From the INSERM U564, Centre Hospitalier Universitaire d'Angers, 4 rue Larrey, 49033 Angers, France
Received for publication, March 27, 2003 , and in revised form, April 15, 2003.
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ABSTRACT |
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INTRODUCTION |
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These cytokines act by the formation of a multimeric receptor complex, including a common receptor unit, gp130 (for a review, see Ref. 2). The common use of gp130 explains in part the overlapping effects of these cytokines (10). IL-6 and IL-11 binding induces dimerization of gp130 (1117). LIF, CNTF, CT-1, and CLC induce heterodimerization of gp130 and of the leukemia inhibitory factor receptor (LIFR) (7, 8, 1822). Human OSM can recruit two kinds of active complexes resulting from the heterodimerization of gp130 with LIFR or with the specific receptor for OSM, OSMR (23, 24). In addition to transducing receptor chains (gp130, LIFR, and OSMR), the active complex can contain specific co-receptor chains conferring high affinity. Specific co-receptor chains were observed for IL-6 (IL-6R) (25) and IL-11 (IL-11R) (14) and for CNTF and CLC, which share the same co-receptor chain (CNTFR) (7, 8, 2628).
The receptor-transducing chains of the IL-6 family, gp130 (13), LIFR (29), and OSMR (23), have a modular organization, with an extracellular domain, a short transmembrane domain, and an intracellular domain. The extracellular domain of gp130 contains an N-terminal Ig-like domain, followed by a cytokine binding domain (CBD) and three fibronectin III domains. The cytokine binding domain is composed of two fibronectin III domains characterized by two conserved disulfide bridges in the N-terminal FnIII domain and a conserved WSXWS motif in the C-terminal FnIII domain (30). This motif is characteristic of class I cytokine receptors and is found for the other class I cytokine receptor family (30). The crystal structure of the gp130 cytokine binding domain indicates that its two FnIII domains have an L-shaped quaternary structure (31). In addition to the modules of gp130, LIFR and OSMR possess an additional N-terminal module. For LIFR, this module is a second copy of a cytokine binding domain (29), whereas for OSMR, it is limited to the C-terminal half of a CBD (23). The co-receptor chains are composed of an Ig-like domain followed by a CBD either linked to the membrane by a transmembrane domain (IL-6R and IL-11R) (14, 25) or through a glycosylphosphatidylinositol linker (CNTFR) (26).
Site-directed mutagenesis studies have shown that the cytokines of the IL-6 family interact with the receptor chains by three binding sites, numerated from I to III by analogy with the growth hormone (32). Cytokines requiring a co-receptor chain (e.g. CNTF, CLC, IL-6, and IL-11) binds to this co-receptor (CNTFR, IL-6R, and IL-11R) through binding site I (C-terminal parts of the AB loop and of helix D (8, 20, 26, 3336)). The glycoprotein gp130 interacts through binding site II, located on the solvent-exposed faces of helices A and C (3, 37, 38). These sites are similar to binding sites I and II of the growth hormone (32). An additional binding site (site III) is located at the N-terminal part of helix D and may include residues from the N-terminal part of the AB loop and from the BC loop. It corresponds to an additional gp130 binding site for IL-6 and IL-11 (36, 39, 40) and to the LIFR binding site for LIF, OSM, CNTF, CT-1, and CLC. These LIFR binding cytokines are characterized by an FXXK motif located at the N terminus of helix D, which is required for LIFR binding and constitutes the signature of this interaction (4145). The sites III are organized as exchangeable modules (45). The recently determined structure of Kaposi sarcoma-associated herpesvirus IL-6 (vIL-6) complexed with gp130 has shown that IL-6 interacts with the cytokine binding domain of gp130 through site II and with the Ig-like domain of a second gp130 molecule through site III in a complex formed by two vIL-6 and two gp130 molecules (46, 47).
Several lines of evidence, based on chimeric receptors, suggest that the Ig-like domain of LIFR is involved in site III binding (4850). The aim of the present study was to determine the LIF binding site of LIFR. For this purpose, we modeled the Ig-like domain of LIFR and analyzed the properties of its surface, to identify an area with physicochemical properties complementary to those of the LIF site III. Two residues of the LIFR Ig-like domain, Asp214 and Phe284, form a mirror image of LIF Phe156 and Lys159, which constitute the LIFR binding hot spots (41). Single and double LIFR mutants, in which Asp214 or/and Phe284 were mutated to alanine, were tested for their ability to induce biological effects in response to LIF stimulation. Stimulation by CT-1 or OSM was also studied, because these two cytokines share the capability to form an active complex with gp130 and LIFR (19, 22, 23, 51). We show that LIF, CT-1, and OSM share overlapping binding sites located in the Ig-like domain of LIFR.
