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PROTEINS: Structure, Function, and Genetics 29:528–544 (1997)
Theoretical Investigation of IL-6 Multiprotein
Receptor Assembly
M.C. Menziani, F. Fanelli, and P.G. De Benedetti*
Dipartimento di Chimica, Università di Modena, Modena, Italy
ABSTRACT
Interleukin-6 (IL-6) is a multifunctional cytokine that regulates cell growth,
differentiation, and cellular functions in many
cell lineages. Recently, evidences for the formation of an active hexameric complex with an
IL-6:IL-6Ra:gp130 stoichiometry of 2:2:2 have
been obtained by different experimental approaches. Analysis of the electrostatic potential complementarity between IL-6 and its receptors has been used, in this study, to guide
the assembly of homology-based 3D models of
the components. The results strongly support a
mechanism whereby the active cytokine (IL-6:
IL-6Ra) associates with the signal transducing
gp130 protein, and the trimeric complex formed
further dimerizes to form the hexameric species. Furthermore, computational simulations
of the multiprotein complexes provide a rationalization of data from mutation experiments
and highlight some key protein–protein interactions which have not yet been the subject of
mutagenesis studies. Proteins 29:528–544,
1997. r 1997 Wiley-Liss, Inc.
Key words: cytokines; IL-6; IL-6 receptor complexes; electrostatic potential; homology modeling; molecular dynamics
INTRODUCTION
Interleukin-6 (IL-6) is a multifunctional cytokine
that regulates cell growth, differentiation, and cellular functions in many cell lineages.1 The biological
effects of IL-6 are initiated by its binding to the
extracellular domain of a specific cell surface receptor (IL-6Ra). This low-affinity (1029 M) complex2
constitutes the active cytokine,3 which associates
with the extracellular domain of the signal trasducin
gp130 protein forming the high-affinity (10210 to
10211 M) ternary complex.4 Signal trusduction requires homodimerization of gp130,5 which takes
place via disulfide linkage in the extracellular portion of the receptor, and consists of the activation of
tyrosine kinases of the Jak/tyk family.6 The information is next relayed by the phosphorylation of STAT
family transcription factors.6
Recently, evidences for the formation of an active
hexameric complex with an IL-6:IL-6Ra:gp130 stoichiometry of 2:2:2 have been obtained by different
r 1997 WILEY-LISS, INC.
approaches7,8 The nucleation of hexameric complexes could be a common biological strategy for
cytokines that share structural or functional similarities with IL-6, using the common signaling chain
gp130, for example, ciliary neutrophic factor (CNTF),
oncostatin M (OSM), leukemia inhibitory factor (LIF),
cardiotropin, and IL-11.9
On the basis of structural comparisons, these
ligands have been predicted to belong to the longchain helical cytokine superfamily,10 which is characterized by four antiparallel a helices of ,25 residues
and the presence of a short helical region in the long
AB loop. The prediction has been confirmed, either
by x-ray crystallography or NMR, for several members of this family, including granulocyte colonystimulating factor (G-CSF), growth factor (GH), and
very recently, LIF, CNTF. The similarities observed
in the structure of the cytokine ligands is mirrored in
the extracellular portion of their cell-surface receptors.11 Both IL-6Ra and gp130 belong to the hemopoietin receptor superfamily (class I). The most striking
feature of these receptors is the conservation of two
similar cytokine binding domains containing a pattern of conserved disulfide bonds and a WSXWs
motif located just outside the TM domain.12 The
crystal structure of some members of this family,
such as growth factor (GH), prolactin (PRL), and
erythropoietin (EPO) receptor complexes,13–15 shows
that both receptor binding domains are related in
the overall topology to fibronectin type III domains,
folding in b barrels of seven antiparallel b strands.
The extracellular region of IL-6Ra consists of one
Ig-like domain and two cytokine-receptor domains,
while the extracellular part of gp130 is composed of
six fibronectin type III modules, but only the second
and the third modules form a cytokine receptor
domain.16
Recently, molecular modeling-guided mutagenesis
studies, based both on the 3D structure of the G-CSF
cytokine and on the GH complex paradigm, allowed
the identification of several regions of protein–
Contract grant support: CNR; Contract grant support: Ministero dell’Università e della Ricerca Scientifica.
*Correspondence to: Dr. P. G. De Benedetti, Dipartimento di
Chimica, Università di Modena, V. Campi 183, 41100 Modena,
Italy.
E-mail: deben@unimo.it
Received 17 January 1997; Accepted 15 May 1997
THEORETICAL MODELS OF IL-6 HEXAMERIC COMPLEXES
protein interactions. According to these findings,
three sites are involved in the interaction between
IL-6 and its receptors:
1. Site 1 is formed by the C-terminal portion of helix
D and by part of the AB loop and interacts with
the IL-6Ra receptor. 17–20
2. Site 2 is formed by a limited number of exposed
residues on helices A and C and interacts with a
gp130 chain.21,22 The location of these sites on the
IL-6 surface corresponds to the one of the functionally equivalent sites 1 and 2 on GH.13
3. Site 3 is a composite binding site for a second
gp130 chain and encompasses residues at the
beginning of helix D and the initial part of the AB
loop.23–28 Furthermore, the IL-6Ra has been proposed to interact with the signaling gp130 chain
by using residues in the E2 strand and AB2
loop.29
Additional protein–protein contacts have been
identified by random30 and rational31 mutagenesis of
the hIL-6Ra receptor, while no experimental information is yet available on the sites of gp130 involved in
the multiprotein receptors assembly.
From these studies, details on the topological
location of each component in the putative multimeric complex emerged, and the hypothesis that two
‘‘GH-like’’ IL-6:IL-6Ra:gp130 trimers flank each other
with inverted orientations, was reported.8
Starting from this hypothesis we constructed a
three-dimensional model of the IL-6 hexameric complex. The assembly of the homology based 3D models
of the different components was guided by a detailed
analysis of the electrostatic potential complementarity. Moreover, molecular simulations of the multiprotein complexes were carried out in order to highlight
molecular determinants for ligand binding and receptor association on the basis of the topological, conformational, and dynamics features of the critical contact residues.
METHODS
Homology Modeling
Sequences of the hormones and receptors were
extracted from the EMBL protein sequence database. The crystallographic structure of the human
(h), bovine (b), and canine (c) G-CSF (PDB entries
1rhg, 1bgc, and 1bgd, respectively), of the mouse (m)
LIF (PDB entry 1lki) and of the hGH receptor
complex (PDB entry ghhr) were obtained from the
Brookhaven Data Bank.32 Secondary structure predictions were achieved with the Rost and Sander
method33 at the Internet address Predict
Protein@Embl-Heidelberg.de.
Homology modeling was performed by using the
program MODELLER.34 The sequence alignment
facility implemented in the program THREADER35
was used to fit the hIL-6 sequence into the 3D
529
structure of b, and cG-CSF. The alignments obtained, together with the structural alignment prepared by multiple least-squares superposition implemented in the MALIGN3D command of the
MODELLER program34 were used to calculate 3D
models for the hIL-6 sequence containing all mainchain and side-chain heavy atoms. Manual modifications of previously reported alignments29 were used
for the IL-6Ra and gp130 receptors.
Briefly, the program MODELLER derives many
distance and dihedral angle restraints on the target
sequence from its alignment with the template 3D
structures. Then, the spatial restraints and the
energy terms enforcing proper stereochemistry are
combined into an objective function, and the final
models are obtained by optimizing the objective
function in cartesian space employing methods of
conjugate gradients and molecular dynamics with
simulated annealing. The generation of several models for the different components of the IL-6 hexameric complex were obtained by randomizing the
cartesian coordinates, allowing a deviation of 64 Å.
