Activation of gp130 by HHV-8 Interleukin-6

doi:10.1016/j.jmb.2003.10.070
J. Mol. Biol. (2004) 335, 641–654
Molecular Mechanisms for Viral Mimicry of a Human
Cytokine: Activation of gp130 by HHV-8 Interleukin-6
Martin J. Boulanger1, Dar-chone Chow1, Elena Brevnova1
Monika Martick1, Gordon Sandford2, John Nicholas2 and
K. Christopher Garcia1*
1
Departments of Microbiology
and Immunology, and
Structural Biology, Stanford
University School of Medicine
Fairchild D319, 299 Campus
Drive, Stanford, CA
94305-5124, USA
2
The Molecular Virology
Laboratories, Sidney Kimmel
Comprehensive Cancer Center
Johns Hopkins University
School of Medicine, Baltimore
MD 21231-1000, USA
Kaposi’s sarcoma-associated herpesvirus (KSHV, or HHV-8) encodes a
pathogenic viral homologue of human interleukin-6 (IL-6). In contrast to
human IL-6 (hIL-6), viral IL-6 (vIL-6) binds directly to, and activates, the
shared human cytokine signaling receptor gp130 without the requirement
for pre-complexation to a specific a-receptor. Here, we dissect the biochemical and functional basis of vIL-6 mimicry of hIL-6. We find that, in
addition to the “a-receptor-independent” tetrameric vIL-6/gp130 complex, the viral cytokine can engage the human a-receptor (IL-6Ra) to
form a hexameric vIL-6/IL-6Ra/gp130 complex with enhanced signaling
potency. In contrast to the assembly sequence of the hIL-6 hexamer, the
preformed vIL-6/gp130 tetramer can be decorated with IL-6Ra, post facto,
in a “vIL-6-dependent” fashion. A detailed comparison of the viral and
human cytokine/gp130 interfaces indicates that vIL-6 has evolved a
unique molecular strategy to interact with gp130, as revealed by an almost
entirely divergent structural makeup of its receptor binding sites. Viral
IL-6 appears to utilize an elegant combination of both convergent, and
unexpectedly divergent, molecular strategies to oligomerize gp130 and
activate similar downstream signaling cascades as its human counterpart.
q 2003 Elsevier Ltd. All rights reserved.
*Corresponding author
Keywords: gp130; molecular mimicry; KSHV/HHV-8; viral pathogenesis;
protein –protein interactions
Introduction
Microbes have an extraordinary capacity to
interfere with host protein functions in order to
create conditions favorable for survival. This process can manifest itself in the form of “molecular
mimicry”, whereby a pathogen, such as a virus,
mimics the structure and/or function of an
endogenous host protein.1 Viruses often express
homologues of host cell signaling proteins, such as
M.J.B., D.C. & E.B. contributed equally to this work.
Present addresses: D. Chow, Department of
Chemistry, University of Houston, 229 Fleming Building,
Houston, TX 77204, USA; E. Brevnova, Department of
Medicine, University of California, San Diego, School of
Medicine, 200 W. Arbor Drive, San Diego, CA 921038382, USA.
Abbreviations used: ITC, isothermal titration
calorimetry.
E-mail address of the corresponding author:
[email protected]
cytokines and cytokine receptors, to either
antagonize a receptor– ligand interaction that may
result in destruction of the virus, or activate a signaling process that will stimulate proliferation of
the infected cell and amplification of the virus.2,3
Despite the importance of such occurrences to
microbial pathogenesis, our knowledge about the
structural biology of molecular mimicry of a viral
receptor– ligand interaction is derived from only a
few examples.4,5 Here, we examine in detail both
the structural and functional basis of mimicry of
the human immunoregulatory cytokine interleukin-6 (hIL-6) by Kaposi’s sarcoma-associated
human herpesvirus 8.
Human herpesvirus 8 (HHV-8) is a g-herpesvirus that has been linked to several lymphoproliferative disorders and malignancies, such as
Castleman’s disease, primary effusion lymphoma,
and Kaposi’s sarcoma.6 – 8 Like some other
g-herpesviruses, HHV-8 has pirated an extensive
array of host genes that are able to hijack various
cellular processes to benefit its viability.9,10 One of
0022-2836/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.
642
these pirated genes in HHV-8 is a homologue of
hIL-6, termed viral IL-6 (vIL-6).11,12 Expression of
vIL-6 in HHV-8 associated malignancies is known
to induce cell proliferation and angiogenesis, and
is thus suspected to play a role in the pathogenesis
of this virus.13 – 15
VIL-6 induced signaling occurs via the shared
cytokine signaling receptor gp130 that is coupled
to the endogenous JAK/STAT pathway.16 – 19 The
crystal structure of the vIL-6 signaling complex
shows that two vIL-6 molecules homodimerize
two gp130 receptors to form a tetramer (Figure
1).20 Each vIL-6 molecule presents two topologically distinct binding epitopes termed sites II and
III. Site II is used to engage the D2D3 domains of
gp130, also known as the cytokine binding homology region or CHR, while site III binds the D1
or IGD domain of an opposing gp130 molecule
completing the tetramer. No structural intermediate of the vIL-6/gp130 complex has been
observed and thus the mechanism of assembly
remains unknown.
The molecular assembly of the hIL-6 signaling
complex is more intricate, requiring an additional
specific a-receptor (IL-6Ra) not required for vIL-6
signaling.16,21 In addition to sites II and III used in
the v-IL6-dependent oligomerization of gp130,
hIL-6 incorporates an additional epitope termed
site I that engages IL-6Ra.22,23 Furthermore, studies
have shown that the assembly of the hIL-6 signaling complex proceeds in a coordinated sequence
with distinct intermediates24,25 (Figure 1(A)). In the
first step, hIL-6 interacts with IL-6Ra through its
site I to form a bimolecular complex, which then
interacts with gp130 CHR domain, resulting in a
trimolecular intermediate.25 The final step in the
assembly of the hexamer is the cross-linking of
these heterotrimers through the D1, or Ig-like
domains (IGD) of gp130. The disposition of all
three receptor binding epitopes, including two
unexpected additional “composite interfaces”
between IL-6Ra and gp130 were revealed in the
recent crystal structure of the hexameric hIL-6
receptor complex26 (Figure 1(C)) and established
the basis for the required pre-complexation
between hIL-6 and IL-6Ra. The conserved core
topology between the vIL-6 and hIL-6 structures
combined with functional data showing that vIL-6
blocks anti-viral interferon-a signaling27 leading to
down-regulation of IL-6Ra surface expression
indicates that vIL-6 has evolved into an effective
mimic of hIL-6.
