in association rates and asimilar magnitude decrease in dissociation rates,resulting in a four orders of magnitude increase inaffinity. The fact that these apparent differenceswere observed when one binding partner wasimmobilized instead of the other strongly suggeststhat they are due to an avidity effect caused by thestoichiometry of binding. That is, the capture ofthe extracellular domain of TbRII appears to resultin two components of the ternary TbRIIED/TGF-b/TbRIIED complex being sequestered in closeproximity to each other at the biosensor surface.This reduced entropy state for the receptor popu-lation would lead to a smaller entropy loss being


required during ligand binding (as compared tothe reverse approach where the ligand is immobi-lized and monomeric TbRIIED is injected).Accordingly, both faster association and slowerdissociation rates would be predicted to occur.The fact that the affinities observed for the type IIreceptor at the cell surface using radioligand com-petition experiments12 are very close to that whichwe determined using the captured receptorapproach in the BIACORE suggests that a similaravidity phenomenon is occurring at the cellsurface.

Although the primary focus of the present studywas to investigate the effect of receptor immobiliz-ation upon the apparent affinity of the TGF-b/TbRIIED interaction, a second goal was to examinethe general utility of a heterodimeric coiled-coildisplay system for surface capture in SPR-basedbiosensor studies. We have shown recently thatthis immobilization approach is effective when thecoil strand was chemically coupled to a peptide.27

In this report, we demonstrate that this capturestrategy is also applicable when recombinantly fus-ing the coil strand to a larger protein. Furthermore,we showed that TGF-b1 binding was equivalentwhen the TbRIIED-E5 fusion protein was capturedwith or without prior purification (Figure 5,Table 3), demonstrating that this approach can beadvantageously applied for the rapid analysis ofprotein–protein interactions without purificationof at least one partner. Other advantages to thiscoiled-coil immobilization system are: (i) the cap-tured fusion protein is displayed in an orientedmanner, thereby eliminating surface heterogeneityartifacts that may result from chemical couplingprocedures; (ii) the regeneration of the surface canbe performed at the coiled-coil level such that thecoil surface can be reused for the loading of differ-ent fusion proteins.

Materials and Methods

Materials

The pcDNA3 vector containing the cDNA encodingthe E5 coil (pcDNA3-E5coil) was a generous gift fromM. Banville (BRI). The pcDNA3 vector containing thecDNA encoding for the N-terminally myc-tagged TGF-btype II receptor (pcDNA3-TbRII) was a generous giftfrom M. Jaramillo (BRI). All the enzymes were fromNew England Biolabs Inc. and were used according tothe manufacturer’s recommendations. All the primerswere purchased from Hukabel Scientific Ltd (Montreal,Que., Canada). Recombinant human TGF-b isoforms(TGF-b1, TGF-b2 and TGF-b3 expressed in ChineseHamster Ovary or NSO cells, carrier-free) were pur-chased from R&D Systems Inc. (Minneapolis, MN). Theexpression vector pTT2 is a derivative of pTT vector17

with extended multiple cloning sites (see Figure 1).The BIACORE 3000, CM5 and Pioneer B1 sensor chips,

N-hydroxysuccinimide (NHS), N-ethyl-N0-(3-diethyl-aminopropyl) carbodiimide hydrochloride (EDC), 2-(2-pyridinyldithio)ethaneamine (PDEA) and 1 M ethanol-

amine (pH 8.5) were purchased from BIACORE Inc.(Piscataway, NJ).