Refinement and Analysis of the 3D Structures
Energy minimization and molecular dynamics
studies were carried out using the software package
QUANTA version 4.1 (Molecular Simulations, Inc,
200 Fifth Avenue, Waltham, MA 02154) implemented on a Hewlett-Packard 720 workstation. Molecular mechanics and molecular dynamics calculations were performed using the program CHARMM
(version 24).36
The minimization procedure consisted of 50 steps
of steepest descent, followed by a conjugate gradient
minimization until the root-mean-square (rms) gradient of the potential energy was ,0.001 kcal mol21
Å21. The united atom force-field parameters, a 12-Å
nonbonded cutoff and a dielectric constant of 80 (see
Results and Discussion section) were used. The
minimized coordinates of the hormones, receptors,
and binary and ternary complexes were used as
starting point for dynamics. During dynamics the
lengths of the bonds involving hydrogen atoms were
constrained according to the SHAKE algorithm,37
allowing an integration time step of 0.001 ps. The a
helices conformation was preserved by means of the
nuclear Overhauser effect (NOE) utility provided by
the CHARMM program, which allows the imposition
of constraints between the backbone oxygen atoms of
residue i and the backbone nitrogen atoms of residue
i 1 4, whereas the b-sheet structure was maintained
by applying NOE constraints between opposite
strands.
The structures were thermalized to 300 K with
5°C rise per 6000 steps by randomly assigning
individual velocities from the gaussian distribution.
After heating, the systems were allowed to equilibrate until the potential energy versus time was
approximately stable (34 ps). Velocities were scaled
530
M.C. MENZIANI ET AL.
by a single factor. An additional 10 ps period of
equilibration with no external perturbation was run.
Time-averaged structures were then determined over
the last 100 ps of each simulation. Data were collected every 0.5 ps.
Interaction energies at the monomer–monomer
interfaces and at the receptor binding site were
computed by using a dielectric constant e 5 4r.
Solvent accessible surface area and buried surfaces
were determined with a probe radius of 1.4 Å. The
fraction of the side chain accessible to the solvent is
defined with respect to the maximum accessibility of
each amino acid side chain calculated for a fragment
GLY-X-GLY.
The Protein Health utility and the 3D Profile
Analysis program38 implemented in QUANTA,
WHAT IF39 and PROCHECK40 programs were used
to verify the models obtained.
Electrostatic Calculations
The electrostatic potentials were calculated by
numerically solving the finite difference linearized
Poisson-Boltzmann equation using an incomplete
Cholesky preconditioned conjugate gradient method
as implemented in the University of Houston Brownian Dynamics (UHBD) program, version 4.0.41 The
charges of the ionized groups were assigned assuming standard amino acid protonation state at pH 7.
His166 of hIL-6Ra was considered in a protonate
state. The OPLS parameter set42 with the radii of
hydrogen atoms set to 1.2 Å, was used. The ionic
strength of 145mM follows a Boltzmann distribution
at 300K, and the dielectric constant for the protein
interior and surrounding solvent were 2 and 78,
respectively. A rolling probe with radius of 1.4 Å was
used to determined the solvent-solute dielectric
boundary and the dielectric boundary smoothing43
was implemented. Electrostatic boundary conditions
were set using the single Debye-Hückel sphere approximation for the hormones receptors.41 A 1003
grid with a 1 Å spacing was used for the calculation
of the molecular electrostatic potentials (MEP) of the
hormone and receptors. Details of the MEP at critical sites were calculated by the focusing method.
According to this method, the potentials from a
coarse (1003 grid, 1 Å spacing, in this work) are used
to set the boundary potentials for a second more
finely spaced grid centered on the region of interest.
Grid spacing was set to 0.25 Å and the grids were
centered at the atomic coordinates of the Ca atom of
residue hIL-6 158 and hIL-6Ra 135 for site 1, hIL-6
100 and hgp130 63 for site 2, hIL-6(A) 33 and
hgp130(B) 47 for site 3, hIL-6(A) 24 and hIL-6(B) 91
for site 4, hIL-6Ra 119 and hgp130 114 for site A and,
finally, hIL-6Ra(A) 114 and hgp130(B) 167 for site B.
RESULTS AND DISCUSSION
Three months after the submission of our manuscript to this Journal the 1.9 Å crystal structure of
hIL-6 was published.44 Although the coordinates are
not yet publicly available, the structure description
given in the paper gave us the opportunity of comparing the results of the homology modeling procedure
with the experimental structure. Therefore, in the
revision of the manuscript we added to the original
sections on modeling, refining and assessment of the
hIL-6 homology model a new section dedicates to the
comparison with the newly determined x-ray structure.
Modeling of hIL-6: Template Selection
Among the long-chain cytokine subgroup the 3D
structure of hGH, bG-CSF, cG-CSF and hG-CSF,
mLIF and hCNTF have been solved by crystallographic or NMR techniques (the coordinates of the
last one are not publicly available). The structural
homology observed between these cytokines is not
echoed in any strict conservation of key residues or
disulfide bonds. However, previously reported21 multiple sequence alignment between IL-6 and G-CSF
from different species shows that the cysteine residues implicates in the disulfide bridges in G-CSF are
conserved in IL-6 as well as the overall pattern of
hydrophobic and hydrophilic amino acids within the
a-helical regions. Moreover, a very similar alignment is obtained by fitting the sequence of IL-6 onto
the 3D structure of G-CSF by means of the program
THREADER.35 The program, principally designed to
solve the inverse folding problem, allows the alignment of a sequence onto a single structure for
modeling purposes by using a set of statistically
determined pairwaise potentials. The alignment of
human IL-6 onto bovine and canine G-CSF significantly differs in the zone of the AB loop, as shown in
Figure 1. The manual alignment of the human
G-CSF 3D structure onto bovine G-CSF and of the
mouse IL-6 sequence onto the human IL-6 sequence
is also reported in Figure 1.
The structural restraints derived by the G-CSF
proteins were exploited for the generation of different 3D models by the program MODELLER.34 Thus,
the sets of models A and B were constructed by
taking the structure of bG-CSF and cG-CSF, respectively, as a template and the alignment of the
sequences reported in Figure 1 as input. The sets of
models C-E were based on a multiple structural
alignment of bG-CSF, cG-CSF and mLIF which was
prepared by multiple least-squares superposition, as
implemented in the MALIGN3D command.34 The
spatial features are transferred from the bG-CSF
and mLIF to the hIL-6 target, in model C and from
the cG-CSF and mLIF, in model D. The number of
restraints on the structure represented by model E is
derived by using bG-CSF cG-CSF and mLIF contemporaneously. The structural alignment obtained for
model C is reported in Figure 2.
The crystal structure of hG-CSF was not considered in the modeling because large parts of the AB
THEORETICAL MODELS OF IL-6 HEXAMERIC COMPLEXES
531
Fig. 1. Threader-based fitting of the sequence of human (h)
IL-6 onto the 3D structure of canine (c) and bovine (b) G-CSF. The
human G-CSF 3D structure is manually aligned onto bovine
G-CSF, and of the mouse IL-6 sequence is aligned onto the human
IL-6 sequence. Coordinate sets of G-CSFs from the PDB were
used, and the entry name are 1rhg, 1bgc, and 1bgd for human,
bovine, and canine, respectively. Helical secondary structure
elements as determined by x-ray are shown as boxes enclosing
the appropiate residues. Helical secondary structure assignments
for IL-6 obtained according to the Rost and Sander methods33 are
shown as shadowed residues. Stretches of residues not resolved
by x-ray are in lowercase.
Fig. 2. Structural alignment of hIL-6 onto bG-CSF and mLIF,
derived by MODELLER multiple least-squares superposition.34 X
represents stretches of residues not resolved by x-ray. Helical
secondary structure elements as determined by x-ray are shown
as boxes enclosing the appropiate residues. Helical secondary
structure assignments for IL-6 obtained according to the Rost and
Sander methods33 are shown as shadowed residues.
and CD loops are yet unresolved. mLIF was added to
the single target model in the attempt of improving
the modeling of the CD loop. In fact, the G-CSF CD
loop is four residues shorter and, being a flexible
region, a stretch of seven amino acids is unresolved
in X-ray structures from different species. Therefore,
a long insertion was necessary in hIL-6 model A,
with negative consequences on the accuracy of the
model in this region. On the contrary, the CD loop in
mLIF has a well-defined single main chain conformation throughout its length.45
Fifteen conformations of hIL-6 were calculated for
each A-E set of models by randomizing the Cartesian
coordinates. The representativity of each randomized conformation is usually judged within the
MODELLER framework by the value of its objective
function. Because of the low percentage of sequence
similarity between the template structures and the
hIL-6 sequence, and because of the presence of
unresolved stretch of structure in the templates this
criterium was not strictly followed for the choice of
the best randomized conformation within each A-E
532
M.C. MENZIANI ET AL.