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MATERIALS AND METHODS |
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The Ig-like domain of LIFR was modeled from the crystal structure of the
Ig-like domain of gp130 in the complex with viral IL-6 (PDB access number:
1I1R
[PDB]
) (46). The Ig-like
domains of LIFR and of gp130 were aligned from a multiple sequence alignment
of the Ig-like domains of the gp130 receptor family with ClustalW
(56). The positions of the
-strands in the Ig-like domain of LIFR were checked with the NNSSP
program (57). Twenty models
were generated with MODELER and refined by simulated annealing. The quality of
the models was checked with Profiles_3D
(58) and the Protein Verify
module of Insight.
The stability of the mutated Ig-like domains of LIFR (D214A, F284A, and D214A/F284A LIFR) was calculated with the FOLD-X program (59) available at fold-x.embl-heidelberg.de. This program predicts the change in the stability of mutated proteins by computing the changes in the free energy of folding upon mutations.
Surface PropertiesA solvent probe radius of 1.4 Å was used to define the protein molecular surfaces (60). Continuum electrostatic calculations were carried out with the DELPHI package (61, 62) under Insight. The formal charge set was used, with an ionic strength of 0. The dielectric constants of the proteins and of the surrounding medium were 2 and 80, respectively. The electrostatic potentials were mapped onto cubic grids with a 0.72-Å point spacing. The percentage grid fill was 50% for LIF (159 x 159 x 159 points/slide) and 40% for the Ig-like domain of LIFR (133 x 133 x 133 points/slide). The boundary potential was full coulombic. The linear Poisson-Boltzmann equation was then solved iteratively. The Eisenberg's hydrophobicity scale (63) was used to display the protein surface hydrophobicity.
Protein DockingThe lowest energy rotameric orientation of
LIFR Phe284 was searched by 1 x
2 isomeric mapping with CHARMM. The dihedrical angles
1 and
2 were defined by the bond
connectivities NC
C
C
and
C
C
C
C
2, respectively. A single
rotameric orientation of Phe284 was stable and corresponded to the
g-, perpendicular orientation. This orientation was used in the starting
structure for the docking procedure. The docking of the Ig-like domain of LIFR
to LIF was carried out with the molecular docking program HEX 2.0 based on
spherical polar Fourier correlations
(64). This program is
available at
www.biochem.abdn.ac.uk/hex.
A filter based on the distance between the rings of LIF Phe156 and
LIFR Phe284 was used to remove false-positive solutions. The best
scoring solution was energy-minimized using the 100 steepest descent steps,
followed by adopted basis Newton-Raphson (ABNR) steps, until a convergence
gradient of 0.001 was reached. A similar procedure was carried out with the
D214A mutant.
Cells and ReagentsTransfected Ba/F3 cells were maintained as previously described (45). The medium was supplemented with hygromycin and neomycin for LIFR/gp130 Ba/F3 cell lines. Purified recombinant human LIF and mouse IL-3 were kindly donated by Drs. K. Turner and M. Stahl (Genetics Institute, Boston, MA). IL-2 was a kind gift of Dr. G. Zurawski (DNAX Research Institute, Palo Alto, CA). Human CT-1, murine CT-1, human IL-6, human OSM, and soluble IL-6R were purchased from R&D Systems (Oxon, UK). IgG1 isotype control and AN-HH1 anti-gp130 and anti-LIFR mAbs (AN-B1, AN-C1, AN-D1, AN-E1, AN-F1, ANG1, AN-H1, ANI1, and AN-J1) were generated in the laboratory as described elsewhere (45, 65). The antibody detecting phospho-STAT3 (Tyr705) was purchased from New England BioLabs (Beverly, MA). Goat anti-mouse peroxidase-labeled immunoglobulins were from Clinisciences (Montrouge, France).
Site-directed Mutagenesis and Cell TransfectionThe pME18S vector containing the cDNA encoding human LIFR was subjected to site-directed mutagenesis using the QuikChangeTM site-directed mutagenesis kit (Stratagene, La Jolla, CA) following the manufacturer's instructions. Mutations were performed on the predicted Ig-like domain of LIFR. Mutations were verified by DNA sequencing with an automatic DNA sequencer (Beckman Coulter) using the Dye Terminator Cycle Sequencing Quick Start Kit (Beckman Coulter).