Here, we report biochemical, biophysical and
functional studies describing the basis of the
“mimicry” of hIL-6 by the viral homologue and
the contested role of IL-6Ra in vIL-6 signaling. We
find that vIL-6 is, indeed, capable of utilizing
IL-6Ra as part of an “enhanced” signaling complex, which appears to contradict a number of
published reports.16,28,29 Our biochemical analysis
of the assembly of the vIL-6 signaling complexes
reveals both a “convergent” strategy utilizing
IL-6Ra to produce a hexameric complex similar to
Viral Mimicry of a Human Cytokine
the human cytokine, and the previously described
“divergent” IL-6Ra-independent strategy. We find,
from detailed comparative analysis of the viral
and human IL-6/gp130 interfaces, that each
cytokine utilizes a unique structural solution for
binding gp130, with vIL-6 having undergone a
“hydrophobic enhancement” of its receptor binding sites in a manner akin to affinity maturation of
an antibody.30 As an aggregate, our studies show
that viral IL-6 has evolved a highly diverse and
unexpected, but extremely effective, set of
strategies to activate gp130, rather than simply
copying the hIL-6 structural determinants.
Results
A key component enabling the studies described
here was the availability of the crystal structures of
the vIL-6/gp130 tetramer20 and the hIL-6/IL-6Ra/
gp130 hexamer,26 which were both determined
recently in our laboratory (Figure 1(C)). These
structures give us a unique opportunity to undertake a comparative analysis of a mammalian cytokine receptor complex and its viral mimic. A
structural superposition of the vIL-6/gp130 tetramer onto the hIL-6 and gp130 components of the
hIL-6 hexamer clearly shows a conserved core
structural scaffold composed of two gp130
molecules lying anti-parallel with each other, and
bridged by two cytokine molecules (Figure 2). A
closer examination reveals that the global fit of the
gp130 structures occurs throughout the site II and
site III regions (Figure 2). However, while the site
I is bound to IL-6Ra in the hIL-6 hexamer, this analogous region is exposed and accessible in the vIL6 tetramer. In principle, it is sterically capable of
binding to IL-6Ra using a topological blueprint
similar to that of the human hexamer. On the
basis of this observation, we asked whether
additional higher-order signaling assemblies exist
for the viral cytokine, as for its human counterpart.
We expressed a series of soluble constructs of
vIL-6, gp130 and IL-6Ra in insect cells and determined the molecular masses by analytical ultracentrifugation. IL-6Ra was expressed as a twodomain construct (D2D3, 30.5 kDa), lacking the
N-terminal domain (D1), which is known to be
unnecessary for complex formation and signaling
of hIL-624 (Figure 3(A)). Although vIL-6 alone can
be expressed at low concentrations for functional
studies, high-level expression of this recombinant
protein has been unsuccessful, as observed
previously,20 likely due to the extensive hydrophobic surface of vIL-6. A soluble vIL-6/gp130
complex formed by co-expression eluted as a
single complex peak from a gel-filtration column
(Figure 3(B)) with molecular mass of 120 kDa, consistent with the formation of a tetramer (theoretical
mass 131.5 kDa) with two copies each of vIL-6 and
gp130 (D1-D3), as observed in the crystallized
complex.20 We then chromatographed the purified
vIL-6/gp130 tetramer with saturating amounts of
Viral Mimicry of a Human Cytokine
643
Figure 1. Assembly mechanism of human and viral IL-6 signaling complexes. (A) hIL-6 initially engages its specific
receptor (IL-6Ra) through its site I to form a binary complex, which then engages gp130 at the elbow formed by the
D2 and D3 domains through its site II binding epitope, generating an intermediate trimolecular recognition complex.
In the final stage, the site III of hIL-6 engages the D1 domain of gp130, resulting in the formation of a hexameric
activation complex. (B) Signaling via the vIL-6 activation complex is independent of an a receptor. The activation complex consists of a tetramer with two copies each of vIL-6 and gp130 D1-D3. (C) Schematic and CPK models of the
atomic structure of vIL-6/gp130 (D1-D3)20 and hIL-6/IL-6Ra/gp130(D1-D3)26 signaling complexes as seen from a
side-view. CPK models were generated with MOLSCRIPT49 and Raster3D.50
IL-6Ra (Figure 3(C)), and the mass of the peak was
determined to be , 197 kDa, consistent with the
formation of a vIL-6 hexamer (theoretical mass of
196 kDa) (two copies each of vIL-6, gp130 and IL6Ra) similar in stoichiometry to the hIL-6 signaling
complex.25,26 Thus, it is quite clear that the vIL-6
tetramer is capable of incorporating IL-6Ra into a
hexameric assembly with high affinity. We then
asked whether this biochemical result is
functionally relevant.
IL-6Ra can influence signaling by vIL-6
To investigate the effect of IL-6Ra on vIL-6 signal
transduction, we carried out transient transfection
acetylation assays in HEK293T cells in which
gp130 and vIL-6 were co-expressed with a STATresponsive CAT reporter, pa2M-CAT. In this assay,
signal transduction is measured as the conversion
of substrate ([14C]chloramphenicol) to acetylated
forms, which are separated by thin-layer chromatography, and radioactivity in the “spots” counted
to obtain the percentage acetylation.31 An increase
in signaling was detected in the three-way
co-expression of vIL-6, gp130 and IL-6Ra relative
to the co-expression of vIL-6 and gp130 alone
(Figure 4(A)). Furthermore, by systematically
increasing the amount of IL-6Ra expressed we
644
Viral Mimicry of a Human Cytokine
Figure 2. Top view of the vIL-6/gp130 (D1-D3) tetrameric20 and hIL-6/IL-6Ra/gp130(D1-D3) hexameric26 signaling
complexes and a least-squares superposition based on the gp130 molecules showing the overall conserved tetrameric
core between the two complexes. A zoomed-in view showing the structural conservation of site II, formed between
the A and C helices of human and viral IL-6 and the D2 and D3 domains of gp130 and site III formed by the tip of
the four-helix bundle of human and viral IL-6 and the D1 domain of gp130. Gp130 in the vIL-6 and hIL-6 complexes
are colored gold and blue, respectively. vIL-6 is colored red, hIL-6 green and IL-6Ra colored cyan.
showed that the activity of vIL-6 was enhanced in
a dose-dependent manner (Figure 4(B)) by IL-6Ra,
consistent with its functional participation in vIL-6
signal transduction.
As it has been reported that vIL-6 can effect an
autocrine induction of hIL-6 in certain cell lines,
which would complicate interpretation of the previous results,32 we tested for vIL-6-induced, or constitutive, hIL-6 expression in HEK293T cells. These
data (Figure 4(C)) confirm that no detectable or
functionally significant levels of hIL-6 were present
in our vIL-6-transfected HEK293T cultures (see
Materials and Methods). Therefore, the ability of IL6Ra to enhance vIL-6 signaling is due to direct effects
on vIL-6-induced signaling complex formation.
The vIL-6 hexamer assembles through a
divergent mechanism
The gel-filtration experiments, combined with
the functional assay, indicate that IL-6Ra is biochemically and functionally incorporated into the
vIL-6 signaling complex. However, neither experiment addresses the question of assembly mechanism. Is IL-6Ra binding to a preformed tetramer of
vIL-6 and gp130? Or is the interaction resulting
from a reorganization and re-equilibration of the
three-component complex, resulting in a hexamer?