TABLE I. Quality Scores for the hIL-6, hIL-6Ra and hgp130 Models and for the Structures Used as Templates
LIGANDS
3D-Profile
Models
hIL-6 model A
hIL-6 model B
hIL-6 model C
hIL-6 model D
hIL-6 model E
Templates
bG-CSF
cG-CSF
mLIF
Scores
WHAT IF
RECEPTORS
Scores
3D-Profile
WHAT IF
PROCHECK
34.52
32.40
34.40
19.54
27.11
20.57
20.62
20.73
20.66
20.73
20.05
0.05
20.01
20.02
20.08
41.36
40.63
38.19
20.36
20.56
20.50
0.10
20.08
0.10
PROCHECK
Models
hIL-6Ra
hgp130
26.92
22.38
21.41
21.20
20.21
20.17
Templates
hGHR
25.97
21.05
20.40
Notes. The minimum 3D-Profile score for a correctly aligned sequence within a given within a given structure is seven. According to
WHAT IF models are classified as follows: perfect . 20.5, very good or good 20.5 4 21.0, normal 21.0 4 22.0, very poor 22.0 4 23.0,
bad ,23.0.
PROCHECK score for a structure solved at 1.5Å resolution is within 20.5 and 0.3.
set of models. Independent checks were preferred
such as: a) evaluation of the overall fold and sidechain packing of the models provided by the 3D
profile program38 in Quanta, b) the quality factor
furnished by the WHAT IF program,39 which assesses how normal or abnormal each side chains
environment is with respect to the average packing
environment for all the residues of the same type in
highly resolved PDB structures, and c) several assessments of the structure’s overall quality offered by
PROCHECK40 and synthesized in the quality geometry factor indicator, which measures how atypical
each residue is as far as its covalent geometry and
dihedral angles are concerned, with respect to the
‘‘typical’’ distribution defined by the PDB structures.
The results for the best randomized conformation of
each set of models are summarized in Table I. A
comparison with the scores obtained for the template
structures, which are also listed in Table I, shows
that pretty good models are, in general, obtained in
this modeling exercises, although the percentage of
similarity between the sequences is very low. According to this analysis, and to the inspection of the
3D-Profile plots, model A is the one with the lowest
chances of occurrence of misfolded regions, furthermore, it achieves the best agreement with the experimental data, as shown in the following section.
Refinement and Evaluation of the hIL-6 Model
All hydrogen atoms were added to the heavy atom
of model A and optimization of the side chain packing
was carried out by CHARMM minimization keeping
the backbone atoms fixed in their original position.
Then the entire molecule was allowed to relax and
the resulting structure was subjected to a 150 ps
dynamic run. The average structure obtained was
further minimized. The refinement procedure followed was previously optimized on the crystal structure of the GM-CSF.46
A substantial improvement in the side-chain packing and hydrogen bonding network is obtained after
refinement. Moreover, in the final model 96% of the
residues resides in the most favoured area of the
Ramachandran plots. Only Arg86 is in a generously
allowed region. This residue contributes to the packing of the BC loop against helix A and helix D by
forming a charge reinforced hydrogen bond with
Ser29 and a salt bridge with Asp142.
Analysis of the Model
Compatibility of the hIL-6 model with the
experimental structural information
available prior to structure determination
Table II summarizes the most significant results
obtained by site-directed mutagenesis studies on
hIL-6. The structural location of the mutated residues, which is also reported in the Table, allows to
distinguish between the probable candidates for
intermolecular interactions and for ligand architecture. Mutated residues, listed in Table II are reported in bold in the text.
Inspection of the fractional solvent accessibility
(SAS) and of the average atomic fluctuations (rms) of
the residues during dynamics gives an estimation of
site exposure, and consequently, of reactivity propensity. Human IL-6 presents two potential glycosilation sites,49 Asn27 and Asn126. They are exposed
residues located, in the 3D model, at the end of helix
A and in the middle of the CD loop, respectively; the
fraction of side chain exposed to the solvent is
greater than 50%, and the average rms fluctuations
of the side chains are of the order of 2 Å.
Furthermore, in chemical modification studies51
the reactivity of Metionines for the oxidation reaction was found to decrease in the order Met31 Met99
Met166 Met143 Met49. These residues are located
in the AB loop (Met31 and Met49) in helix C (Met99)
and in helix D (Met166 and Met143) and, a part for
THEORETICAL MODELS OF IL-6 HEXAMERIC COMPLEXES
533
TABLE II. Binding Properties of hIL-6 Mutants and Structural Location of Mutated
Residues. Residues Listed in This Table are in Bold in the Text
Residues
Arg12A
Glu33A
Lys36A
Leu133V
Leu140V
Met143A
Leu147V
Leu149V
Arg150M
Phe152L
Phe155A
Leu156V
Gln157D/N/A
Ser158R
Ser159R/K/P/I
Leu160R/Q/Y
Arg161K/N/G/A/P/S/W
Ala162T/R/P/F/L
Leu163P/V
Arg164G/L/W/A
Gln165H/E
Gln157W,Gln165H
Gln157I,Ser158R,Gln165A (IRA)
Met166K
Residues
Tyr13D,Gly17F,Ser100R,Val103D
(DFDR)
Gln141E, Thr144P
Leu39A
Leu39D,Glu41F,Asn42W,IRA
DFRD,Leu39D,Glu41F,Asn42W,
Gln57Y,Ser58K,IRA
Trp139R,Asp142R
Thr144D
IL-6Ra
binding
Ref.
<
<
<
5
5
<
5
<
<
>
<
<
<
>
<
<
<
<
<
<
<
<
>
<
24
24
24
19
19, 47
18, 47
19, 47
19
18
48
49
19
17
17
48, 17
48, 17
48, 18, 17, 49
48, 17
17, 19
48, 49
20
20
20
18
sgp130
sgp130
binding dimerization
activity
activity
Met31, the amount of SAS decreases in a parallel
degree with that of the oxidation reactivity. The
reactivity features of Met31 can be explained by the
high mobility (average rms fluctuations: 2.26 Å) of
its side chain during dynamics which compensates
for the small amount of SAS found in the minimized
average structure of hIL-6.
Moreover, 1H-NMR studies provided information
on the mutual spatial positions of Trp139, Met143,
His146 and Met166.47 Trp139 is positioned, in our
model, at the beginning of helix D, it packs against
the sulfurs of Met31 and Met143 and the hydrophobic microenvironment is completed by Leu44 and
His146.
Recently, a comparative NMR study on folding
topologies of hIL-6 and its mutants allowed the
identification of residues involved in hydrophobic
packing of the four helix bundle on the basis of the
exposed
exposed
exposed
vs. T144 and I148 (HD)
vs. L44 (AB loop)
exposed vs. AB loop
vs. AB loop
core
exposed
core
inter-helix
core
exposed
exposed
core
inter-helix
exposed
inter-helix
inter-helix
exposed
exposed
C-terminal
Ref.
Structural position
<
<
<
21, 22
50, 26
24
27
HA, HC
HD
vs. CD and BC loop
AB loop vs. L147 (HD)
AB loop
<
<
<
28
8
8
AB loop
HD
HD
vs. L133 (CD loop)
<
<
Structural position
HA
AB loop
AB loop
CDloop
HD
HD
HD
HD
HD
HD
HD
HD
HD
HD
HD
HD
HD
HD
HD
HD
HD
HD
HD
HD
structural changes observed by conservative amino
acid replacement of Leu residues with Val residues.19
Consistently, our 3D model predicts that Ile11 and
Ile18 in helix A, Leu73, Phe76, Tyr79, and Tyr82 in
helix B, Val97 in helix C, and Ile148, Leu149,
Phe152, Phe155, and Leu156 in helix D contribute
to the hydrophobic core of the cytokine, most of them
being completely buried and displaying no or very
low mobility during dynamics. Moreover, no significant structural changes in the NMR data are observed for conservative amino acid replacement of
Leu140, Leu147 and Leu163 with a Val residue.