Ba/F3 cell lines stably expressing the gp130 receptor were transfected with cDNA encoding mutated LIFR using electroporation (960 microfarads and 230 V) with 40 µg of the mutated plasmid. Transfected cells were selected for growing in the presence of 300 ng/ml hygromycin and IL-3 (1 unit/ml).
Flow Cytometry AnalysisLIFR expression on transfected Ba/F3 cell lines was verified by flow cytometry analysis using nine different monoclonal antibodies (AN-B1, AN-C1, AN-D1, AN-E1, AN-F1, ANG1, AN-H1, ANI1, and AN-J1) directed against different conformational epitopes to ensure the correct folding of the protein. Gp130 expression was verified using the AN-HH1 anti-gp130 mAb. Cells were successively incubated for 30 min at 4 °C with the appropriate primary antibody or isotype control antibody (10 µg/ml) and a phycoerythrin-conjugated anti-mouse antibody. Fluorescence was subsequently analyzed with a FACScan flow cytometer from BD Biosciences (Mountain View, CA).
Proliferation AssaysBa/F3 cell lines expressing gp130 and wild type or mutated LIFR were seeded in 96-well plates at a concentration of 5 x 103 cells/well in RPMI 1640 medium containing 5% fetal calf serum. Serial dilutions of the cytokines tested were performed in triplicate. After a 72-h incubation period, 0.5 µCi of [3H]thymidine was added to each well for the last 4 h of the culture, and the incorporated radioactivity was determined by scintillation counting.
STAT3 Tyrosine Phosphorylation AnalysisAfter a 36-h cytokine and serum starvation, cells were stimulated for 10 min in the presence of the indicated cytokine. Then cells were lysed in 10 mM Tris-HCl, pH 7.6, 5 mM EDTA, 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, 1 mM sodium orthovanadate, and proteinase inhibitors (1 µg/ml pepstatin, 2 µg/ml leupeptin, 5 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride). After pelleting insoluble material and protein standardization, the supernatants were submitted to SDS-PAGE and transferred onto an Immobilon membrane (Millipore, Bedford, MA). The membranes were subsequently incubated with the phospho-STAT3 (Tyr705) polyclonal antibody before being incubated with the second antibody labeled with peroxidase for 60 min. The reaction was visualized on an x-ray film using the ECL reagent (Amersham Biosciences, Les Ullis, France) according to the manufacturer's instructions.
Protein Radiolabeling and Binding ExperimentsBecause iodination of human CT-1 completely inactivated the biological activity of the cytokine,2 binding assays were carried out using radioiodinated murine CT-1. Comparison of mCT-1 with hCT-1 on the proliferative response of the gp130/LIFR Ba/F3 cells gave a similar specific activity of 106 units/mg. hLIF, hOSM, and mCT-1 were iodinated by the two-phase method as previously described (66). The specific activity of radiolabeled products was 100,000300,000 cpm/ng. Cells (56 x 106) were incubated with the indicated concentration of radiolabeled ligand, and the nonspecific binding component was measured by including a 100-fold excess of unlabeled cytokine. After a 90-min incubation at 4 °C, cell-bound radioactivity was separated from the unbound fraction. Determination of affinity binding constants was performed according to Scatchard (67).
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RESULTS |
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The deleted regions are part of the structural epitope of vIL-6 binding to
gp130 through site III. The -strands F and G and the linking loop
contain several residues directly involved at the binding interface
(Gln78, Thr80, Asn82, Ile83,
Asn92, Val93, Tyr94, Gly95, and
Ile96). The interaction between vIL-6 and gp130 at site III is
stabilized by seven H-bonds
(46). Four H-bonds involve
backbone-backbone interactions. Two of these H-bonds involve gp130
Asp4 and Cys6, located in the N-terminal tail of gp130,
with no equivalent residues in LIFR. Three H-bonds (vIL-6
Trp144:N
1
gp130 Asn92:O; vIL-6
Tyr32:O
gp130 His49:N
2; and vIL-6
Thr34:O
Gln78:N
2) involve side
chain-backbone interactions and participate in the specificity of the binding.
None of these H-bonds can be conserved in the LIF·LIFR complex. The
residue homologous to Trp144 is Phe, implying the loss of this
H-bond. Gp130 His49 is part of the deleted helix and
Gln78 is mutated to Val in LIFR.
The large structural reorganization at the binding epitope due to the
deletion in the -strands F and G and the loss of H-bonds prevented
straightforward prediction of residues involved in LIF binding by homology
with vIL-6. These findings prompted us to perform an analysis of the surface
properties of LIF and LIFR.