Construction of the TbRIIED-E5 expression vector

For construction of pTT2 E5coil, the cDNA encodingfor the E5 coil was PCR amplified using the pcDNA3-E5coil as template and the following primers, Efor: 50-TAGAGCGGCCGCGGTGGCGAGGTATCC-30 (Not Irestriction site underlined) and Erev: 50-TAGGATCCCTAATGGTGATGATGGTGATGACCGCCCTTCTCAAGTG-30 (BamHI restriction site underlined). The resultingfragment was digested with Not I/BamHI and ligated topTT2 digested with the same enzymes. For constructionof pTT2 TbRIIED-E5, the cDNA encoding for the myctagged TbRIIED was PCR amplified using the pcDNA3-TbRII as template and the following primers TbRIIfor: 50-AAGCTTCACCATGGAGGCGGCG-30 (HindIII restric-tion site underlined) and TbRIIrev: 50-TAGAGCGGCCGCCGTCAGGATTGCTGGTGTT-30 (Not I restriction siteunderlined). The resulting fragment was digested withHindIII/Not I and ligated to pTT2 E5coil digested withthe same enzymes. The pTT2 TbRIIED-E5 was thenamplified in Escherichia coli (DH5a), and the plasmidpurified using MAXI prep columns (QIAgen,Mississauga, Ont., Canada). Each construct was verifiedby sequencing. For quantification, plasmids were dilutedin 50 mM Tris–HCl (pH 7.4) and the absorbances at260 nm and 280 nm measured. Only plasmidpreparations with A260/A280 ratios between 1.8 and 2.0were used for transient transfection.

Transient transfections

Transient tranfection was performed in shaker flasksas described elsewhere17 except that a 293SF cell line28

derivative stably expressing the EBNA1 protein and anew serum-free medium formulation (P.L.P. et al.,unpublished results) were used. The recombinant pro-tein was expressed by the transientlly transfected cellsand secreted into the medium. The culture was har-vested five days after transfection and the medium wasclarified by centrifugation at 3500g for ten minutes.

TbRIIED-E5 fusion protein purification

The pH of the supernatant containing the TbRIIED-E5fusion protein was adjusted to 7.4. The purification wasdone using a Ni-NTA agarose affinity column (2 ml bedvolume, QIAgen) by loading the supernatant by gravityflow. The column was then washed two times with25 ml buffer A (50 mM sodium phosphate, 300 mMNaCl, pH 7.4). Elution was achieved with buffer B (buf-fer A þ 150 mM imidazole, pH 7.4, 8 ml collected). Theflow-through sample was reloaded and eluted twiceusing the same conditions as above. Elution fractions(8 ml each) were then individually concentrated, fol-lowed by buffer exchange for PBS, by using a Centriprep10 device (Amicon), according to the manufacturer’s rec-ommendations. The concentration of the purifiedTbRIIED-E5 fusion protein (three aliquots of 500 ml) wasdetermined with Coomassie Plus Protein Assay ReagentKit (Pierce), using BSA as standard.

The yield of TbRIIED-E5 from 500 ml of conditionedmedia was approximately 766 mg. However, some frac-tions were observed by non-reducing SDS-PAGE to con-tain higher order aggregates of TbRIIED-E5. Thesefractions were not used in this study.


Electrophoresis, Western blotting, Coomassieblue staining

The purity of the fusion protein was estimated byCoomassie blue staining after resolving the proteins(20 ml loaded) on a reducing Novex Nupage 4–12%(Invitrogen) gel. The purified TbRIIED-E5 protein wasalso detected by Western blot (anti-myc 9E10, SantaCruz)following protein separation on non-reducing 11% SDS/polyacrylamide gels.

SPR experiments

The running buffer (HBS; 20 mM Hepes (pH 7.4),150 mM NaCl, 3.4 mM EDTA, 0.05% Tween20) was usedfor diluting all the species injected in BIACORE experi-ments (the analytes in BIACORE terminology).

Immobilization of TGF-b on CM5 sensor chips

TGF-b1 surfaces and control dextran surfaces on CM5sensor chips were prepared as described elsewhere11

using the standard amine coupling procedure.

