From the
Activation of the cell surface receptors for tumor necrosis
factor (TNF) is effected by the aggregation of cytoplasmic domains that
occurs when the extracellular domains of two or three receptors bind to
trimeric TNF
Tumor necrosis factors (TNF
Antibodies that recognize and cross-link the extracellular domain of
the type I receptor elicit a signaling response
(4) . Conversely,
expression of a deletion mutant of the human TNF receptor that lacks
most of the cytoplasmic domain results in the suppression of signaling
(5). These experiments provide strong evidence that signal transduction
is triggered by TNF-mediated dimerization or trimerization of free
monomeric receptors. The 2.9-Å resolution structure of the
extracellular domain of the type I receptor (sTNF-R1) complexed with
TNF
Here, we
report that in crystals, the soluble extracellular domain of the human
type I TNF receptor forms two distinct types of dimeric structures. We
suggest that intact receptors may form one or both types of dimer on
the plasma membrane. These dimers could down-regulate signaling in the
absence of TNF and, in one case, perhaps enhance TNF-induced receptor
aggregation.
The type I receptor for TNF (TNF-R1) is composed of an
N-terminal extracellular ligand binding domain linked by a single
transmembrane segment to a C-terminal cytoplasmic signaling
domain
(9, 10) . The receptor belongs to the nerve growth
factor receptor superfamily, which includes the type II TNF receptor,
CD40, and the Fas antigen
(11) . Members of this family are
characterized by extracellular domains that comprise repeats of a
Human sTNF-R1, consisting of residues 11-172 of the
type I receptor, was purified and crystallized as described
previously
(13) . The crystal belongs to the tetragonal space
group P4
Human sTNF-R1 was expressed in Escherichia coli,
refolded to form active protein, and crystallized at pH
8.0
(13) . Using single isomorphous replacement/optimized
anomalous scattering (SIROAS), we have determined the structure of
non-glycosylated sTNF-R1 to 2.25 Å (, Fig. 1).
The unit cell contains two molecules of sTNF-R1 related by a
non-crystallographic diad axis. The final refined model includes
residues 12-150 of one monomer and residues 14-155 of the
second. Thus, in both molecules, from 17 to 22 residues of the C
termini are disordered and cannot be located in the electron density.
The two molecules adopt similar conformations; superposition of
corresponding C
In crystals of
unliganded receptor, the two independent molecules of sTNF-R1 are in
extensive contact with each other, generating two distinct
monomer-monomer interfaces. In contrast, apart from nonspecific crystal
contacts, individual receptor extracellular domains do not contact each
other in crystals of the sTNF-R1
Comparison of the structure of the unliganded sTNF-R1 with
that crystallized as a complex with TNF
We have described two types of
TNF-receptor dimer that appear in crystals of unliganded sTNF-R1; in
each of these, monomers associate at interfaces that are comparable in
area and composition to other protein-protein complexes. It is
therefore possible that such dimers could form between holoreceptors at
the membrane surface. Although dimerization of TNF-receptor
extracellular domains has not, to our knowledge, been observed in
dilute solution, the limited solubility of sTNF-R1 (2-3 mg/ml in
PBS buffer at pH 8.0)(
The induction of apoptosis in the absence
of TNF by expression of the TNF-R1 cytoplasmic domain suggests that the
extracellular domain of the receptor is not required to initiate
signaling and that self-association of cytoplasmic domains can occur in
its absence
(7) . Rather, the extracellular domain of the type I
receptor may enforce a non-signaling state, possibly by inhibiting
productive self association of intracellular domains. In accordance
with the crystal structure, an antiparallel association between the
second disulfide-rich motifs of two receptors would cause their
signaling domains to be separated by more than 100 Å. It is
possible that an interaction of this type has steric consequences as
well, so as to prevent productive contacts between cytoplasmic domains
in different receptor dimers. The immediate effects of TNF binding
would be, first, to disrupt inhibitory interactions between receptors,
and second, to promote productive interactions between cytoplasmic
domains. The mechanism of receptor-mediated signal inhibition proposed
here requires a specific set of interactions that would not, in
general, be mimicked by the extracellular domains of other receptors.
Accordingly, Bazzoni et al.(25) found that chimeric
receptors, containing the extracellular domain of erythropoietin
receptor fused to the TNF-R1 cytoplasmic domain, are cytotoxic even in
the absence of erythropoietin.