Determination of the Putative LIF Binding SiteProtein-protein complexes result from electrostatic, polar (H-bond), and hydrophobic interactions (68). These interactions require complementary shapes and physicochemical properties. To determine the putative complementary site III in the Ig-like domain of LIFR, we computed the molecular surface properties (electrostatic potential and hydrophobicity) of the LIF site III and of the LIFR Ig-like domain and searched for complementary areas (Fig. 2).
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The LIF receptor binding site III is located at the N terminus of helix D, on the "top" of the cytokine opposite to the N and C terminals. Site-directed mutagenesis has shown that residues from the N-terminal of the AB loop and from the BC loop are also part of the binding epitope (41). The core of the cytokine top corresponds to the protruding Lys159 side chain of the FXXK motif and has a very positive potential (Fig. 2A). This positively charged core is surrounded by two distinct patches of residues forming a rim around it (Fig. 2B). The first patch contains Pro51, Phe156, Val155, Leu104, Ile103, and Pro106 and forms a very hydrophobic area with a horseshoe shape. The second patch is composed of three hydrophilic residues: Glu50, Gln48, and Asn105. Among these residues, mutations of Pro51 and Pro106 to Ala have been shown to alter LIF binding to LIFR (41). Phe156 is in the trans rotameric state and is held in this orientation by neighbor Pro51 and Phe52. These three residues form a cluster of interacting residues.
The strategy used to determine the LIFR site III binding epitope was to
search an area of the LIFR Ig-like domain with physicochemical properties
complementary to those of the LIF site III, i.e. a negative core,
surrounded by two patches of hydrophobic and hydrophilic residues. The
electrostatic potential and the hydrophobicity pattern of the LIFR molecular
surface were computed and carefully analyzed. The side of the upper
sheet displays a complementary image of the LIF site III
(Fig. 2, C and
D). The negative core corresponds to Asp214.
This residue is surrounded by a hydrophobic patch with a horseshoe shape
composed of Val216, Phe284, Val282,
Gly280, and Phe279 and by a hydrophilic patch composed
of Gln213, Thr281, and Lys215. LIFR
Phe284 and Asp214 form a mirror image of LIF
Phe156 and Lys159 and are thus putative hot spots of the
binding interface. This putative binding site is located on the same sheet of
the Ig-like domain as that of the binding site of gp130. However, the two
sites do not overlap. Equivalent residues of gp130 are Ser13 and
Ile98. Only Ser13 is marginally involved in viral IL-6
site III binding (46).
Site-directed Mutagenesis of the LIFR Ig-like DomainResidues 214 and 284 in the Ig-like domain of LIFR were substituted with alanine to experimentally verify the putative binding site of LIFR. Single or double mutations were introduced in the receptor cDNA. cDNAs encoding mutated proteins were stably transfected in Ba/F3 cell lines already transfected with gp130 to generate high affinity LIF receptor. Mutant expression was then analyzed using a set of anti-LIFR monoclonal antibodies, which bind to different conformational epitopes. An example using the AN-E1 mAb is given in Fig. 3. No significant change in flow cytometry analysis could be detected, and the expression of the LIFR mutants was comparable to that of the wild type protein. Similar results were obtained using eight additional mAbs (not shown). These results indicate that the mutations did not alter the folding and did not introduce significant changes in the structure of LIFR.
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Ba/F3 Proliferation AssaysThe function of the mutated LIF receptors was assessed by determining proliferation of Ba/F3 cell lines expressing gp130 and LIFR in response to LIF (Fig. 4 and Table I). LIF induced a robust proliferation of Ba/F3 cells expressing gp130 and wild type LIFR. Introducing the F284A mutation in LIFR impaired the proliferative response of Ba/F3 cells upon stimulation by LIF with a 10-fold increase in EC50 (Table I). The D214A mutation did not impair the proliferation of Ba/F3 cells and did not significantly alter EC50. Unlike single mutations, which had no or moderate effects, the double mutation had a dramatic effect on Ba/F3 cell proliferation with an increase in EC50 larger than 50,000-fold.
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To provide further evidence about the importance of Asp214 and Phe284 for receptor function, proliferation assays were performed upon stimulation of Ba/F3 cell lines expressing gp130 and wild type or mutated LIFR by cardiotrophin-1 or oncostatin M (Fig. 4 and Table I). These cytokines use the same functional receptor as LIF. As previously observed, cardiotrophin-1 was less efficient than LIF for stimulating Ba/F3 cells transfected with wild type LIFR (Table I) (69). However, the effects of the D214A and/or F284A mutations in the LIFR Ig-like domains were similar to those observed with LIF. The D214A mutation did not induce any significant change in the proliferative response of the Ba/F3 cells induced by CT-1, whereas the F284A mutation markedly impaired cellular proliferation (100-fold increase in EC50). The double mutation totally abrogated the proliferative response (at least 10,000-fold increase in EC50).