Immobilization of K5 coil on PIONEER B1 sensor chips

The synthesis and purification of the K5 coil peptide,which contains a N-terminal Cys-Gly-Gly extension, isdescribed elsewhere.21 The K5 coil was covalentlyimmobilized on the surfaces of PIONEER B1 sensorchips using the standard ligand thiol coupling pro-cedure. Specifically, surface carboxylic acid groups wereactivated by the injection of 30 ml of a mixture containing0.05 M NHS and 0.2 M EDC, followed by PDEA injection(30 ml of 80 mM PDEA in 100 mM boric acid, pH 8.5).The K5 coil (100 nM K coil freshly dissolved in 10 mMacetic acid, pH 4.0) was injected under manual controluntil the desired amount of peptide was coupled (20–500 RUs, depending on the experiment). Finally, freshlyprepared 0.05 M L-cysteine (40 ml, in 0.1 M sodium for-mate, 1 M NaCl, pH 4.3) was injected to block anyremaining activated sites on the sensor chip surface.Control surfaces, when required, were prepared simi-larly, except that running buffer (10 ml) was injectedinstead of K5 coil. All steps in the immobilization pro-cess were carried out at a flow rate of 5 ml/minute.When low amounts of K5 coil were coupled (less than300 RUs), all the K coil coupling steps were preceededby blocking some of the carboxylic acid groups by suc-cessive 20 ml NHS/EDC and 20 ml etholamine injections.

Kinetic assays on the BIACORE

Injections of TbRIIED-E5 fusion protein over TGF-b1. Allthe kinetic experiments were carried out at 25 8C with aflow rate of 5 ml/minute. Different concentrations ofTbRIIED-E5 (0–225 nM) were randomly injected induplicate over the TGF-b1 surface as well as over a con-trol surface (15 ml injections), following which theanalyte solution was replaced by buffer for 180 seconds.Regeneration of the sensor chip for subsequent injectionswas accomplished by two pulses of HCl (20 mM, 120seconds), followed by an EXTRACLEAN procedure per-formed according to the BIACORE manual.

Injections of TbRIIED-E5 fusion protein over K5 coil. Allthe kinetic experiments were carried out at 25 8C with aflow rate of 100 ml/minute. Different concentrations ofTbRIIED-E5 (0–100 nM) were randomly injected in

duplicate over a K5 coil surface (RUs) as well as over acontrol surface for 90 seconds, following which theanalyte solution was replaced by buffer for 270 seconds.Regeneration of the sensor chip for subsequent injectionswas accomplished by two pulses of 5 M guanidinehydrochloride (25 ml each), followed by an EXTRA-CLEAN procedure and an IFC wash performed accord-ing to the BIACORE manual.

Injections of TGF-bs over K5-captured TbRIIED-E5 fusionprotein. Purified TbRIIED-E5 or cell supernatant contain-ing TbRIIED-E5 (1/10 dilution in HBS) was injectedover different K5 coil surfaces (flow rate 5 ml/minute,injections of 10 ml or 15 ml, depending on the experi-ments). After TbRIIED-E5 capture, the flow rate wasthen set up to 100 ml/minute and TGF-b isoform injec-tions (TGF-b1 from 0 nM to 10 nM, TGF-b2 from 0 nMto 60 nM, TGF-b3 from 0 nM to 3.33 nM; stock solutionsof at least 1 mM TGF-b in 4 mM HCl were freshly dilutedin running buffer) were performed (90 seconds or 120seconds injection, dissociation 180 seconds at least). Acontrol experiment was performed by repeating thesequential injections using running buffer in lieu ofTbRIIED-E5. Regeneration of the K5 coil surface wasachieved by two pulses of 5 M guanidine hydrochloride(25 ml each), followed by an EXTRACLEAN procedureand an IFC wash done according to the BIACOREmanual.

Data preparation and analysis

Sensorgrams were prepared and globally fit usingnon-linear least squares analysis and numericalintegration of the differential rate equations using theSPRevolutionq software package. The data preparationwas done as described elsewhere.19 Each sensorgramgenerated using a control surface was subtracted fromthe corresponding experimental sensorgrams and theresulting curves were transformed to concentrationunits. Finally, the curve corresponding to buffer injectionwas subtracted from the control surface-correctedcurves. Each data set, which consists of sensorgramsfrom injections of different analyte concentrations overthe same surface, was then analyzed using several differ-ent kinetic models that are available in the SPRevolutionq

software.