Our previous experiment (Figure 3) suggests that
assembly of the vIL-6 hexamer does not follow the
sequential pathway used by hIL-6 (Figure 1(A)),
as the viral tetramer lacking IL-6Ra is stable: hIL-6
has no measurable interaction with gp130 unless
initially pre-complexed with IL-6Ra. Despite
several attempts to co-express vIL-6 with IL-6Ra,
we were unable to demonstrate bimolecular complex formation (data not shown), which would represent the analogous first step (IL-6 þ IL-6Ra) in
the assembly of the hIL-6 hexamer. To test whether
IL-6Ra was able to “decorate” a pre-formed vIL-6/
gp130 tetramer, we used gel mobility-shift assays
with native gel electrophoresis (Figure 5(A)). On
the native gel, the vIL-6/gp130 tetramer migrates
as a focused band, while IL-6Ra runs as a series of
diffuse bands possibly due to conformational or
glycosylation heterogeneity. The addition of
increasing amounts of IL-6Ra to the vIL-6/gp130
tetramer resulted in a distinct band shift that is
saturated at a twofold molar excess of IL-6Ra, consistent with an assembly mechanism in which two
molecules of IL-6Ra decorate a preformed vIL-6/
gp130 tetramer to form the hexamer (Figure 3(C)).
Dissecting the vIL-6 hexamer: a
thermodynamic characterization
of
To further solidify this unexpected mechanism
IL-6Ra interaction, we characterized the
Viral Mimicry of a Human Cytokine
645
Figure 3. Biochemical and biophysical characterization of IL-6Ra, vIL-6/gp130 (D1-D3), and vIL-6/IL-6Ra (D2-D3)/
gp130 (D1-D3). Each panel contains a chromatogram from an FPLC Superdex200 gel-filtration column, SDS-PAGE of
the peak fractions and an analytical ultracentrifugation plot. (A) IL-6Ra (D2-D3) was purified as a single homogeneous
peak with molecular mass of 30.5 kDa. (B) Co-expression of vIL-6 and gp130 (D1-D3) resulted in a purified complex
with molecular mass of 120 kDa, consistent with a tetramer with two copies each of vIL-6 and gp130. (C) Mixture of
IL-6Ra and tetramer of vIL-6 and gp130 (D1-D3) produced a complex with molecular mass of 197 kDa consistent
with a hexamer with two copies each of IL-6Ra, vIL-6 and gp130 (D1-D3).
thermodynamics of IL-6Ra interaction with the
vIL-6 tetramer, using isothermal titration calorimetry (ITC), which measures the free energy of an
interaction ðDGÞ and the component enthalpy
ðDHÞ; from which entropy can be derived. Figure
5(B) shows representative ITC traces obtained by
titrating the tetrameric vIL-6/gp130 complex
against IL-6Ra at different temperatures. At 5 8C
(278 K), the interaction between IL-6Ra and the
pre-formed vIL-6/gp130 tetramer shows an
unfavorable enthalpy of þ 6.2 kcal/mol (1 cal ¼
4.184 J), and favorable entropy of þ 58 cal/(mol K).
The large favorable entropy is consistent with the
expulsion of water molecules that would be
expected from the hydrophobic surface of vIL-6.
Overall, the interaction has a Gibbs free energy
ðDGÞ of 2 10 kcal/mol and a binding affinity of
KD , 22 nM: In order to assess the extent of
enthalpy – entropy compensation, we increased the
temperature to 26 8C (299 K), which shifts the
thermodynamic profile of the reaction to be
enthalpy driven ðDH ¼ 27:1 kcal=molÞ with a
significantly smaller, yet still favorable, entropic
contribution ðDS ¼ 12:3 cal=ðmol KÞÞ: Importantly,
the binding stoichiometry of IL-6Ra titrated into
the tetrameric complex of vIL-6/gp130 shows that
two IL-6Ra receptors bind to each vIL-6/gp130
tetramer consistent with our previous biochemical
646
Viral Mimicry of a Human Cytokine
Figure 4. Influence of IL-6Ra on signal transduction by vIL-6. STAT-CAT reporter based transient transfection assays
in HEK293T cells were used to measure signal transduction by vIL-6. (A) The increase in signaling of vIL-6 in the presence of IL-6Ra. (B) The dose-dependency of increasing IL-6Ra expression vector (0.25, 0.50 and 0.75 mg; empty vector
(pEFBOS) was used as the negative control and to normalize DNA totals) on vIL-6 signaling. (C) Western blot analysis
for the detection of hIL-6 in transfected cell culture media. Human IL-6 levels were correlated with reporter activation
by transfecting cells with 0.25, 0.75 and 1.50 mg (lanes b, c and d; lane a represents the empty vector) of cytokine
expression vector and quantifying hIL-6 in cell media (40 ml) against rhIL-6 standards (0.1, 1.0 and 10.0 ng; lanes h, i
and j). Functionally significant levels of hIL-6 were not present in the vIL-6-expressing cell cultures, ruling out the
possibility that effects of IL-6Ra are due to induced hIL-6 (lanes d, e and f).
data (Figures 3 and 5(A)). Thus, the viral hexamer
assembles into a faithful mimic of the human
hexamer.
To determine whether protonation/deprotonation events occur during assembly of the vIL-6
hexamer, as has been suggested for hIL-6 and
IL-6Ra, we measured the interaction between the
vIL-6/gp130 tetramer and IL-6Ra in several
different buffer systems that display a range of ionization enthalpies. The plot of the apparent
enthalpies versus buffer ionization enthalpies
shows a linear relationship with a slope of approximately 2, consistent with two protons liberated or
absorbed per hexamer, or one per unique site I
binding epitope (Figure 5(C)). These measurements
are consistent with structural observations where a
conserved arginine residue is centrally disposed on
helix D (Figure 7(A), blue residue) of the site I
binding epitope of vIL-6.
These titrations were also measured at several
different temperatures in order to calculate the
heat capacity DCp (Figure 5(D)), which is a measure
of exposure, or burial, of hydrophobic surface in a
polar solvent. In the absence of complete structural
information, buried surface can be calculated
based on the empirical relationships between heat
capacity DCp ¼ 0:45Aapol 2 0:26Apol and enthalpy
DH ¼ 28:44Aapol þ 31:4Apol :33 Using these parameters, we calculated the total buried surface
area between the site I binding epitope of the
˚ 2. The
vIL-6 tetramer and IL-6Ra to be , 2000 A
overall buried surface is somewhat higher compared to normal buried surface area for typical
rigid body interactions.34 This suggests that IL6Ra contacts additional epitopes on the tetramer
other than vIL-6, possibly in a similar fashion
observed in the hIL-6 hexamer26 where the upper,
or D2 domain, of IL-6Ra contacts (buried surface
˚ 2) the D1 domain of gp130 to form
area , 500 A
site IIIb (Figure 6(C)). Importantly, titrations of IL6Ra against free soluble gp130 (D1-D3) showed no
detectable interaction (data not shown). Hence,
any additional contact between IL-6Ra and gp130
is only enabled by the presence of the cytokine.
Therefore, we devised a strategy to test the
possible role of this IL-6Ra – gp130 interaction,
within the viral hexamer, using functional assays
and mutagenesis.