These residues are buried in our model, but show a
significant mobility during the time evolution of the
trajectories (average rms fluctuations of 1.39 Å, 1.29
Å and 1.12 Å, respectively).
Finally, an important role in packing the AB loop
against the four helix bundle is assigned to the
534
M.C. MENZIANI ET AL.
Fig. 3. hIL-6 x-ray structure44 versus hIL-6 homology model. Secondary structure elements and
other relevant features revealed by the crystal structure (first row) and by the homology model
(second row) of hIL-6 are highlighted on the sequence as boxes enclosing the appropriate residues.
hydrophobic residues Leu44, Leu46, Pro47 and Met49
on the basis of the low tolerance to mutation of these
residues highlighted in a recent study where phagedisplayed libraries of mutants were generated by
fully randomizing four different amino acids in the
predicted AB loop.27 The role of Leu44 has been
described above, Pro47 faces Leu39 and Leu147 in
helix D, while Leu46 and Met49 are part of an
extended hydrophobic cluster together with Ile148 in
helix D and Leu 80 and Thr79 in helix B.
Compatibility of the model with the crystal
structure
A quantitative comparison of the hIL-6 model
obtained (hIL-6 model A) with the crystal structure
newly determined can not be presented here since
the IL-6 coordinates are not yet available. However,
a qualitative evaluation of consistencies and inconsistencies can be attempted.
In Figure 3, the secondary structure elements and
other relevant features revealed by the crystal structure of hIL-6 are highlighted on its sequence (first
row) as boxes enclosing the appropriate residues. In
the second row the secondary structure features of
the final IL-6 3D selected model (hIL-6 model A) are
reported. Again, boxes enclosing appropriate residues highlight important secondary structure elements.
A good agreement in the position and length of the
four principal helices is observed. The stretch of
residues at the beginning of loop B, predicted to form
a minihelix on the basis of the structure of G-CSF
from different species and of secondary structure
prediction methods,33 shows no interpretable electron density. A similar situation is observed in the
crystal structure of CNTF.52 However, in this case
the Authors forward the hypothesis that the stretch
of unresolved residues might fold in helical conformation on the basis of the comparison with the 3D
structure of LIF45 and of the amino acids conservation in their sequences. The structural elements of
the rest of the AB loop appears to be very similar in
the model and crystal structure, although the first b
turn is misplaced by one residue.
The difficulty of predicting insertions or region
with little sequence similarity with know structure
is evident in loop CD. In fact, the crystal structure
presents in the C-terminal portion of this loop a
short helix which, being an unique feature of IL-6, is
missing in the homology model. Since the target
structure is characterized by a long stretch of unresolved residues in this region (see Figure 1) the loop
has been constructed from scratch in hIL-6 model A,
whereas the structural restraints of mLIF were
exploited in hIL-6 model C (obtained by multiple
least-square superposition of bG-CSF and mLIF, see
previous section). Retrospectively, the second choice
was not correct because the CD loop of mLIF is
tightly packed against the four-helix bundle and
presents an extended conformation.45 On the contrary, a better agreement with the experimental
structure would have been obtained by considering
the secondary structure prediction results,33 shown
in Figure 1.
The structural comparison reported by Somers et
al.44 between the newly determined hIL-6 structure
and G-CSF revealed a good agreement in the packing arrangements and interhelix angles. However,
IL-6 presents a kink in the middle of helix B due to a
break in the hydrogen bonding which is not conserved in G-CSF and, as a consequence, in our
homology model.
Thus, notwithstanding differences between the
model and the experimental structure might be
THEORETICAL MODELS OF IL-6 HEXAMERIC COMPLEXES
observed in several regions once the coordinates will
be available, a careful inspection of the side chain
contacts as described in the Somers’s paper shows a
gratifying agreement. In particular consistency with
experiment is achieved in the following features: a)
the hydrophobic core is formed by residues I7, I11,
I14, I18, L21, T25, I69, L73, L80, L83, F87, A94, V97,
T101, L104, L108, T145, L149, F152, L156, S159,
and L163 (see Figure 3, for numbering system
comparison). b) Hydrogen bonds are observed between the following amino acid side chains: K111
and S4, S90 and E24, E77 and K102, R86 and D142.
c) The side chains of T101, T145 and S159 are at
hydrogen bonding distance with the main-chain
oxygen atoms of V97, Q141 and F155, respectively. d)
Hydrophobic interactions are observed between L66
and M166 and the side chains of L44, L46, P47 and
M49 point into a cleft between helix B and D (see
previous section for an extensive description).
As expected, inconsistency with the experiment is
mainly observed for the cluster of hydrophobic side
chains stabilizing the position of the E minihelix,
which corresponds to a misfolded region in the IL-6
homology model.
In summary, the following conclusions can be
drawn on the basis of a qualitative comparison
between the homology model and the newly determined 3D structure: a) the sequence alignment
obtained by means of the program Threader gives, in
this case, a satisfactory result. Although a recent
analysis of the Thread algorithm performance suggests that sequence alignments implicit in the ‘‘correct’’ threading of a sequence through a structure are
frequently incorrect,53 confidence on the alignment
result can be gained by the agreement with previously published21,45,51 multiple alignment which included sequences of other members of this family of
cytokines and by the capability of the model derived
to fit the available experimental data. b) According to
the site-directed mutagenesis information available
at present the misfolded region in loop CD is not
involved in binding or dimerization regions, on the
contrary, region of the AB loop putatively involved in
the interaction with IL-6Ra and in complex dimerization seems to be satisfactorily predicted.
Modeling of the hIL-6Ra and hgp130
Receptors
The hGH receptor was used as reference structure
for modeling the portions of the hIL-6Ra and hgp130
receptors putatively involved in the IL-6 binding and
signal transduction. The presence of four cysteine
residues in the N-terminal domains and a structural
PXPP motif in the interdomain linker, conserved in
all the three receptor chains facilitated the alignment of the first cytokine binding domain. The
alignment of the second domain can be based only on
535
motives such as VRXR and WSXWS12 not strictly
conserved among different receptors, and on secondary structure predictions. Several 3D models of the
hIL-6Ra and hg130 receptors were obtained by
randomizing the initial cartesian coordinates. Basically, the alignment recently presented by Salvati et
al.29 was used as input. Manual modifications of this
alignment were necessary in order to improve the
quality of the models in the EF loop regions of the
receptors. This was done by a trial-and-error procedure till no stretches of negative scores were found in
the Profile 3D plots. The sequence alignment which
furnishes the best models is reported in Figure 4.
The overall quality of the models obtained is
comparable to the resolution of the template hGH
receptor as shown by the 3D profiles, WHAT IF and
PROCHECK scores, reported in Table I.
The initial model of the IL-6Ra and gp130 receptors were subjected to the same refinement procedure used for the ligands.
All backbone angles of the final models obtained
after energy refinement have trans-planar v angles
and fall into allowed region of f_ c space.