The effect of the LIFR mutations on the response of Ba/F3 cells was markedly different when these cells were stimulated by oncostatin M. The single D214A and F284A mutations had no significant effect upon stimulation by OSM. On the other hand, the double mutation impaired cell proliferation with a 20-fold increase in EC50 (Table I). This indicates that OSM binds LIFR through the Ig-like domain.
STAT3 PhosphorylationThe dimerization of gp130 and LIFR upon binding of LIF, CT-1, or OSM induces cell activation via the recruitment of Janus kinases. Tyrosine-phosphorylated gp130 and LIFR in turn serve as docking proteins for signal transduction molecules such as STAT3 (4, 18, 70, 71). To test whether the mutations could influence the levels of STAT3 phosphorylation, we used a Western blot assay to monitor STAT3 activation in transfected Ba/F3 cells (Fig. 5). The results corroborated the data obtained with the Ba/F3 cells proliferation assay. The D214A mutation did not affect STAT3 phosphorylation in response to LIF, CT-1, or OSM. The F284A mutation affected STAT3 phosphorylation in response to CT-1, but not to LIF or OSM. This is consistent with the larger EC50 in proliferation required upon stimulation by CT-1 than by LIF (Fig. 4). The double mutation altered STAT3 phosphorylation in response of CT-1 and LIF but did not impair the response to OSM, in agreement with proliferation assays.
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Binding AffinityThe effect of the mutations in the LIFR Ig-like domain on the biological response induced by LIF, CT-1, or OSM could be due to impaired binding or signal transduction. To answer this question, we determined the dissociation constants of these cytokines for the mutated receptors. Binding of radioiodinated LIF, CT-1, and OSM to Ba/F3 cells expressing gp130 and wild type or mutated LIFR was thus carried out, and the results were analyzed according to Scatchard (67) (Table I).
The high affinity dissociation constant of LIF binding to the gp130/LIFR heterocomplex expressed in Ba/F3 cells (Kd = 250 pM) was in agreement with previously published data (51). The D214A mutation did not induce a significant change in Kd. On the other hand, the decrease in the affinity upon the F284A mutation or upon the double mutation prevented measurement of the affinity constant, indicating a Kd > 10 nM. Similar results were observed with CT-1. The high affinity dissociation constant of CT-1 was consistent with previous data (69) and was not significantly altered by the D214A mutation. The decrease in the affinity upon the F284A and the D214A/F284A mutations did not allow the dissociation constant to be measured (Kd>10 nM) (Table I).
The affinity of OSM for the gp130·LIFR heterocomplex expressed in Ba/F3 cells was similar to previously published values (72). The high affinity dissociation constant was not significantly altered upon the D214A or the F284A single mutations, with less than 2-fold changes in Kd values found (Table I). However, the D214A/F284A double mutation yielded a decrease in the affinity sufficient to prevent measurable binding, indicating Kd > 10 nM.
Molecular Docking of the LIF·LIFR ComplexThe initial assumption of an interaction between LIF Phe156 and LIFR Phe284 was consistent with the effect of the F284A mutation. However, the assumption of an interaction between LIF Lys159 and LIFR Asp214 was challenged by the experimental results obtained with the D214A LIFR mutant. Molecular docking of LIF to the Ig-like domain of LIFR using the HEX program (64) was thus carried out to get better insight into the molecular details of the interaction.
Energetically favorable Phe-Phe interactions require edge-to-face
orientations of the phenylalanine rings
(73). The rotameric
orientations of the Phe side chains are thus crucial for proper binding. The
orientation of LIF Phe156 in the crystal structure corresponds to
the trans, p rotamer (1 = -165°,
2 =
78°) and is stabilized by interactions with Pro51 and
Phe52. In this rigidly held orientation, Phe156 lies on
the protein surface. In the MODELER-built model of the Ig-like domain of LIFR,
Phe284 is positioned in the g-, p rotameric orientation
(
1 = -60°,
2 = -88°), with the
phenylalanine ring perpendicular to the protein surface.
1
x
2 isomeric mapping of LIFR Phe284 indicates
that this orientation corresponds to the single rotameric orientation of
Phe284 possible (not shown). The relative orientations of LIF
Phe156 and LIFR Phe284 are favorable for interacting.