Rescue of site III mutation by IL-6Ra
Our strategy was to mutate a central site III
interface residue on vIL-6, and then ask whether
the addition of IL-6Ra would “rescue” this interaction, presumably through site IIIb. Our experiments utilized functionally abrogated, site IIImutated, vIL-6, vIL-6.15 (Trp144 mutated to
Gly). Trp144 forms the major hydrophobic contact with gp130 D1 (Figure 6(A)),20 and therefore
is required for stable interactions between vIL-6
Viral Mimicry of a Human Cytokine
647
Figure 5. (A) Native gel binding characterization of IL-6Ra with the preformed vIL-6/gp130 (D1-D3) tetramer. A
constant amount (3 mg) of vIL-6/gp130 (D1-D3) complex was mixed with an increasing amount of IL-6Ra. The leftmost and rightmost lanes serve as controls containing only monomeric IL-6Ra and the vIL-6/gp130 tetramer, respectively. The second lane from the right shows that most of the tetramer complex was shifted to the higher molecular
mass complex at a stoichiometric ratio of vIL-6/gp130 to IL-6Ra (D1-D3) of approximately 23 pmol:43 pmol or 2:1. Isothermal calorimetric titrations of the preformed vIL-6/gp130 (D1-D3) tetramer complex against IL-6Ra. (B) Titration
curves are shown for the vIL-6/gp130 complex into IL-6Ra at 5 8C (278 K) and 26 8C (299 K). The curves are fit with a
least-squares algorithm for a one-site binding model with the program ORIGIN 5.0. (C) A plot of apparent enthalpy
versus the buffer ionization enthalpy at 26 8C showing that approximately two protons are liberated when two molecules of IL-6Ra bind the preformed vIL-6/gp130 tetramer. The ionization enthalpy for the different buffers are as follows: Hepes buffer (filled triangle), 4.99 kcal/mol; a mixture of 10 mM cacodylate and 20 mM Tris buffer pH 7.6 (filled
diamond) has a calculated composite ionization enthalpy of 3.2 kcal/mol; Pipes buffer (open triangle), 2.72 kcal/mol;
cacodylate buffer pH 7.2 (filled circle), 2 0.47 kcal/mol. (D) A plot of DH versus temperature for experiments carried
out in three different buffer systems with the same data point designations described above. The average change in
heat capacity ðDCp Þ derived from the slope of each curve is 2 510 cal/mol (contact site)/K.
site III and gp130 IGD. We reasoned that if
IL-6Ra can form part of the signaling complex
and stabilize vIL-6/gp130 hexamers, then IL-6Ra
co-expression with gp130 and the inactive
vIL-6.15 site III mutant in cell culture might
enable significant signal transduction. Consistent
with previous data,19,35 vIL-6.15 was unable to
effect efficient activation of a STAT-CAT reporter
in HEK293T cells over-expressing gp130, whereas
wild-type vIL-6 activated the reporter by around
19-fold (Figure 6(B)). However, over-expression
of IL-6Ra along with gp130 and vIL-6.15 allowed
activation of STAT signaling to around 50% of
that achieved with vIL-6 under the same conditions (Figure 6(B)). These data suggest
strongly that IL-6Ra can form part of the vIL-6induced signaling complex and compensates in
large part for the weakened site III-IGD interactions affected by the Trp144Gly substitution in
vIL-6.15.
648
Viral Mimicry of a Human Cytokine
Figure 6. (A) Ribbon diagram of the vIL-6 site III:gp130 IGD interaction interface showing the position of the altered
Trp residue in site III-altered variant vIL-6.15 (Trp144Gly). (B) Reporter-based transfection assays were carried out using
vIL-6.15 to determine the functional effects of IL-6Ra on signal transduction through over-expressed gp130: 0.5 mg of
each receptor subunit expression vector was used per transfection. Rescue of vIL-6.15 by IL-6Ra indicated stabilization
of gp130:vIL-6.15 complexes by the a-subunit. (C) Zoomed-in view showing site IIIb in the hIL-6 hexamer26 formed by
the D1 domain of gp130 and the D2 domain of IL-6Ra.
Discussion
The ability of viruses to mimic host cell signaling
proteins, such as cytokines, has emerged as an
important paradigm in viral pathogenesis. HHV-8
encodes a homologue of human IL-6, known as
viral IL-6 that mediates its effects through the
shared signaling receptor gp130,21,35 but does not
require pre-association with an a receptor.
Although some studies have suggested a role for
human IL-6Ra in vIL-6 signaling,18,19,35,36 several
recent studies have been unable to show functional
participation of IL-6Ra association with vIL-6 and
gp130.16,28,29 From the biochemical and biophysical
analysis presented here, we have clearly shown
that IL-6Ra can be incorporated into a vIL-6/IL6Ra/gp130 hexameric complex and that the
addition of IL-6Ra enhances vIL-6 induced
signaling.
With these biochemical and functional data in
hand, we can revisit the crystal structures to
establish the structural basis for the vIL-6 molecular mimicry of hIL-6. Using our superimposed
model of the vIL-6 tetramer20 onto the hIL-6
hexamer,26 we were able to identify structurally
conserved residues of the site I epitopes of human
and viral IL-6 (Figure 7(A)). The most striking
observation is a conserved centrally disposed arginine residue (Arg179 on hIL-6, and Arg166 on
vIL-6, blue residues),37 – 39 which, when mutated in
hIL-6, is known to ablate receptor binding.39
Furthermore, mutation of Arg166 (blue residue) in
vIL-6 abrogates signaling of an engineered “IL6Ra-dependent” vIL-6 variant.19 The participation
of Arg166 on the site I of vIL-6 in the binding to
IL-6Ra is also consistent with the thermodynamic
data presented here showing that a proton is
liberated or absorbed per site I binding interface.
Interestingly, the affinity between IL-6Ra and the
vIL-6/gp130 tetramer ðKD , 20 nMÞ is similar to
that of IL-6Ra and hIL-6 ðKD , 21 nMÞ; consistent
with the viral and human cytokines sharing a key
similar centrally disposed hotspot residue involved
in IL-6Ra-binding.
Chemistry of the cytokine mimicry
The overall surface chemistries of the receptorbinding epitopes of human and viral IL-6 are, however, very different from one another, despite the
contact residues occupying similar positions in the
sequence alignment (Figure 8(A)). Thus, the two
cytokines appear to have evolved unique binding
chemistries to interact with gp130, rather than the
virus simply copying the hIL-6 receptor binding
mode, as seen for a cmvIL-10 homologue.5 The
site II residues of vIL-6 are strikingly hydrophobic
(Figure 7(B), green surface). Amidst the vIL-6 site
Viral Mimicry of a Human Cytokine
Figure 7. Structural comparison showing the contact
residues of the site I, II and III binding epitopes of
human and viral IL-6. (A) vIL-6 cytokine is shown in
green and hIL-6 in purple. For vIL-6, the site I residues
predicted from our IL-6Ra docking studies are shown.