Unlike antiparallel dimers, parallel
TNF-R1 dimers might form in the presence of TNF, because the
self-association and TNF-binding surfaces are located on opposite sides
of the receptor extracellular domain. For the same reason, both
interactions might occur simultaneously, thus generating an aggregate
of TNF-R1
Since it is difficult to
assess the specific binding energies associated with interacting
protein-protein surfaces, it is not possible to predict whether
parallel or antiparallel dimers (if either) would predominate at the
plasma membrane in the absence of TNF. Neither are we able to formulate
a simple mechanism by which parallel dimer formation, in the absence of
TNF, would down-regulate signaling through the cytoplasmic domains of
the receptor. We have presented evidence that the type I TNF receptor
dimerizes in the absence of TNF, and might do so in in its presence,
through interactions between the receptor extracellular domains.
Whether or not such effects will be found in vivo and whether
they occur generally among the NGF class of receptors remains to be
determined.
On-line formulae not verified for accuracy (119)
Theatomic coordinates and structure factors (code 1NCF) have been
deposited in the Protein Data Bank, Brookhaven National Laboratory,
Upton, NY.
We thank Albert Berghuis, Sean McSweeny, and Peter
Lindley for help with data collection at Daresbury; Lynn Rodseth, Karin
Hale, and Michael Eck for help at the start of the project; Bruce
Beutler and Flavia Bazzoni for helpful discussions; and Elizabeth
Goldsmith for a critical reading of the manuscript. We are grateful to
David Banner and Werner Lesslauer of Hoffman La Roche for providing
coordinates of the sTNF-R1
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
or TNF
. The structure of the type I TNF receptor
extracellular domain (sTNF-R1), crystallized in the absence of TNF, has
now been determined at 2.25-Å resolution. The receptor itself is
an elongated molecule comprising four disulfide-rich domains in a
nearly linear array. Contrary to expectations, the unliganded domains
are found to associate into dimers of two distinct types, in which
monomers are related by local two-fold axes of symmetry. In one case,
the receptors are antiparallel to each other and associate through an
interface that overlaps the TNF binding site. If intact receptors were
capable of such an association, their cytoplasmic domains would be
separated by over 100 Å. This interaction could inhibit signaling
in the absence of TNF. Parallel dimers are also observed in which the
dimer interface is well separated from the TNF binding site.
Associations among TNF-bound parallel dimers could cause receptor
clustering. Both dimers bury substantial areas of protein surface and
are formed by polar and non-polar interactions.
, TNF
)(
)
are potent cytokines and immunomodulators that mediate many of
the inflammatory and immune responses to infection and
cancer
(1) . Two cell surface receptors, type I (55 kDa) and type
II (75 kDa), have been identified as transducers of TNF
activity
(2) . It is of crucial importance for cell viability
that receptor signaling be tightly regulated and, in the absence of
TNF, prevented. Excessive TNF-mediated receptor signaling may be
responsible in part for the deleterious effects of diseases such as
septic shock, malaria, and bacterial meningitis
(1, 3) .
is consistent with this hypothesis
(6) . However, it was
recently shown that expression of a truncated form of the receptor
lacking the extracellular and transmembrane domains also leads to
signaling events that result in apoptosis
(7) . These effects may
be initiated by dimerization or multimerization of the cytoplasmic
signaling domains (7, 8) of the truncated receptor. From these
experiments, it might be surmised that under normal circumstances, the
extracellular domains of the TNF receptor inhibit signaling in the
absence of TNF. A possible inhibitory mechanism might involve the
formation of specific contacts between the extracellular (ligand
binding) domains of the receptor in the absence of ligand.
40-residue cystine-rich motif; TNF-R1 has four such repeats, each
containing three internal disulfide bridges. The 161-residue
extracellular domain of the type I TNF receptor (sTNF-R1) is found in
serum and urine
(12) , where it may sequester free TNF, thus
down-regulating TNF signaling. In the crystal structure of the
sTNF-R1
TNF
complex
(6) , TNF
binds three sTNF-R1
molecules, each at an intersubunit site on the trimeric cytokine. The
second and third motifs (numbering from the N terminus) of the TNF-R1
extracellular domain form virtually all of the contacts with TNF
.
Presumably this complex brings the receptor intracellular domains
together below the membrane in the correct steric manner for signal
initiation.