Acknowledgements

This work was supported by the Protein Engin-eering Network of Centres of Excellence (PENCE)and the National Research Council of Canada.G.D.C. is supported by a PENCE studentship. Theauthors acknowledge Drs Sulea (BRI, NRCC) andChao (Sensium Technologies, Inc) for fruitful dis-cussion during the writing of this manuscript.

References

1. Massague, J., Blain, S. W. & Lo, R. S. (2000). TGFbetasignaling in growth control, cancer, and heritable dis-orders. Cell, 103, 295–309.

2. Massague, J. & Wotton, D. (2000). Transcriptionalcontrol by the TGF-beta/Smad signaling system.EMBO J. 19, 1745–1754.


3. Roberts, A. B., McCune, B. K. & Sporn, M. B. (1992).TGF-beta: regulation of extracellular matrix. KidneyInt. 41, 557–559.

4. Sporn, M. B. & Roberts, A. B. (1992). Transforminggrowth factor-beta: recent progress and newchallenges. J. Cell Biol. 119, 1017–1021.

5. Wakefield, L. M., Colletta, A. A., McCune, B. K. &Sporn, M. B. (1992). Roles for transforming growthfactors-beta in the genesis, prevention, and treatmentof breast cancer. Cancer Treat. Res. 61, 97–136.

6. Massague, J. & Chen, Y. G. (2000). Controlling TGF-beta signaling. Genes Dev. 14, 627–644.

7. Wrana, J. L., Attisano, L., Carcamo, J., Zentella, A.,Doody, J., Laiho, M. et al. (1992). TGF beta signalsthrough a heteromeric protein kinase receptor com-plex. Cell, 71, 1003–1014.

8. Blobe, G. C., Schiemann, W. P., Pepin, M. C.,Beauchemin, M., Moustakas, A., Lodish, H. F. &O’Connor-McCourt, M. D. (2001). Functional rolesfor the cytoplasmic domain of the type III transform-ing growth factor beta receptor in regulating trans-forming growth factor beta signaling. J. Biol. Chem.276, 24627–24637.

9. Lopez-Casillas, F., Wrana, J. L. & Massague, J. (1993).Betaglycan presents ligand to the TGF beta signalingreceptor. Cell, 73, 1435–1444.

10. Cheifetz, S., Hernandez, H., Laiho, M., ten Dijke, P.,Iwata, K. K. & Massague, J. (1990). Distinct trans-forming growth factor-beta (TGF-beta) receptor sub-sets as determinants of cellular responsiveness tothree TGF-beta isoforms. J. Biol. Chem. 265,20533–20538.

11. De Crescenzo, G., Grothe, S., Zwaagstra, J., Tsang, M.& O’Connor-McCourt, M. D. (2001). Real-time moni-toring of the interactions of transforming growthfactor- beta (TGF-beta) isoforms with latency-associ-ated protein and the ectodomains of the TGF-betatype II and III receptors reveals different kineticmodels and stoichiometries of binding. J. Biol. Chem.276, 29632–29643.

12. Massague, J. (1990). The transforming growth factor-beta family. Annu. Rev. Cell Biol. 6, 597–641.

13. Chao, H., Houston, M. E., Jr, Grothe, S., Kay, C. M.,O’Connor-McCourt, M., Irvin, R. T. & Hodges, R. S.(1996). Kinetic study on the formation of a de novodesigned heterodimeric coiled-coil: use of surfaceplasmon resonance to monitor the association anddissociation of polypeptide chains. Biochemistry, 35,12175–12185.

14. De Crescenzo, G., Grothe, S., Lortie, R., Debanne,M. T. & O’Connor-McCourt, M. (2000). Real-timekinetic studies on the interaction of transforminggrowth factor alpha with the epidermal growth fac-tor receptor extracellular domain reveal a confor-mational change model. Biochemistry, 39, 9466–9476.