Residues colored in blue contribute the most buried
surface area at each molecular interface as calculated
from the Protein – Protein Interaction (PPI) server
(http://www.biochem.ucl.ac.uk/bsm/PP/server). The
remaining residues that form the molecular interface are
colored yellow. The site I residues shown for vIL-6 are
colored orange because they were identified on the
basis of a docked model with IL-6Ra following a leastsquares superposition of the conserved cores of the
vIL-6/gp130 tetramer20 and the hIL-6/IL-6Ra/gp130
hexamer.26 (B) Same view as in (A) but a surface
representation showing the chemical nature of the
interface residues of human and viral IL-6 with
hydrophobic residues colored as green surface and
polar residues colored as red surface.
II epitope, two bulky tryptophan residues (Trp18
and Trp21, Figure 7(A), blue residues) contribute
30% of the total buried surface area in complex
with gp130, and form a broad hydrophobic patch
on helix A (Figure 7(A) and (B)). The site II epitope
of hIL-6 is significantly more polar (Figure 7(B), red
surface) with a single, centrally disposed tyrosine
residue (Tyr31 Figure 7(A), blue residue), which,
from mutational studies is known to play an
important role in receptor binding.40 Despite different surface chemistries of the site II epitopes of
human and viral IL-6 (Figure 7(B)), these cytokines
contact primarily the same core shared region on
gp130 (Figure 8(B), red surface mapped on gp130
D2D3). The most prominent buried contact residue
on gp130 engaged by the site II of human and viral
649
IL-6 is Phe169, which is known to be critical to
ligand engagement for all gp130-cytokines,36,41 – 43
followed by the shared residues Val170 and
Trp142 for vIL-6,33 and Ser165 for hIL-6
(Figure 8(B)). Overall, the unique surface area of
gp130 utilized by each cytokine amounts to no
more than 10% of the total buried surface in the
site II molecular interfaces (i.e. 90% shared).
The site III of both human and viral IL-6 incorporates tryptophan (hIL-6, Trp157; vIL-6, Trp144;
Figure 7(A), blue residues) and leucine (hIL-6,
Leu57; vIL-6, Leu41; Figure 7(A), blue residues) as
the most buried residues when bound to gp130,
with Trp144 having been our target residue to
establish the functional incorporation of IL-6Ra
into the vIL-6 hexamer (Figure 6). Unlike the site
II binding site, however, the site III binding inter˚ 2) bury sig˚ 2) and vIL-6 (484 A
faces of hIL-6 (789 A
nificantly different amounts of surface area when
bound to the D1 domain of gp130 (Figure 8(B)).
Overall, hIL-6 buries an additional 50% of surface
area relative to vIL-6. Furthermore, although both
cytokines use a core shared region on the gp130
D1 domain, they rely more heavily on unique
patches of gp130, especially for hIL-6, where more
than one-third of the buried surface area maps to
unique contact residues on gp130 (Figure 8(B),
gp130 D1 red surface shared, blue surface unique
to hIL-6, and green surface unique to vIL-6). Thus,
while the site II of gp130 is largely shared, the site
III on the D1 domain is largely unique in their
interactions with the viral and human cytokine.
From these structural observations (Figures 7
and 8), we can conclude that the site II and III
epitopes of vIL-6 use extensive hydrophobic surfaces to engage gp130. In fact, the percentage
hydrophobic content of site II in going from hIL-6
to vIL-6 increases from 32% to 75%, respectively,
and increases from 49% to 84% at site III. Hence,
“IL-6Ra independence” of vIL-6 is undoubtedly
due to the dramatic hydrophobic enhancement of
the site II and III binding epitopes relative to
hIL-6. Thus, the parsing of energetics between the
viral and human cytokine are unique, and vIL-6
does not simply “mimic” hIL-6. This situation is
quite different from a relevant comparative
example, the complex of human cytomegalovirus
IL-10 bound to soluble human IL-10Ra.5 In that
complex, the viral cytokine uses essentially the
same structural epitope as hIL-10. The cytokine
residues contacting the receptor are identical
between the viral and human cytokines. Thus, the
cmvIL-10 is a clear case of molecular mimicry,
whereas the vIL-6 mechanism of mimicry appears
much less obvious.
In conclusion, we find that HHV-8 vIL-6 has an
unexpectedly complicated molecular mechanism
to achieve mimicry of hIL-6. Evolutionary
biologists use the terms “convergent” and “divergent” evolution to reflect whether an organism
uses a similar or different pathway to achieve a
similar functional endpoint to a related organism
or species. In the case of vIL-6, there appear to be
650
Viral Mimicry of a Human Cytokine
Figure 8. (A) Structure-based sequence alignment of human and viral IL-6.51 Green shaded boxes indicate identical
residues and residues that form the site I, II and III epitopes are designated with a 1, 2, or 3, respectively. (B) Contact
residues of the site II and III interfaces of gp130 displayed graphically on a vertical histogram showing relative buried
˚ 2). Residues colored in blue are exclusive to hIL-6 binding, residues in red are shared between hIL-6
surface area (in A
and vIL-6 and residues in green are exclusive to vIL-6. Residues listed in the histogram are also mapped onto the surface of gp130 domains D2 D3 for site II and domain D1 for site III using the same color scheme.
651
Viral Mimicry of a Human Cytokine
Figure 9. Assembly pathways of vIL-6 and hIL-6 signaling complexes. (A) Divergent signaling solution of the vIL-6
tetramer. (B) Divergent and convergent assembly pathways to form the vIL-6 hexamer. (C) Assembly pathway for the
hIL-6 hexamer.
two divergent pathways to activate gp130 relative
to human IL-6 (Figure 9): one involves vIL-6/
gp130 tetramer formation (Figure 9(A)), the other
involves the post-tetramer recruitment of IL-6Ra
into a hexamer (Figure 9(B)), which contrasts to
the cytokine pre-complexation required for human
IL-6. The possibility remains that vIL-6 also uses a
convergent mechanism during assembly of the
hexamer by complexing with IL-6Ra prior to
engagement of gp130 (Figure 9(B)). The multiplicity of assembly pathways of the viral cytokine
versus the human also endows vIL-6 with great
flexibility to activate gp130 on cells bearing IL-6Ra
in a “regulated”, or tissue-specific, fashion as well
as cells that do not express this receptor. The role
of vIL-6 in the pathogenesis of Kaposi’s sarcoma,
as well as other lymphoproliferative disorders, is
gaining increasing attention. Ultimately, the structural information about this cytokine may have
utility in the design of antagonists in an effort to
diminish the unregulated proliferative effects of
vIL-6 on tumor cells.
Materials and Methods
Expression constructs and baculovirus preparation
of vIL-6 and IL-6Ra
The DNA fragment corresponding to the vIL-6 mature
fragment was generated by PCR using the forward
primer 50 ATG CAA GGA TCC CCT CCT GGT AGA
652
ATT CCC CCC C (Bam HI site) and reverse primer 50
ATG CAA TCT AGA TCA GTG ATG GTG ATG GTG
ATG CAT TTG CCG AAG AGC CCT CAG (Xha I site).