2
2 (cell dimensions a =
69 Å, c = 185.2 Å). Diffraction data
measured in a nitrogen cryostream (95 K) at Station PX9.5 of the
Daresbury Synchrotron () were processed with DENZO and
SCALEPACK
(14) . Heavy atom sites for a single
chloromercurynitrophenol derivative were found in difference and
anomalous Patterson maps. The platinum hexaiodoplatinate derivative
reported earlier
(13) proved to be nonisomorphous with the
native crystals at 95 °C and consequently could not be used in
phasing. The initial phases were calculated using MLPHARE
(15) and refined and extended to 2.25 Å using
SQUASH
(16, 17) . To aid in map interpretation, the
phases from SQUASH were used to compute a sulfur anomalous difference
Fourier map with native data (data set native2) measured at a
wavelength of 1.54 Å. All other calculations were performed with
the CCP4 program package
(15) . Using the anomalous map to locate
disulfide bonds, a model was built into the SQUASH
(16, 17) map using O
(18) . The atomic model of the receptor
from the coordinates of the sTNF-R1
TNF
complex were not used
to interpret the experimental electron density, nor was any phase
information derived from that model. Fig. 1shows the final
2F
- F
map and the anomalous map. The model was refined using X-PLOR
using the stereochemical restraint set cof Engh and Huber (19). To
follow the progress of refinement using the free R-factor as a
criterion
(19) , 10% of the data (1904 of a total of 19,343
reflections), were randomly selected and excluded from further model
building and refinement. Non-crystallographic symmetry restraints were
used during the initial stages, but were released in the final cycles
of refinement and model adjustment. Removal of the crystallographic
restraints resulted in a reduction of the free R-factor.
Ordered water molecules were included in the model if: 1) they
corresponded to peaks with magnitudes greater than three standard
deviations above mean absolute value (3
) of the
F
- F
difference Fourier map, 2) they formed potential hydrogen bond
contacts with protein (or other water) molecules with reasonable
stereochemistry, 3) they were visible in a 2F
- F
map at the 1.5
level, and 4) their inclusion resulted in a reduction of the free
R-factor. The model includes 2094 protein atoms and 260
solvent atoms, and is refined to a free R-factor of 24.9% and
an R-factor of 19.3% for 19,343 reflections with F > 3
F in the resolution range 6-2.25
Å. The R-factor for all data (20,714 reflections) in
this resolution range is 20.8%. The root mean square deviation from
ideality of bond lengths is 0.009 Å, and from bond angles is
1.6°. PROCHECK version 3.0
(20) shows all stereochemical
parameters to be better than or equal to the average for structures at
2.25 Å. No non-glycine residues are outside allowed regions in a
Ramachandran plot. Atomic coordinates have been deposited with the
Protein Data Bank (21) with accession code 1NCF and will be available 6
months after the date of this publication.
Figure 1:
The electron density map is shown in
the region near the disulfide bonded residues Cys-52 and Cys-33. The
2F - F (phases from final model) electron
density map shown in blue is contoured at 2 standard
deviations ( above the mean absolute value of the map), and the
anomalous density map (phases from SQUASH (16, 17) in red is
contoured at 4
.
carbons results in a root mean square deviation of
1.03 Å. In contrast, individual motifs can superimposed with root
mean square deviations ranging from 0.2 Å for C
atoms in
domain one to 0.4 Å for atoms in domain two, indicating segmental
flexibility. The overall fold of the rod-shaped molecule is identical
to that observed in the 2.85-Å crystal structure of sTNF-R1
complexed to TNF
(6) (Fig. 2). Operation of
crystallographic symmetry elements upon the local two-fold axes
generates two types of dimer in the unit cell as described below.
Figure 2:
The protein is an elongated rod of four
domains arranged linearly along the long axis of the molecule. The
domains are numbered consecutively from the N terminus. The disulfide
bonds are arranged as rungs of a ladder (step 10 Å) along the
length of the long molecular axis; the sulfur atoms are shown as
space-filling yellowspheres. Domains 1, 2, and 3 are
each composed of 41-45 amino acids and are structurally similar.
Only part of domain 4 has been traced and is similar to the other three
domains; the remainder of domain 4 is disordered. This and subsequent
illustrations were created with the program MOLSCRIPT (28) and rendered
for the Silicon Graphics Iris with the program RASTER3D
(29).
Free sTNF-R1 is slightly more curved than in the complex with
TNF. Pairwise superposition of corresponding motifs at
-carbon positions gives an average root mean square deviation of
0.65 Å; superposition of the entire molecule as a rigid unit
gives a root mean square deviation of 1.70 Å. The greatest
differences between the liganded and unliganded receptors appear in the
structure of loop and turn regions. As in the complex, only half of the
residues of the of the fourth motif are ordered.