The structure consistency with the available experimental data was checked. The side chains of
Asn107 and Asn131 in IL-6Ra54 and Asn4, Asn30
and Asn100 in gp1304 are putative N-linked glycosylation sites and are exposed to the solvent in the
models. Several hIL-6Ra residues involved in ligand
binding and signal transduction have been identified
by means of random30 and rational29,31 mutagenesis
studies and are listed in Table III. Mutated residues
listed in Table III are in bold in the text. A rationalization of the role of some amino acids might be
attempted on the basis of the 3D model presented. In
fact, in virtue of the 3D model obtained, a structural
role might be hypothesized for several mutants
which show impaired ligand binding. Thus, for example, mutation of Lys10 to Gln or Asp103 to Val
prevents the establishment of an electrostatic interaction between the two residues which may participate in the stabilization of the relative orientation
between the two cytokine binding domains of IL6Ra. A similar interaction is also present in the x-ray
structure of the GHR, the PRLR and EPOR complexes.13-15 Substitution of Val76 to glycine was
reported to result in decreased IL-6 binding and in
loss of biological activity. In our model Val76, located
in the F strand, is part of an hydrophobic zone
composed of Phe39, and three residues of the EF loop
Phe73, Tyr74 and Ile75. The interaction of Ser72
with the backbone of Ser77 helps to maintain the EF
loop local architecture, in agreement with the results
obtained from point mutations of the Ser71, Ser72,
Phe73, Tyr74 motif, very recently reported.31
A large hydrophobic patch forms the core of the
second domain of IL-6Ra. To this cluster contribute:
536
M.C. MENZIANI ET AL.
Fig. 4. Sequence alignment of the cytokine binding domains of hIL-6Ra and hgp130 onto
hGHR. b-Strand regions, as determined by x-ray are boxed in hGHR, and secondary structure
assignments obtained according to the Rost and Sander method33 are shown as shadowed
residues.
Trp130, Pro127, Pro102, Leu137, Ala180, Pro105,
Phe139, Trp124, Tyr143, Leu178, Ile108, Leu141,
Val122, Val176, Ile165, Val110, Leu120, Pro200
and Trp119. Ten of these residues have been mutated
and, according to the 3D model, structural disturbance might be responsible for the decreased ligand
binding capability showed by the mutants.
A low tolerance to mutation of the WSXWS motif is
common to several members of the cytokine receptor
family. Very recently the crystal structure of the EPO
15
receptor complex provided detailed insight into the
role of these residues in structure stabilization. The
b bulge of the WSXWS sequence interacts with the
F2 b-sheet of the VRXR consensus sequence, and a
stack of the aromatic side chains interleaved by
positive charged residues is formed. These p cation
system interactions are observed also in the GHR
and PRLR complexes.13,14
In our 3D model WSXWS takes part in an extended p cation system interactions consisting of
three aromatic groups (Phe151, Trp189 and Trp192)
that stack between five charged residues (Glu140,
Arg142, Arg144, Arg179 and Glu191). Two glutamine residues, Gln181 and Gln177, hydrogen bond
and orient the arginines, while Ser190 and Ser193
form hydrogen bonds with the main chain of residues
Pro104 and Pro105. Most of these residues have
been mutated and the mutants showed no IL-6
binding capability, furthermore, some of them presented a very low expression level.
Structure–function relationships of the gp130 are,
as yet, poorly characterized. The hydrophobic residues participating in the core of the second domain of
IL-6Ra and those responsible for the p cation system
interactions are largely conserved in the hgp130
receptor. Thus, a very similar packing is observed in
this portion of the two receptor models. On the
contrary, significant differences are observed in the
domain-domain interface. In particular, the absence
of the electrostatic interaction between residue 10 in
module I and residue 103 in module II, which restrict
the domain association in IL-6Ra, is only partially
compensated by a number of hydrogen bonding and
hydrophobic intermodule interactions involving
Asn10, Lys13, Tyr63, Val90, Tyr91, Val93, Ser124,
Val125, Lys176 and Lys180. Moreover, Lys92 is
completely buried at the interface and makes hydrogen bonds to main-chain oxygen atoms of residue
Cys7 and Asp88 and a p cation interaction with
Phe87. A similar situation has been recently observed in the crystal structure of the human Tissue
Factor.55
IL-6 Ternary Complex
The assembly of the trimeric complex 3D model is
based on the assumption of a ligand binding mode
similar to the one revealed by the crystal structure of
the hGH complexed with its receptor. Thus, a least
squares fit procedure was used to obtain a superposition of the conserved b strands Ca backbone of the
IL-6Ra and gp130 receptors onto the b-stands of the
hGH receptor. The Ca-carbon atoms of the IL-6 ahelices were superpositioned onto the aligned residues of the hGH. In this way, a rough overlap of the
main axis of the two hormones can be achieved, but
adjustments in the orientation of the IL-6 ligand
537
THEORETICAL MODELS OF IL-6 HEXAMERIC COMPLEXES
TABLE III. Binding Properties of hIL-6Ra Mutants and Structural Position
of Mutated Residues. Residues Listed in This Table are Reported
in Bold in the Text
Residue
Arg9Q,Lys10S
Trp20L
Pro67L,Glu68Q
Ser71Y
Ser72I/L/PR/V
Phe73H/I/Y
Tyr74/A/G/R/V
Val76G
Asp103V
Pro104A,Pro105H
Asn116D
Val122S, Trp124L
Ser133G/I/R/V
Phe134S/V
Tyr135A/I/R/V
Ser133A,Phe134V
Arg136S,Leu137I
Glu140V,Leu141H
Arg142S,Tyr143D
Val164G,Ile165M
Ile165D
His166S,Asp167G/V
His166S
Asp167V,Ala168D
Asp167G
His166S,Asp167V,Ala114D,Asn116D
Gly171D
His174Y,Val175L
Arg173A,His174I
Val176M,Gln177H
Arg179G
Arg179G,A180T
Glu183A,Phe184I
Gly187V,Glu188D
Trp189H,Ser190A
Ser190A,Ser193A
Glu191D, Trp192R
Thr199K,P200L
with respect to the two receptors were necessary to
remove the major steric overlap. Furthermore, the
mutual orientations of the three components was
adjusted in order to maximize the complementarity
of the electrostatic potentials computed on IL-6 and
IL-6Ra for the binary complex, and on IL-6:IL-6Ra
and gp130 for the ternary complex (Fig. 5). Recently,
electrostatic analysis was successfully used to distinguish between homo and hetero-oligomeric receptor
binding properties of IL-4 and G-CSF.56
Assuming standard amino acid protonation state
at pH 7, the hIL-6 is globally neutral. The net
charges of hIL-6Ra and hgp130 are 11.0e and -1.0e,
respectively, by considering only His166 of hIL-6Ra
in a protonate state and all the other histidine
IL-6
binding
<
<
<
5
5
5
5
<
<
<
5
<
5
<
<
<
<
<
<
<
<
5
5
5
<
5
<
5
<
<
<
<
<
<
<
<
<
<
IL-6
signaling
5
5<
5
<
<
<
5
<
<
<
<
<
<
<
<
<
Ref.
Structural
position
15a
15a
15a
17a
17a
17a
17a
15a
15a
15a
16a
15a
17a
17a
17a
15a
15a
15a
15a
15a
15a
15a, 16a
15a
15a
15a
16a
15a
15a
15a
15a
15a
15a
15a
15a
15a
15a
15a
15a
AB loop
B sheet
EF loop
EF loop
EF loop
EF loop
EF loop
EF loop
G-A2
G-A2
A2-B2 loop
B2 sheet
B2-C2 loop
B2-C2 loop
B2-C2 loop
B2-C2 loop
B2-C2 loop
C2 sheet
C2 sheet
E2
E2
E2
E2
E2
E2
E2
E2F2 loop
E2F2 loop
E2F2 loop
F2
F2
F2
F2-G2 loop
F2-G2 loop
F2-G2 loop
F2-G2 loop
F2-G2 loop
G2
residues as neutral. The electrostatic potential surrounding the hIL-6 ligand is asymmetric and, a part
from a region of negative potential confined to the
end of helix A, part of the AB loop and the N and
C-terminal of helix B, the rest of the surface is
covered by positive potential. The IL-6Ra receptor
presents a large region of negative potential surrounding the AB, CC’, C’E loops, the N-terminal part
of the EF loop and the hinge region connecting the
two cytokine binding domains. The second domain,
on the contrary, is mainly surrounded by a positive
potential. Intriguing is the electrostatic potential
distribution of gp130 which reflects the extreme
flexibility of its interaction surface. It presents two
different faces with respect to the main plane on
538
M.C. MENZIANI ET AL.