The construct of human IL-6Ra domains D2-D3 was constructed in similar fashion with forward primer 50 ATG
CAA GGA TCC CCT CCT GGT AGA ATT CCC CCC C
(Bam HI site) and reverse primer 50 ATG CAA TCT AGA
TCA GTG ATG GTG ATG GTG ATG CAT TTG CCG
AAG AGC CCT CAG (Xha I site). All PCR products were
sequenced to verify that no mutation had been introduced.
The PCR products were digested with Bam HI and
Xha I, and subcloned into pAcGP67A (Pharmingen). Production of the recombinant baculovirus was performed
as described.20 Briefly, Spodoptera frugiperda (Sf9) cells
(Invitrogen) were transfected with a combination of the
recombinant transfer vector and linearized AcNPV
DNA (BaculoGold, Pharmingen) using CELLFectin
(Invitrogen) as described by Pharmingen (San Diego,
CA). Baculovirus containing recombinant DNA was
amplified in Sf9 cells in serum-containing Sf900 medium.
Protein expression, purification and characterization
Typical infection with baculovirus was carried out in
Insect Express medium (Biowhitaker, Walkersville, MD)
at a cell density of 1.5 £ 106/ml for three days. Baculovirus was titrated in small scale (2 ml) to optimize infection parameters prior to the large-scale (1– 6 l)
production. The medium containing the expressed
recombinant proteins was centrifuged to pellet the cellular debris, concentrated and dialyzed using tangential
flow filtration, and allowed to “batch” bind overnight
with Ni-NTA beads (Qiagen) at 4 8C on a rotating shaker.
Protein was eluted from the Ni-NTA beads with 350 mM
imidazole and the fractions visualized by SDS-PAGE.
The eluant was further purified by gel-filtration
(Amersham Pharmacia Biotech) on a Sephadex 200
column calibrated with apoferritin (443 kDa), a amylase
(200 kDa), albumin (67 kDa), carbonic anhydrase
(29 kDa), and cytochrome c (12.4 kDa). Protein concentration was determined with the bicinchninic acid
(BCA) assay (Pierce, Rockford, Il) using bovine serum
albumin as the standard. Native gel analysis of proteins
was carried out at pH 8.0 using 10 –15% (w/v) polyacrylamide gels and native gel buffer strips on the PhastSystem (Pharmacia, NJ) described by the manufacturer’s
protocol. Gels were stained with Coomassie brilliant blue.
Analytical ultracentrifugation analysis
Sedimentation equilibrium analysis was carried out in
a Beckman Optima XL-A analytical ultracentrifuge
equipped with a four-position An-50 Ti rotor and sixchannel centerpieces. Each sample was loaded at three
different concentrations with initial A280 nm of about 0.3,
0.6, and 1.2. All samples were centrifuged at 20 8C in
Hepes-buffered saline (HBS; 10 mM Hepes (pH 7.2),
150 mM NaCl). The samples were centrifuged to equilibrium at series of incremental speeds between 7000
and 25,000 rpm. The samples were centrifuged at a specified speed for 20 hours and distribution profile scans
were taken. After three hours, distribution scans were
again taken to ensure that equilibrium had been reached.
The data were analyzed using Winnolin 1.06, Windows
version of program NONLIN,44 and the details of the
data analysis have been described.25
Viral Mimicry of a Human Cytokine
Isothermal titration calorimetry (ITC)
ITC measurements were carried out using a VP-ITC
microcalorimeter (MicroCal LLC. Northampton, MA) as
described.26,45 All the protein preparations were purified
and exchanged into appropriate buffers with gelfiltration chromatography to control for heat of dilution
effects. Protein concentration was determined with the
bicinchninic acid (BCA) assay (Pierce, Rockford, Il)
using bovine serum albumin as the standard. Each
sample was degassed for ten minutes prior to loading
into the calorimeter. The binding site molar concentrations of the samples in the sample cell were in the
range of 0.5– 10 mM. The equivalent binding site molar
concentrations in the injection syringe were at least
sevenfold greater than that of the cell. The curves are fit
with a least-squares algorithm for a one-site binding
model using the software Origin 5.0 (OriginLab, MA).
Measurements of ionization enthalpies were carried out
as described above but in the following buffers; (Hepes,
4.99 kcal/mol;
Pipes,
2.72 kcal/mol;
cacodylate,
2 0.47 kcal/mol; a mixture of cacodylate and Tris at pH
7.55 with a composite enthalpy of 3.4 kcal/mol).
Cell culture, transfections and plasmids
HEK293T cells were grown in minimum essential
medium supplemented with 5% (v/v) fetal calf serum.
Cells were passaged 12 –24 hours before transfection to
obtain subconfluent monolayers (40 – 60% confluency) in
six-well tissue culture plates. Transfections were carried
out using standard calcium phosphate/DNA coprecipitation with HBS; medium-containing precipitate was
changed with fresh medium after eight hours, and cells
were harvested 48 hours after medium change. Assays
of chloramphenicol acetyltransferase (CAT) activities in
cell lysates were carried out as described.31 Generally,
1 mg of effector (pSG5-vIL-6, pSG5-vIL-6.15, or pSG5
(negative control)) and 0.5 mg of receptor expression
plasmid(s) (pEFBOS-hIL-6R and/or pEFBOS-hgp130),
pEF-BOS (negative control) and pa2MCAT were used.
For pEFBOS-hIL-6R and pSG5-hIL-6 dose-response
experiments, DNA totals were kept constant by addition
of pEF-BOS or pSG5, respectively. Expression vectors for
vIL-6 and vIL 6.15 have been described;35 pEFBOS-hIL-6
and pEFBOS-hgp130 vectors comprise the coding
sequences of the a and b receptor subunits cloned into
pEF-BOS,46 and were provided by Narazaki &
Kishimoto; the pa2MCAT reporter has been described47
and was provided by Shaefer.
In order to test for autocrine induction of human IL-6
by viral IL-6 (Figure 4(C)), we co-transfected HEK293T
cells with gp130 and IL-6Ra expression vectors, the
pa2M-CAT reporter, and either vIL-6 or hIL-6 expression
constructs (the latter used at different doses). Cells and
media were subsequently harvested for determinations
of CAT activities in cell extracts, and for detection and
quantification of hIL-6 in the culture medium, using
Western analysis (Figure 4(C)). In this way, we could correlate hIL-6 levels with reporter activation and determine
if functionally significant levels of hIL-6 were present in
vIL-6-expressing cultures.
Western analysis for detection of hIL-6
Samples of transfected cell medium were analyzed
for the presence of hIL-6 by Western blotting. Proteins
were size fractionated by SDS-PAGE and transferred
653
Viral Mimicry of a Human Cytokine
electrophoretically to nitrocellulose membranes. These
were blocked in PBS containing 5% (v/v) non-fat milk
prior to the addition of primary antibody to hIL-6 (R &
D Systems, Minneapolis, MN; cat no. AB-206-NA) at
0.1 mg/ml and incubation at 4 8C overnight. The membranes were then washed in PBS prior to addition of
horse radish peroxidase-conjugated anti-goat IgG detection antibody (Santa Cruz Biotech, Santa Cruz, CA; cat.
no. sc-2020) diluted 1:2000 (v/v). Recombinant hIL-6
used as a positive control for Western analysis and to
allow quantification of hIL-6 in transfected cell medium,
was obtained from R & D Systems (cat. no. 206-IL).