TNF
complex (6). We refer to
the two dimeric species observed in crystals of unliganded sTNF-R1 as
parallel and antiparallel. The antiparallel dimer is
shown in Fig. 3A and is formed by a head to tail
arrangement of the receptors. The second motifs of the two monomers are
joined by a ladder of hydrogen bonds, forming a pseudo
-ribbon
(Fig. 3B). The 1475 Å
of protein
solvent-accessible surface area
(22) buried by the antiparallel
dimer formation (737 Å
/monomer) is comparable with
that observed in stable dimeric protein associations (23). The
interaction surface possesses the high degree of complementarity
typical of protein antibody-antigen complexes
(24) and of the
sTNF-R1
TNF
complex itself (). Antiparallel
dimers are stabilized by an extensive network of van der Waals and
hydrogen bond interactions (). If such dimers were formed
by intact receptors on the cell surface, the cytoplasmic domains of the
two monomers would be separated in space by more than 100 Å
(Fig. 3A). TNF would be expected to disrupt antiparallel
dimers because the TNF binding surface completely overlaps the area of
monomer-monomer contact.
Figure 3:
Antiparallel dimers are generated by
non-crystallographic dyad symmetry between the two independent
molecules in the unit cell. One monomer (A) is colored red and salmon to distinguish individual motifs as in Fig. 2,
and the other (B) is colored blue and cyan.
The extracellular domain of the intact, unliganded receptor is proposed
to form antiparallel dimer interactions on the surface of the plasma
membrane (shown as a surface of bluespheres). In
panel A, the dimer is viewed along the plane of the membrane;
the vertical non-crystallographic axis (not shown) is in the plane of
the diagram. The C-terminal half of the fourth (carboxyl-terminal)
sTNF-R1 motif is depicted as a solidgreenbar extending into the membrane. The 16 amino acid residues separating
the last disulfide bond and the transmembrane sequence could serve as a
flexible tether allowing free rotation of the extracellular domain
about its membrane attachment site. As a consequence of this
interaction, the cytoplasmic domains (not shown) would be separated by
>100 Å. PanelB shows the hydrogen bonding
contacts between A and B monomers around the non-crystallographic dyad
axis.
Parallel dimers are formed by a head to
head arrangement of the sTNF-R1 monomers (Fig. 4A). The
subunit interface is formed almost entirely between the first motifs of
the two molecules. The 2143 Å of total surface area
withdrawn from solvent by dimer formation (1071
Å
/monomer) is comparable with that buried by the
interaction of a single sTNF-R1 monomer with TNF
in crystals of
the complex. Like the antiparallel dimer, the parallel interface is
characterized by a high degree of spatial complementarity and is
stabilized by a large number of non-bonded interactions
(). The three glycosylation sites at Asn-25, Asn-116, and
Asn-125 are remote from the interface and would seem unlikely to
disrupt this, or the antiparallel dimer. Formation of parallel dimers
by the intact receptors would place the cytoplasmic domains in close
juxtaposition. A molecular docking experiment demonstrates that
formation of parallel dimers would not interfere with TNF binding.
Residues 64-116 (which contain the ligand binding surface) of the
free and complexed sTNF-R1 can be superimposed at C
positions with
a root mean square deviation of 0.74 Å. The small increase in
curvature of the free receptor in the dimerized state does not generate
steric conflicts when docked into the TNF binding site. Thus, this
dimer would seem likely to survive exposure to TNF.
Figure 4:
Panel
A shows the parallel dimer, using the coloring scheme adopted
in Fig. 3. Molecule A is in contact with B`, where B` is related to B
(Fig. 3) by a crystallographic symmetry transformation. The
extracellular domain of the intact, unliganded receptor is proposed to
form parallel-dimer interactions on the surface of the plasma membrane
(shown as a surface of sage-green spheres). The dimer
interactions principally involve domain 1 of each monomer. The TNF
binding surface is exposed in these dimers, and cytoplasmic domains
would be closely juxtaposed. Parallel dimer formation between
TNF-ligated receptor dimers could result in receptor/TNF clustering as
depicted schematically in panel B with TNF trimers shown as
triangles and receptor monomers as
semicircles.
demonstrates that TNF
binding does not induce global changes in the tertiary structure of the
receptor. Instead, it appears that TNF binding may disrupt at least one
type of TNF receptor dimer.
)
indicates that it easily
forms aggregates. In vivo, the receptors are restricted to the
two-dimensional surface of the membrane in which self-association is
entropically more favored.
TNF complexes on the cell surface
(Fig. 4B). Such an array would increase the local
concentration of TNF signaling complexes at the cell surface
(26) and could also play a role in receptor internalization.
Favorable interactions between cytoplasmic domains
(9) would
enhance the stability of parallel dimers.
Table: 543582574p4in
Chloromercurynitrophenol.
TNF
complex prior to their general
release.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.