Fig. 5. Electrostatic isopotential surfaces for the IL-6 receptor complex and its components. The
blue and red contours represent the isopotential surface at 10.5 kcal (e·mol) and 20.5 kcal (e·mol),
respectively. Important sites for intermolecular interactions are highlight by arrows on ribbon
diagrams of the molecules.
which the two domains lie. One face (shown in Fig. 5)
is characterized by spots of positive and negative
potentials, while on the other side a large spine of
positive potential is contoured by zones of negative
potential, stretching over the F strand and the CC’
loop.
In order to characterize, in a more detailed way,
the MEP distribution on the receptor and on ligand
putative binding sites, we used the focusing method41 to describe the electrostatics in the important
regions. Table IV reports the values of the potentials
(EP), the solvent accessible surface area (SAS), the
resulting density of the potential (r) and the list of
the residues contributing to the generation of the
surfaces putatively involved in the ligand–receptor
and receptor–receptor recognition.
Site 1. Interaction between hIL-6 and hIL-6Ra
The site 1 interface of IL-6 and IL-6Ra models
shows the most significant complementarity of the
electrostatic potentials. This argues for the funda-
mental importance of the electrostatic interaction
for IL-6 receptor recognition and supports the hypothesis of the sequential assembly of the ternary complex model proposed by Taga et al..3 A description of
the interactions established by the charged residues
responsible for the complemetarity of the electrostatic potential during the dynamics simulation of
the binary and ternary complex is given in Table V.
Several intermolecular interactions involve residues
(in bold) identified to be important for binding and
dimerization activity (Tables II and III, and following text). A good agreement between the experimental evidences and the role assigned to critical contact
residues in our model is observed. Moreover, a recent
study27 aimed at improving the interaction of the
IL-6 AB loop residues with hIL-6Ra proposed, on the
basis of the consensus sequence derived from fully
randomized phage libraries, Asp53 and Phe56 to be
direct contact points to the receptor. In fact, these
residues are not tolerant to substitution. On the
contrary, a 20-fold improvement in binding of the
IL-6Ra is observed for the Lys48V, Ala50R, Glu51M,
Lys52E quadruple mutant. Accordingly, in our model,
THEORETICAL MODELS OF IL-6 HEXAMERIC COMPLEXES
539
TABLE IV. Electrostatic Potential (EP), Solvent Accessible Surface (SAS) and Density of the Potential
(r 5 EP/SAS) at the Surface Generated by the Residues Putatively Involved in Ligand-Receptor and
ReceptorReceptor Recognition
EP
kcal/(e · mol)
Site 1
hIL-6
hIL-6Ra
Site 2
hIL-6
hgp130
Site A
hIL-6Ra
hgp130
SAS
(Å)2
r
kcal/(e · mol)Å2
Residues
3480.88
1350
2.58
23497.29
1607
22.18
12, 15, 36, 48, 52–53, 56–57, 60, 150–151, 154–155, 157–158, 161–162,
164–165
38–39, 67–68, 73–76, 95, 97, 131–136, 158–159, 183–186
21450.58
2565.34
1030
1034
21.41
2.48
7, 10, 13–14, 16–17, 20–21, 91–93, 95–96, 98–100, 102–103, 105–107
11–14, 35–38, 46, 58–60, 62–65, 124
2060.11
2365.62
871
793
2.36
20.46
113–114, 116–119, 154, 163–164, 166–167, 169
104, 106–107, 112, 114, 118, 153, 154, 155–158, 160
Asp53 makes intermolecular charge reinforced hydrogen bonding interactions with the Arg9 and
Ser14 receptor residues, while Phe56 packs against
the side chains of the Pro12 and Leu13 receptor residues. In addition, the interactions established by Lys48
and Glu51 can easily be compensated and improved
by the charged mutants in position 50 and 52.
It is worth noting that, although the hydrophilic
groups are required to give specificity, the interaction is dominated by the hydrophobic forces, in fact,
more than 60% of the total interaction energy between the hormone and the IL-6Ra receptor (-364.20
kcal/mol), computed on the minimized average structure of the binary and ternary complexes, is due to
the dispersion forces as represented by the vdW
component of the total interaction energy.
A salt bridge is observed in the final model between the Arg164 residue of the IL-6 cytokine and
the Asp178 residue of the gp130 receptor. The loss of
ligand binding capability toward the IL-6Ra, observed for the Arg164A mutant (Table II) seems to be
due to structural disturbance,48 in fact, it reduces the
a-helical content by 25% as compared to that of the
wild-type molecule. In our model, besides contributing to the stabilization of the interaction with the
gp130 receptor, Arg164 interacts with Glu5, in helix
A, strengthening the packing of the four helix bundle.
Site 2. Interaction between hIL-6 and hgp130
Ligand receptor complementarity of the MEP at
site 2 is less marked but the dipolar nature of the
interface putatively involved in the interaction
showed by both ligand and receptor might be exploit
to guide the docking. At the 0.5 kcal/(e·) isopotential
surface, the gp130 receptor shows to the ligand a
positive pole around the CC’ and the second half of
the EF loops, while the AB loop, the interdomain
linker GA2, and the B2C2 loop constitute a negative
pole. On the contrary, the ligand offers to the receptor a negative surface around the middle of helix A
and a positive surface surrounds the N-terminal part
of helix A and helix C (Fig. 5).
Analysis of the final ternary complex, as resulted
from the minimized average structure, reveals just a
few specific contact points between polar residues
and, among these, Tyr13 and Ser100 have been
shown to be important for gp130 binding,21 once
combined in the quadruple mutant Tyr13D,Gly17F,
Ser100R,Val103D (DFRD). However, by taking into
account the synergistic effect of the combined mutations (the only single mutant showing a detectable
and reproducible decrease in activity is Tyr13D)21
and their nonconservative nature, it seems to be
reasonable to impute the loss of activity to structural
changes or to global modifications of the electrostatic
potential (the r values for the DFRD mutant at this
site is 1.82 kcal/(e·mol)), which prevent the correct
orientation of the active cytokine IL-6:IL-6Ra complex with respect to gp130. In this way, because of
the generous tolerant nature of the gp130 receptor,
the binding of the gp130 is not totally impaired,28 but
its efficient association with the IL6-Ra receptors is
prevented.
Site A. The hIL-6Ra:hgp130 dimerization
interface
Following the hypothesis formulated in the previous paragraph, the bridging role of the IL-6 ligand
with respect to the two receptors is a key factor for
the correct orientation of the two receptors which
results in a productive receptor–receptor interaction, as also confirmed by the complementarity of the
r index at this interface. Residues in the A2B2 loop
and E2 strand of the IL-6Ra have been identified to
be involved in stabilizing the heterodimeric receptor
on the base of a rational site-directed mutagenesis
study recently published.29
IL-6Ra presents a positive surface at this interface
with the only exception of a small spot of negative
potential around the Asp167 residue, on the contrary, gp130 presents a negative surface at this site,
which surrounds a large zone of positive potential
around the Lys114 residue. This scenario is in full
agreement with the results obtained by rational
540
M.C. MENZIANI ET AL.