7.
8.
9.
Structure analysis
Structural analysis including structural superposition
of the Ca backbone (Figure 2) of gp130 in the hIL-6 hexameric and vIL-6 tetrameric complexes were carried out
with the program O.48 Docking of IL-6Ra onto the core
vIL-6/gp130 tetramer was performed following an initial
least-squares superposition of the hIL-6/gp130 core onto
the vIL-6/gp130 core. The coordinates for the D2D3
domain construct IL-6Ra were then simply appended to
the vIL-6/gp130 tetramer to simulate the vIL-6/IL-6Ra/
gp130 hexamer. Buried surface analysis was carried out
using the Protein – Protein Interaction (PPI) server†.
10.
11.
12.
13.
14.
Acknowledgements
This work was supported by NIH grant RO1AI51321 and Pew Scholars, Cancer Research
Institute, and Keck Foundation grants to K.C.G., and
NIH R01-CA76445 to J.N. M.J.B. is supported by a
National Science and Engineering Research Council
(NSERC) of Canada post-doctoral fellowship.
15.
16.
References
1. Stebbins, C. E. & Galan, J. E. (2001). Structural
mimicry in bacterial virulence. Nature, 412, 701– 705.
2. Alcami, A. (2003). Viral mimicry of cytokines,
chemokines and their receptors. Nature Rev. Immunol.
3, 36 –50.
3. Tortorella, D., Gewurz, B. E., Furman, M. H., Schust,
D. J. & Ploegh, H. L. (2000). Viral subversion of the
immune system. Annu. Rev. Immunol. 18, 861– 926.
4. Gewurz, B. E., Gaudet, R., Tortorella, D., Wang, E. W.,
Ploegh, H. L. & Wiley, D. C. (2001). Antigen presentation subverted: structure of the human
cytomegalovirus protein US2 bound to the class I
molecule HLA-A2. Proc. Natl Acad. Sci. USA, 98,
6794–6799.
5. Jones, B. C., Logsdon, N. J., Josephson, K., Cook, J.,
Barry, P. A. & Walter, M. R. (2002). Crystal structure
of human cytomegalovirus IL-10 bound to soluble
human IL-10R1. Proc. Natl Acad. Sci. USA, 99,
9404–9409.
6. Chang, Y., Cesarman, E., Pessin, M. S., Lee, F.,
Culpepper, J., Knowles, D. M. & Moore, P. S. (1994).
Identification of herpesvirus-like DNA sequences in
† http://www.biochem.ucl.ac.uk/bsm/PP/server
17.
18.
19.
20.
21.
22.
AIDS-associated Kaposi’s sarcoma. Science, 266,
1865 –1869.
Cesarman, E., Chang, Y., Moore, P. S., Said, J. W. &
Knowles, D. M. (1995). Kaposi’s sarcoma-associated
herpesvirus-like DNA sequences in AIDS-related
body-cavity-based lymphomas. N. Engl. J. Med. 332,
1186– 1191.
Soulier, J., Grollet, L., Oksenhendler, E., Cacoub, P.,
Cazals-Hatem, D., Babinet, P. et al. (1995). Kaposi’s
sarcoma-associated herpesvirus-like DNA sequences
in multicentric Castleman’s disease. Blood, 86,
1276 –1280.
Ploegh, H. L. (1998). Viral strategies of immune
evasion. Science, 280, 248– 253.
Raftery, M., Muller, A. & Schonrich, G. (2000).
Herpesvirus homologues of cellular genes. Virus
Genes, 21, 65 – 75.
Moore, P. S., Boshoff, C., Weiss, R. A. & Chang, Y.
(1996). Molecular mimicry of human cytokine and
cytokine response pathway genes by KSHV. Science,
274, 1739 –1744.
Nicholas, J., Ruvolo, V. R., Burns, W. H., Sandford,
G., Wan, X., Ciufo, D. et al. (1997). Kaposi’s sarcomaassociated human herpesvirus-8 encodes homologues of macrophage inflammatory protein-1 and
interleukin-6. Nature Med. 3, 287– 292.
Cannon, J. S., Nicholas, J., Orenstein, J. M., Mann,
R. B., Murray, P. G., Browning, P. J. et al. (1999).
Heterogeneity of viral IL-6 expression in HHV-8associated diseases. J. Infect. Dis. 180, 824– 828.
Aoki, Y., Jaffe, E. S., Chang, Y., Jones, K., TeruyaFeldstein, J., Moore, P. S. & Tosato, G. (1999). Angiogenesis and hematopoiesis induced by Kaposi’s
sarcoma-associated
herpesvirus-encoded
interleukin-6. Blood, 93, 4034– 4043.
Jones, K. D., Aoki, Y., Chang, Y., Moore, P. S.,
Yarchoan, R. & Tosato, G. (1999). Involvement of
interleukin-10 (IL-10) and viral IL-6 in the spontaneous growth of Kaposi’s sarcoma herpesvirusassociated infected primary effusion lymphoma
cells. Blood, 94, 2871– 2879.
Mullberg, J., Geib, T., Jostock, T., Hoischen, S. H.,
Vollmer, P., Voltz, N. et al. (2000). IL-6 receptor independent stimulation of human gp130 by viral IL-6.
J. Immunol. 164, 4672– 4677.
Molden, J., Chang, Y., You, Y., Moore, P. S. &
Goldsmith, M. A. (1997). A Kaposi’s sarcoma-associated herpesvirus-encoded cytokine homolog (vIL-6)
activates signaling through the shared gp130
receptor subunit. J. Biol. Chem. 272, 19625– 19631.
Burger, R., Neipel, F., Fleckenstein, B., Savino, R.,
Ciliberto, G., Kalden, J. R. & Gramatzki, M. (1998).
Human herpesvirus type 8 interleukin-6 homologue
is functionally active on human myeloma cells.
Blood, 91, 1858– 1863.
Li, H., Wang, H. & Nicholas, J. (2001). Detection of
direct binding of human herpesvirus 8-encoded
interleukin-6 (vIL-6) to both gp130 and IL-6 receptor
(IL-6R) and identification of amino acid residues of
vIL-6 important for IL-6R-dependent and-independent signaling. J. Virol. 75, 3325– 3334.
Chow, D., He, X., Snow, A. L., Rose-John, S. & Garcia,
K. C. (2001). Structure of an extracellular gp130
cytokine receptor signaling complex. Science, 291,
2150 –2155.
Osborne, J., Moore, P. S. & Chang, Y. (1999). KSHVencoded viral IL-6 activates multiple human IL-6
signaling pathways. Hum. Immunol. 60, 921– 927.
Paonessa, G., Graziani, R., De Serio, A., Savino, R.,
654
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
Viral Mimicry of a Human Cytokine
Ciapponi, L., Lahm, A. et al. (1995). Two distinct and
independent sites on IL-6 trigger gp 130 dimer formation and signalling. EMBO J. 14, 1942 –1951.