Docking of the LIF·LIFR complex with HEX was thus carried out with the
side chains of LIF Phe156 and LIFR Phe284 in their most
stable rotameric orientation. The energy-minimized structure of the best
scoring solution is shown in Fig.
6A. In this three-dimensional model, LIFR
Phe284 is involved in
-
interactions with LIF
Phe156, with an edge-to-face geometry. The distance between the Phe
ring centroids is equal to 5.0 Å. LIFR Asp214 forms a salt
bridge with LIF Lys159. The distance between LIF Lys159
N
and LIFR Asp214 O
1 or O
2 is 2.6 Å. LIF
Glu50 interacts with LIFR Lys215 to form a second salt
bridge. Several H-bonds between the two proteins are also formed at the
interface (LIF Leu83:O with LIFR Lys209:N
; LIF
Ala26:O and Gln27:O with LIFR
Gln213:N
2) (not shown).
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When the same procedure was carried out with the D214A LIFR mutant, the
best scoring solution was very similar (root mean square deviation = 0.37
Å). Its energy-minimized structure is shown in
Fig. 6B. In this case,
LIF Lys159:N is involved in a H-bond interaction with LIFR
Thr281:O
1. This indicates that an alternative interaction
can stabilize the complex. The other interactions are conserved.
It is worth noting that HEX is a rigid body-docking program and that its success for the docking of the Ig-like domain of LIFR to LIF may be related to the fact that the interface does not involve flexible parts, but rigid surfaces requiring only minor side chain rearrangements. In particular, the side chains of LIF Phe156 and LIFR Phe284 have a single rotameric orientation possible. Their relative orientation is favorable to edge-to-face interaction without any side-chain rearrangement.
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DISCUSSION |
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These basic principles underlying complex formation were used to search the complementary site III of LIFR, in a two-step procedure. First, the putative structural binding epitope was determined by searching a surface area with physicochemical properties complementary to those of the LIF site III. Second, two residues of this area, Asp214 and Phe284, forming a mirror image of the two known hot spot residues of LIF, Phe156 and Lys159, were the best candidates for the functional binding epitope. They were mutated to alanine to verify this assumption by assaying the biological activity and the binding affinity of the single and double mutants.
The LIFR site-directed mutagenesis study presented here clearly establishes that the F284A mutation in the Ig-like domain of LIFR impairs LIF binding to the gp130·LIFR heterocomplex. The decreased efficiency of LIF for inducing the proliferation of Ba/F3 cells expressing the F284A mutant is consistent with the decreased affinity of the cytokine for the receptor. These results are in agreement with the observation that a LIFR mutant bearing the double mutation F284T and A285S had at least a 30-fold loss in affinity (49). The F284T/A285S double mutation is very disruptive, because it replaces two hydrophobic residues, Phe and Ala, by two polar residues, Thr and Ser.
The D214A mutation did not impair binding or biological activity. This
indicates either that Asp214 did not contribute to binding affinity
or that the interactions disrupted by this mutation were replaced by
alternative interactions of similar strength
(|G|
0.5 kcal/mol). The D214A/F284A
double mutation had a dramatic effect on the proliferative response of Ba/F3
cells upon stimulation by LIF. This might be due either to a gross disruption
of the LIFR structure or to the prevention of alternative interactions
possible upon the single D214A mutation.
The former explanation can be ruled out for several reasons. First, the double mutant was correctly expressed and recognized by monoclonal antibodies directed against different conformational epitopes as well as wild type or single mutant proteins (Fig. 3). Second, we verified the stability of the mutated proteins by computational mutagenesis using FOLD-X (59). The results (Table II) indicate that the mutations do not significantly alter the stability of the Ig-like domain and that the effect of the double mutation is just additive as compared with the single mutations. Third, the double mutation, albeit dramatically impairing the biological activity induced by LIF or CT-1, did not disrupt the biological response induced by OSM. The proliferative response of Ba/F3 cells expressing gp130 and the D214A/F284A LIFR mutant was still efficient upon stimulation by OSM (20-fold increase in EC50). Moreover, weak STAT3 phosphorylation upon LIF stimulation of Ba/F3 cells transfected with gp130 and the D214A/F284A LIFR mutant could be detected. This indicates that, although the affinity was strongly reduced by the double mutation, a slight recruitment of the signaling cascade remained. In turn, this corroborates the assumption that the structure of the Ig-like domain was not altered.