TABLE V. Hydrogen Bonding Pattern for the IL-6 Trimeric Complex. Residues Identified to be Critical for
Binding by Site Directed Mutagenesis Stdies are Reported in Bold (see text)
IL-6
ARG
ARG
ARG
SER
ARG
LYS
LEU
ALA
ASN
LYS
LYS
GLU
GLU
GLU
GLU
GLU
ASP
ASP
CYS
GLN
GLN
GLN
GLN
SER
LYS
ARG
LYS
GLU
GLN
GLN
GLN
ARG
ARG
ARG
IL-6Ra
12
12
12
19
22
36
39
40
42
48
48
51
51
51
51
51
53
53
55
57
57
57
57
58
68
150
153
154
157
157
157
161
161
161
aDistances
HH11
HH12
HH21
OG
O
HZ3
O
O
OD1
H
HZ3
H
OE2
OE2
OE1
OE2
OD1
O
O
H
HE22
HE22
HE22
H
HZ2
HH21
HZ2
OE2
OE1
OE1
HE22
HE
NH2
NH2
GLU
ASP
GLU
ARG
GLN
CYS
GLN
GLN
GLN
ASN
TYR
GLU
ARG
ARG
GLN
TYR
ARG
SER
ASN
SER
PRO
HIS
SER
SER
GLU
GLY
GLU
ARG
ARG
GLU
GLU
PHE
PHE
GLU
183
158
183
138
186
97
95
95
95
41
74
68
37
37
40
74
9
14
131
11
127
128
132
11
68
185
183
9
136
183
183
134
134
183
OE1
OD2
OE2
HH21
HE21
O
HE21
HE22
HE21
O
OH
OE2
HH11
HH21
HE21
HH
HH21
HG
HD22
O
O
ND1
OG
O
OE1
O
O
HH11
H
H
OE2
O
O
OE2
DHAa
d(D-A)a
IL-6
171.50
156.57
167.30
171.45
169.40
174.56
99.51
162.90
169.36
99.44
168.54
172.65
155.07
154.71
158.44
162.02
162.70
164.11
158.66
164.49
174.07
103.43
100.29
170.59
166.42
148.60
169.78
176.75
171.86
156.33
174.02
154.10
157.21
170.86
2.07
2.38
2.05
1.90
2.10
1.93
2.57
2.25
2.06
2.77
1.92
2.14
2.30
2.38
2.57
2.10
2.05
2.19
2.18
2.11
2.05
2.91
2.53
2.46
2.03
2.43
1.92
1.99
1.99
2.11
2.29
2.19
2.09
2.20
GLU
ARG
LYS
GLN
TYR
TYR
ASP
GLU
MET
SER
SER
SER
LYS
GLN
GLN
GLN
GLN
LYS
THR
ARG
ARG
IL-6Ra
ASN
TRP
TRP
TRP
TRP
HIS
HIS
HIS
HIS
HIS
HIS
HIS
ASP
ASP
gp130
5
6
9
10
13
13
16
92
99
100
100
100
102
106
106
106
109
110
124
164
164
O
HH12
HZ2
HE21
OH
OH
OD1
O
O
OG
HG
HG
HZ1
HE22
OE1
OE1
OE1
HZ3
HG1
HE
HH22
LYS
PHE
GLU
TYR
TYR
LYS
LYS
LYS
LYS
LYS
ASP
TYR
HIS
SER
GLU
TRP
TRP
GLU
HIS
ASP
ASP
gp130
116 HD22 GLN
119 H
THR
119 HE1
GLU
119 NE1
ARG
119 NE1
ARG
161 H
ASP
162 H
ASP
162 O
ASP
166 HD1 THR
166 NE2
ARG
166 ND1 THR
166 ND1 THR
167 H
THR
167 OD2
LYS
180
64
177
63
63
176
13
46
41
13
58
59
40
35
36
37
37
36
40
178
178
HZ3
O
O
O
OH
HZ3
H
HZ3
HZ2
NZ2
O
O
O
OG
H
H
HE1
OE2
NE2
OD2
OD2
DHAa
d(D-A)a
175.1
169.5
159.8
167.4
130.4
159.6
173.5
174.8
172.7
130.5
92.2
177.9
177.3
176.7
162.1
167.4
168.7
171.4
159.0
158.7
160.6
2.02
2.22
1.95
2.33
2.90
2.94
2.16
1.99
1.90
2.90
2.98
2.04
1.98
2.10
2.30
2.08
2.06
1.98
2.55
2.35
2.22
DHAa d(D-A)a
160 O
106.0
2.57
158 O
108.1
2.75
148 OE2
157.1
2.34
154 HH21
90.9
2.74
154 HH22 110.2
2.43
149 OD1
174.7
2.23
149 OD1
165.3
2.12
149 H
167.2
2.05
158 OG1
173.0
2.02
154 HE
133.4
2.80
158 H
169.0
2.42
158 H
169.0
2.42
158 OG1
177.3
2.46
114 HZ3
167.3
2.14
(Å) and angles (deg) are averaged throughout the molecular dynamics analysis period.
site-directed mutagenesis study at this site.29 In fact,
double mutation of the His166S, Asp167V prevents
the binding with specific residues of gp130 (see Table
V), while the introduction of negative charged residues at position 114 and 116 (Ala114D, Asn116D)
might result in a significant alteration of the local
electrostatic potential (the r values for the SVDD
mutant at this site is 0.66 kcal/(e·mol)). This suggests that local, rather than global, charge complementarity controls productive interaction at this
site.
The Hexameric Complex
Rules for the assembly of the hexameric complex
are dictated by the following experimental evidences: a) contact between the two gp130 molecules
in the hexameric complex has been inferred by the
observation that tyrosine kinase activity was associated with the disulfide-linked dimeric protein;5 b) In
the examer, the IL-6 ligand establishes interactions
with two distinct gp130 chains. The first chain
interacts with ligand residues in the helix A and
C,the second chain interacts with a composite binding site formed by residues at the beginning of helix
D and the initial part of the AB loop.22–28 An equivalent region is involved in the binding of LIF and
CNTF to the LIFR chain responsible for signal
transduction through heterodimezation of gp130;57,58
c) a very recent study on the influence of IL-6
dimerization on formation of the high affinity hexameric complex shows that the increased binding
affinity of the IL-6 dimer (IL-6D) is due to its ability
to crosslink two IL-6Ra molecules, while its reduced
biological potency results from a decreased ability of
THEORETICAL MODELS OF IL-6 HEXAMERIC COMPLEXES
the IL-6D:(IL-6Ra)2 complex to obtain a productive
coupling with the soluble portion of gp130, in fact the
residual biological activity of IL-6D seems to be due
to the dimer dissociation in active monomers.59
These results strongly supports a mechanism
whereby monomeric IL-6 crosslinks IL-6R and gp130
to form a trimeric complex, two such complexes
further dimerizing to form the hexameric species.59
The tentative model of the hexameric receptor
complex assembly reported in Figure 6 best conciliates most of the experimental information available
and the constraints imposed by the complementarity
of the electrostatic potentials on the surface of the
trimeric complexes (Fig. 5). In this assembly one
trimer is rotated by an angle of about 45° with
respect to each other and the two IL-6 molecules are
faced in a head to head position (opposite the adjacent amino and carboxy termini). Although this
bears a superficial resemblance with the dimerization mode of the M-CSF and SCF cytokines10,46 the
long helices of the IL-6 are more tightly packed in
structure therefore, the interpenetration of the dyad,
which stabilized the dimeric structure of the two
short-chain cytokines,46 is prevented in this case and
only a small amount of each IL-6 surface results
buried. This confirms the hypothesis that IL-6 in
stable dimers are oriented differently to those purported to occur in the hexameric complex.59
The electrostatic potential surface computed on
the minimized average structure of the trimer
presents significant differences with respect the ones
computed on the single components. The most striking features of the electrostatic potential of the
trimer is a more diffuse positive potential over the
IL-6Ra with respect to the dimeric complex and a
significant increase of the negative potential area
associated with the complexed gp130 with respect to
the isolated one. This might constitute the driving
force for the hexameric receptor assembly and furnishes one more argument to support a dimerization
mechanism involving the two preformed trimeric
complexes.
The electrostatics in the important regions of
protein–protein interactions are reported in Table VI
and the pairs of residues which are mainly involved
in the stabilization of the complex are listed in Table
VII. Due to the size of the hexameric complex,
molecular dynamics simulations were not carried
out on the system, and the hydrogen bonding pattern
reported in Table VII refers to the minimized structure.
The interaction at site 3 occurs between IL-6 of the
first trimer (IL-6(A)) and the gp130 chain of the
second trimer (gp130(B)) and concerns, in our model,
a very limited area. However, several residues predicted to be responsible of the second gp130 chain
recruitment by site-directed mutagenesis experiments are involved in electrostatic interactions with
residues of the gp130 receptor mainly located in the
541
Fig. 6. Stereo view of the IL-6 hexameric receptor complex.