Simpson, R. J., Hammacher, A., Smith, D. K.,
Matthews, J. M. & Ward, L. D. (1997). Interleukin-6:
structure-function relationships. Protein Sci. 6,
929– 955.
Yawata, H., Yasukawa, K., Natsuka, S., Murakami,
M., Yamasaki, K., Hibi, M. et al. (1993). Structurefunction analysis of human IL-6 receptor:
dissociation of amino acid residues required for IL6-binding and for IL-6 signal transduction through
gp130. EMBO J. 12, 1705– 1712.
Chow, D., Ho, J., Nguyen Pham, T. L., Rose-John, S.
& Garcia, K. C. (2001). In vitro reconstitution of
recognition and activation complexes between interleukin-6 and gp130. Biochemistry, 40, 7593– 7603.
Boulanger, M. J., Chow, D. C., Brevnova, E. E. &
Garcia, K. C. (2003). Hexameric structure and
assembly of the interleukin-6/IL-6 alpha-receptor/
gp130 complex. Science, 300, 2101– 2104.
Chatterjee, M., Osborne, J., Bestetti, G., Chang, Y. &
Moore, P. S. (2002). Viral IL-6-induced cell proliferation and immune evasion of interferon activity.
Science, 298, 1432– 1435.
Naka, T., Nishimoto, N. & Kishimoto, T. (2002). The
paradigm of IL-6: from basic science to medicine.
Arthritis Res. 4, S233 – S242.
Aoki, Y., Narazaki, M., Kishimoto, T. & Tosato, G.
(2001). Receptor engagement by viral interleukin-6
encoded by Kaposi sarcoma-associated herpesvirus.
Blood, 98, 3042– 3049.
Li, Y., Li, H., Yang, F., Smith-Gill, S. J. & Mariuzza,
R. A. (2003). X-ray snapshots of the maturation of
an antibody response to a protein antigen. Nature
Struct. Biol. 10, 482– 488.
Gorman, C. M., Moffat, L. F. & Howard, B. H. (1982).
Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol. Cell.
Biol. 2, 1044– 1051.
Mori, Y., Nishimoto, N., Ohno, M., Inagi, R.,
Dhepakson, P., Amou, K. et al. (2000). Human
herpesvirus 8-encoded interleukin-6 homologue
(viral IL-6) induces endogenous human IL-6
secretion. J. Med. Virol. 61, 332–335.
Murphy, K. P. & Freire, E. (1992). Thermodynamics
of structural stability and cooperative folding
behavior in proteins. Advan. Protein Chem. 43, 313–361.
Lo Conte, L., Chothia, C. & Janin, J. (1999). The
atomic structure of protein– protein recognition
sites. J. Mol. Biol. 285, 2177– 2198.
Wan, X., Wang, H. & Nicholas, J. (1999). Human
herpesvirus 8 interleukin-6 (vIL-6) signals through
gp130 but has structural and receptor-binding
properties distinct from those of human IL-6. J. Virol.
73, 8268– 8278.
Li, H. & Nicholas, J. (2002). Identification of amino
acid residues of gp130 signal transducer and gp80
alpha receptor subunit that are involved in ligand
binding and signaling by human herpesvirus
8-encoded interleukin-6. J. Virol. 76, 5627– 5636.
37. Leebeek, F. W., Kariya, K., Schwabe, M. & Fowlkes,
D. M. (1992). Identification of a receptor binding site
in the carboxyl terminus of human interleukin-6.
J. Biol. Chem. 267, 14832 –14838.
38. Leebeek, F. W. & Fowlkes, D. M. (1992). Construction
and functional analysis of hybrid interleukin-6
variants. Characterization of the role of the C-terminus for species specificity. FEBS Letters, 306, 262– 264.
39. Kalai, M., Montero-Julian, F. A., Grotzinger, J.,
Fontaine, V., Vandenbussche, P., Deschuyteneer, R.
et al. (1997). Analysis of the human interleukin-6/
human interleukin-6 receptor binding interface at
the amino acid level: proposed mechanism of interaction. Blood, 89, 1319– 1333.
40. Savino, R., Ciapponi, L., Lahm, A., Demartis, A.,
Cabibbo, A., Toniatti, C. et al. (1994). Rational design
of a receptor super-antagonist of human interleukin6. EMBO J. 13, 5863– 5870.
41. Bravo, J., Staunton, D., Heath, J. K. & Jones, E. Y.
(1998). Crystal structure of a cytokine-binding region
of gp130. EMBO J. 17, 1665– 1674.
42. Horsten, U., Muller-Newen, G., Gerhartz, C.,
Wollmer, A., Wijdenes, J., Heinrich, P. C. &
Grotzinger, J. (1997). Molecular modeling-guided
mutagenesis of the extracellular part of gp130 leads
to the identification of contact sites in the interleukin-6 (IL-6) IL-6 receptor gp130 complex. J. Biol.
Chem. 272, 23748– 23757.
43. Kurth, I., Horsten, U., Pflanz, S., Dahmen, H., Kuster,
A., Grotzinger, J. et al. (1999). Activation of the signal
transducer glycoprotein 130 by both IL-6 and IL-11
requires two distinct binding epitopes. J. Immunol.
162, 1480– 1487.
44. Johnson, M. L., Correia, J. J., Yphantis, D. A. &
Halvorson, H. R. (1981). Analysis of data from the
analytical ultracentrifuge by nonlinear least-squares
techniques. Biophys. J. 36, 575– 588.
45. Boulanger, M. J., Bankovich, A., Kortemme, T., Baker,
D. & Garcia, K. C. (2003). Convergent mechanisms
for recognition of divergent cytokines by the shared
signaling receptor gp130. Mol. Cell, 12, 577– 589.
46. Mizushima, S. & Nagata, S. (1990). pEF-BOS, a
powerful mammalian expression vector. Nucl. Acids
Res. 18, 5322.
47. Shaefer, T. S., Sanders, L. K. & Nathans, D. (1995).
Cooperative transcriptional activity of Jun and
Stat3b, a short form of Stat3. Proc. Natl Acad. Sci.
USA, 92, 9097– 9101.
48. Jones, T. A., Zhou, J. Y., Cowan, S. W. & Kjeldgaard,
M. (1991). Improved methods for building models
in electron density maps and the location of error in
these models. Acta Crystallog. sect. A, 47, 110 – 119.
49. Kraulis, P. (1991). MOLSCRIPT: a program to produce both detailed and schematic plots of protein
structures. J. Appl. Crystallog. 24, 946– 950.
50. Merritt, E. A. & Murphy, E. P. M. (1994). Raster3D
version 2.0. A program for photorealistic molecular
graphics. Acta Crystallog. sect. D, 50, 869– 873.
51. Barton, G. J. (1993). ALSCRIPT: a tool to format
multiple sequence alignments. Protein Eng. 6, 37 – 40.
Edited by I. Wilson
(Received 27 August 2003; received in revised form 23 October 2003; accepted 28 October 2003)

Download Report