15. Fisher, R. J. & Fivash, M. (1994). Surface plasmon res-onance based methods for measuring the kineticsand binding affinities of biomolecular interactions.Curr. Opin. Biotechnol. 5, 389–395.

16. Morton, T. A., Myszka, D. G. & Chaiken, I. M. (1995).Interpreting complex binding kinetics from opticalbiosensors: a comparison of analysis by linearization,

the integrated rate equation, and numerical inte-gration. Anal. Biochem. 227, 176–185.

17. Durocher, Y., Perret, S. & Kamen, A. (2002). High-level and high-throughput recombinant proteinproduction by transient transfection of suspension-growing human 293-EBNA1 cells. Nucl. Acids Res.30, E9.

18. Guimond, A., Sulea, T., Pepin, M. C. & O’Connor-McCourt, M. D. (1999). Mapping of putative bindingsites on the ectodomain of the type II TGF-betareceptor by scanning-deletion mutagenesis andknowledge-based modeling. FEBS Letters, 456,79–84.

19. Khaleghpour, K., Kahvejian, A., De Crescenzo, G.,Roy, G., Svitkin, Y. V., Imataka, H. et al. (2001). Dualinteractions of the translational repressor Paip2 withpoly(A) binding protein. Mol. Cell. Biol. 21,5200–5213.

20. Myszka, D. G. (1999). Improving biosensor analysis.J. Mol. Recognit. 12, 279–284.

21. De Crescenzo, G., Litowski, J. R., Hodges, R. S. &O’Connor-McCourt, M. D. (2003). Real-time monitor-ing of the interactions of two-stranded de novodesigned coiled-coils: effect of chain length on thekinetic and thermodynamic constants of binding.Biochemistry, 42, 1754–1763.

22. Matsuzaki, K., Kan, M. & McKeehan, W. L. (1996).Reconstitution of a pentameric complex of dimerictransforming growth factor beta ligand and a type I,II, III receptor in baculoviral- infected insect cells. InVitro Cell Dev. Biol. Anim. 32, 345–360.

23. Hart, P. J., Deep, S., Taylor, A. B., Shu, Z., Hinck, C. S.& Hinck, A. P. (2002). Crystal structure of the humanTbetaR2 ectodomain–TGF-beta3 complex. NatureStruct. Biol. 9, 203–208.

24. Chao, H., Bautista, D. L., Litowski, J., Irvin, R. T. &Hodges, R. S. (1998). Use of a heterodimeric coiled-coil system for biosensor application and affinitypurification. J. Chromatog. B Biomed. Sci. Appl. 715,307–329.

25. Tripet, B., Yu, L., Bautista, D. L., Wong, W. Y., Irvin,R. T. & Hodges, R. S. (1996). Engineering a de novo-designed coiled-coil heterodimerization domain offthe rapid detection, purification and characterizationof recombinantly expressed peptides and proteins.Protein Eng. 9, 1029–1042.

26. Myszka, D. G., Morton, T. A., Doyle, M. L. &Chaiken, I. M. (1997). Kinetic analysis of a proteinantigen–antibody interaction limited by mass trans-port on an optical biosensor. Biophys. Chem. 64,127–137.

27. Tripet, B., De Crescenzo, G., Grothe, S., O’Connor-McCourt, M. & Hodges, R. S. (2002). Kinetic analysisof the interactions between Troponin C and theC-terminal Troponin I regulatory region and vali-dation of a new peptide delivery/capture systemused for surface plasmon resonance. J. Mol. Biol..

28. Cote, J., Garnier, A., Massie, B. & Kamen, A. (1998).Serum-free production of recombinant proteins andadenoviral vectors by 293SF-3F6 cells. Biotechnol.Bioeng. 59, 567–575.

Edited by M. Yaniv

(Received 26 August 2002; received in revised form 24 January 2003; accepted 27 January 2003)


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