The picture was prepared by using the MOLSCRIPT program60
according to secondary structure assignment.
C’E loop and B sheet. These are the zones of the
gp130 receptor which display the greater variability
of residues among different species (human, mouse
and rat).
The double mutant Trp139R, Asp142R significantly reduces the potential density (the r values is
-1.9 kcal/(e·mol)) at this site with devastating consequences on the gp130 dimerization activity (Table
II). Thr144 is a buried residue in our model, and as
previously hypothesized,61 its substitution with a
negative charge residue might induce local distortions which, indirectly, impair the binding with
gp130(B).
Substitution of the hIL-6 stretch of residues Thr25Glu37 with the corresponding mouse residues give
rise to a IL-6 chimera with reduced biological activity. It shows selective impairment of the in vitro
binding to gp130 but not to IL-6Ra.61
According to our model, this substitution could
reduce binding both at site 3 and site 4. In fact, in the
chimera Glu33 is changed to Met, and Lys36 and
Glu37 to Asp. Thus, the ionic pairs interactions
between the Glu33 and Lys36 IL-6 residues and the
Arg16 and Glu18 gp130 residues and the charge
reinforced hydrogen bonding between the Glu37
ligand residues and the Lys48 gp130 residue are
destroyed. It is worth noticing that the nonconservative mutations which occur in position 33 and 36 of
the mouse AB loop of IL-6 are paralleled by nonconservative mutations of residues in position 16 and 18
(Arg16 to Leu, and Glu18 to Gln) in the mouse gp130
receptor, while the conservative mutation in position
37 (Glu37 to Asp) is mirrored by the conservation of
residue Lys48 in the mouse receptor.
The effect of the IL-6 chimera on the interaction
between IL-6(A) and IL-6(B) might be due, according
to our model, to the substitution of residues Lys28
and Asn30, which are involved in interactions with
Gly and Ser residues of the IL-6 partner BC loop,
respectively. Moreover, also the complementarity of
the electrostatic potential, observed at this site is
markedly reduced.
542
M.C. MENZIANI ET AL.
TABLE VI. Electrostatic Potential (EP), Solvent Accessible Surface (SAS) and Density of the Potential
(r 5 EP/SAS) at the Surface Generated by the Residues Putatively Involved in Ligand-Receptor
and Receptor-Receptor Recognition
EP
kcal/(e · mol)
SAS
(Å)2
r
kcal/(e · mol)Å2
23901.08
2581.3
558
747
26.99
3.45
30–33, 37, 41, 139, 142–143
14, 16, 47–52
2633.92
21860.52
1153
850
2.28
22.18
19–20, 22–24, 27–30, 86–93
24, 28, 84–85, 87–89, 91–94
2342.17
21951.44
1100
1115
2.13
21.75
142, 144–148, 151, 173, 175, 177, 192–194, 195–198
136–139, 165–167, 169, 189–195
Site 3
hIL-6(A)
hgp130(B)
Site 4
hIL-6(A)
hIL-6(B)
Site B
hIL-6Ra(A)
hgp130(B)
Residues
TABLE VII. Hydrogen Bonding Pattern for the IL-6 Exameric Complex. Residues
Identified to be Critical for Binding by Site Directed Mutagenesis Studies
are Reported in Bold
IL-6(A)
CYS
GLU
GLU
SER
LYS
LYS
GLU
GLU
TRP
ASP
ASP
TRP
IL-6(A)
LYS
LYS
LYS
LYS
ASN
SER
SER
GLN
GLN
SER
SER
SER
SER
IL-6Ra(A)
GLU
ARG
ARG
ARG
LYS
LYS
GLU
aDistances
DHAa
d(D-A)a
O
O
HH12
HH11
OE2
O
HZ1
HZ3
HH11
HH11
HH12
HH21
140.81
175.91
162.36
170.2
170.9
170.10
167.48
127.39
156.13
103.81
96.51
148.50
2.73
2.10
2.25
3.00
2.50
2.49
2.07
2.18
1.97
2.69
2.80
2.16
d(D-A)a
1.98
2.13
2.06
2.01
2.05
1.95
2.13
2.42
2.36
2.59
2.23
2.14
2.29
d(D-A)a
2.11
2.03
2.04
2.16
1.92
2.02
2.53
gp130(B)
32
33
33
34
36
36
37
41
139
142
142
139
H
H
OE1
OG
HZ2
HZ1
OE2
OE2
O
OD1
OD1
O
ASP
ASP
ARG
ARG
GLU
PRO
LYS
LYS
ARG
ARG
ARG
ARG
50
50
16
16
18
52
48
48
49
49
49
49
23
23
28
28
30
89
89
93
93
89
89
89
89
HZ1
HZ2
HZ2
HZ2
HD22
HG
H
H
HE21
OG
HG
OG
OG
IL-6(B)
SER
GLU
GLU
GLU
GLU
PHE
SER
GLU
GLU
SER
GLU
SER
GLU
89
24
92
92
92
87
89
88
88
89
24
90
91
OG
OE1
OE1
OE1
OE2
O
O
OE1
OE1
H
OE1
H
H
DHAa
171.88
169.58
173.73
172.98
171.30
177.72
171.66
164.33
118.21
102.99
178.30
175.33
163.93
45
142
142
144
149
149
195
OE2
HH11
HH21
HH21
HZ2
HZ3
OE2
gp130(B)
THR
GLU
GLU
GLU
VAL
TYR
LYS
25
188
188
167
103
195
137
HG1
OE2
OE1
OE1
O
OH
HZ1
DHAa
166.21
177.83
174.86
164.28
173.10
177.05
159.47
(Å) and angles (deg) are computed on the minimized structure.
The predicted contact residues of the IL-6Ra of the
first trimeric complex IL-6Ra(A) at the interface
with the gp130 chain of the second gp130(B) (site B)
are located on strand C2, loop C2C2’ and loop F2G2.
Some of the IL6Ra(A) residues involved in the
interaction have been mutated. Double mutation of
residues Arg142S, Tyr143D gives ambiguous results
probably due to structural disturbance,30 while a
THEORETICAL MODELS OF IL-6 HEXAMERIC COMPLEXES
triple mutant Arg147G, Ser148A, Lys149G shows
decreased signal trunsduction, ,50% of normal.
Moreover, an additional contact point, established
between residues Glu45 and Thr25 in the first
cytokine binding domains, is observed between the
two receptors.
It is interesting to note that all the residues of the
gp130 chains involved in the receptor–receptor interactions in both site A and B are conserved among
species (human, rat and mouse).
The assembly does not allow direct contact between the two gp130 chains. Thus, the hypothesized
homodimerization of the receptor,5 according to our
model should take place in one of the three membral
proximal fibronectin type III modules. Indeed three
cysteine residues are found in this portion of the
sequence: Cys419, Cys458, and Cys466 (the numbering corresponds to the full sequence).54
6.
7.
8.
9.
10.
11.
CONCLUSIONS
Three-dimensional molecular modeling and computational simulations on hIL-6, hIL-6:hIL-6Ra and
hIL-6:hIL-6aR:hgp130 allow the identification and
the quantitative analysis of the molecular determinants for structure stabilization and ligand–receptor
recognition. Confidence in the models obtained is
provided by the concurrence between key residues
highlighted by the models and results from mutagenesis studies.
Moreover, analysis of the electrostatic potential
complementarity of the complex components supports a stepwise mechanism whereby IL-6 recognizes and binds the IL-6Ra to form the active
cytokine IL-6:IL-6Ra. This associates with the signal
transducing gp130 protein and the trimeric complex
formed further dimerizes to form the hexameric
species.
Furthermore, the model of the hexameric complex
provides a rationalization of data from mutation
experiments and predicts some key protein–protein
interactions which might be the targets of future
experimental investigations.
12.
13.
14.
15.
16.
17.
18.
19.
ACKNOWLEDGMENT
Financial support from CNR and Ministero
dell’Università e della Ricerca Scientifica (funds
40%) is acknowledged.
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