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The latter explanation is based on the possibility of alternative interactions upon the single D214A mutation. Examples of alternative interactions yielding no apparent effect of a single mutation were reported in the literature (78, 80). This phenomenon is usually connected to superadditivity of multiple mutations, i.e. the effect of multiple mutations is larger than the sum of the individual mutations. A single mutation may be compensated for by neighboring residues making alternative contacts at the interface. However, upon two mutations, these compensatory contacts should no longer be possible, leading to greater conformational perturbation in the complex than single mutations.
Molecular modeling of the complex between LIF and the Ig-like domain of
wild type LIFR (Fig.
6A) corroborates the initial hypothesis of a salt bridge
between LIFR Asp214 and LIF Lys159 and of -
interactions between LIFR Phe284 and LIF Phe156. During
the writing of this report, a study reporting mutations in the Ig-like domain
of LIFR increasing or decreasing its affinity for LIF was made available
(81). The three-dimensional
model of the complex between LIF and the Ig-like domain of LIFR that we
computed was used to analyze the effect of the reported mutations. LIFR
Phe279 should be involved in van der Waals interaction with LIF
Leu104, and LIFR Val282 should participate with the
hydrophobic cluster involving LIFR Phe284 and LIF
Phe156. The identified residues complete the present work and the
definition of LIFR binding site III. Analysis of the interface involving LIF
and the D214A LIFR mutant (Fig.
6B) indicates that, upon the Asp214 to Ala
mutation, the salt bridge between LIF Asp214 and LIFR
Lys159 can be replaced by a H-bond interaction between LIF
Lys159:N
and LIFR Thr281:O
1. The difference
in the dissociation constant of LIF to wild type or D214A LIFR corresponds to
a change in the binding free energy of <0.5 kcal/mol. The strength of a
salt bridge at a protein-protein interface is difficult to evaluate because of
the entropic cost of desolvation. Salt bridges may be stabilizing or
destabilizing. At protein-protein interfaces, the global balance is generally
positive with a
G of about 2 kcal/mol for correct geometry
(8284).
The strength of the H-bond is usually in the 0.52 kcal/mol range
(8486).
The similar binding free energy of salt bridge or H-bond involving LIF
Lys159 could be due to a higher desolvation energy of the charged
Asp214 and Lys159 in the salt bridge that would offset
the more favorable enthalpy. The interaction of the two Phe rings makes a
geometry favorable to the formation of the LIF
Lys159:N
LIFR Thr281:O
1 H-bond. Under
the double mutation, the absence of
-
interaction should prevent the
correct geometry and thus the formation of this H-bond, yielding a LIFR mutant
with strongly reduced binding affinity, which is indeed observed.
Comparison with Oncostatin M and
Cardiotrophin-1Cardiotrophin-1 and oncostatin M share with LIF the
capability to induce biological response through the gp130·LIFR
heterocomplex. However, the sequential process yielding complex formation is
different. LIF and CT-1 first bind LIFR, forming the so-called "low
affinity" complex (22,
51). This first event is
followed by the recruitment of gp130, which induces the formation of the
"high affinity" complex. On the other hand, OSM first binds gp130,
forming a low affinity complex before recruiting LIFR or OSMR in a high
affinity complex (19,
23). The inability of OSM to
directly bind LIFR indicates that its affinity for LIFR is much lower than
that of LIF or CT-1. The difference in the dissociation constants of the low
affinity (Kd 10-8 M)
and the high affinity complexes (Kd
10-10 M) is about 100. Thus, the free energy of binding
is about 10 kcal/mol for the first interacting receptor, but only 3 kcal/mol
for the second interacting receptor.
The CT-1 response to LIFR mutations was similar to that of LIF. The activity and the binding constant were not altered upon the D214A mutation, whereas the biological response was impaired upon the F284A mutation and totally abrogated upon the D214A/F284A double mutation. These results clearly indicate that CT-1 binds LIFR through the Ig-like domain and that the binding mechanism is similar to that of LIF. This is consistent with the very similar sites III of the two cytokines (Fig. 7). In addition to the conserved FXXK motif (Phe168 and Lys171), most residues of the AB loop N-terminal part (Leu51, Gln52, Gly53, Asp54, and Pro55) and of the BC loop (Leu111, Asn112, and Pro113) are conserved or type-conserved. Phe56 is also conserved. Conservation of Pro55 and Phe56 allows CT-1 Phe168 to be held in the same orientation as LIF Phe156. Nonconserved residues are at the periphery of the interface (Glu110, Arg114, Gly166, and Pro169). This strongly suggests that the formation of the cytokine·LIFR complex with subnanomolar affinity requires very conserved structural properties of the interface. Site-directed mutagenesis of LIF has shown the involvement of some of these residues in the interaction (Pro51 and Pro106) (41). The effect of these mutations on the affinity or on the biological activity is, however, reduced (<10-fold), as compared with the effect of the F156A and K159A mutations. This suggests that these residues may have an indirect role for the correct positioning of residues involved in the interaction rather than a direct role in the interaction.
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The effect of the D214A/F284A double mutation on the biological activity of oncostatin M and on its affinity for its receptor clearly indicates that OSM interacts with the Ig-like domain of LIFR. However, the binding mechanism should be different from that of LIF or CT-1, because the D214A and F284A single mutations of LIFR had no significant effect on the affinity or the biological activity of OSM and that the double mutated receptor was still able to induce efficient BAF proliferation and STAT3 phosphorylation, upon OSM stimulation. Fig. 7 shows residues conserved in oncostatin M as compared with LIF. In addition to the FXXK motif (Phe160 and Lys163), the few conserved or type-conserved residues are located at the N-terminal part of the AB loop (Gln38 and Gly39), at the C-terminal part of the CD loop (Asp158 and Ala159), and at the N-terminal part of helix D (Gln161 and Arg162). The large insertion in the BC loop yielding an additional helix (Ala95-Gly102) should make steric hindrance to binding. This is consistent with the much smaller free energy of binding observed for the second interacting receptor in the high affinity complex (LIFR binding to the OSM·gp130 complex) than for the first interacting receptor (LIFR binding to LIF or CT-1). In a study aimed to find the binding epitope of the Ig-like domain of G-CSF receptor, Layton et al. (37) observed that single mutations in the Ig-like domain of the granulocyte-colony stimulating factor receptor did not impair binding or biological activity but that double mutations were required to observe an effect. They explained this behavior by the low additional change in free energy upon the second receptor binding, which should be shared by several residues at the interface, corresponding to weak interactions.
The interaction of OSM with the Ig-like domain of LIFR corresponds to a weak affinity binding interface, and the molecular details must be different from those observed for CT-1 or LIF, corresponding to a much stronger affinity interface. Both the F284A and the D214A mutations do not impair the interaction of OSM with LIFR. Contrary to LIF or CT-1, LIFR Phe284 does not constitute a hot spot for OSM binding, which is consistent with weaker interactions (19). OSM Phe160 is not held in an efficient geometry for interacting with LIFR Phe284 by a cluster of aromatic residues, which may weaken the free energy of binding due to entropic cost. The additional helix in the BC loop of OSM (44) should also be destabilizing. The effect of the double mutation, however, indicates that Asp214 and Phe284 contribute to the binding free energy and are part of the OSM binding epitope in the Ig-like domain of LIFR.
In conclusion, LIF, CT-1, and OSM share an overlapping binding epitope
located in the Ig-like domain of LIFR and involving the Phe284 and
Asp214 residues. The behavior of OSM can be related to its lower
affinity for LIFR, implying a binding interface where interacting residues
have not a geometry optimized for strong interactions. On the other hand, for
LIF and CT-1, the Phe284 and Asp214 pair corresponds to
the binding hot spot. These residues should be involved in salt bridge and
-
interactions with the site III FXXK motif. Such
interactions are frequently observed in cytokine-receptor complexes and, more
generally, in protein-protein complexes. Examples are given by the
IL-4·IL-4R
complex
(87,
88) and by the p35·p40
complex (89). These
interactions yield high free energy of binding when their geometry is
optimized by favorable environment of neighbor residues.
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FOOTNOTES |
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Supported by a fellowship from the City of Angers.
Supported by a fellowship from Ministère de l'Education Nationale,
de la Recherche et de la Technologie.
¶ To whom correspondence should be addressed. Tel.: 33-2-41-35-47-31; Fax: 33-2-41-73-16-30; E-mail: gascan{at}univ-angers.fr.
1 The abbreviations used are: IL-6, interleukin-6; IL-11, interleukin-11;
LIF, leukemia inhibitory factor; CT-1, cardiotrophin-1; OSM, oncostatin M;
CNTF, ciliary neurotrophic factor; CLC, cardiotrophin-like cytokine; LIFR, LIF
receptor; OSMR, OSM receptor; Ig-like, immunoglobulin-like; CBD, cytokine
binding domain; FnIII, fibronectin type III; mAb, monoclonal antibody; STAT3,
signal transducers and activators of transcription; m, mouse; h, human.
2 H. Plun-Favreau, E. Lelièvre, and H. Gascan, unpublished
results.
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ACKNOWLEDGMENTS |
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REFERENCES |
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