©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Crystallographic Evidence for Dimerization of Unliganded Tumor Necrosis Factor Receptor (*)

James H. Naismith (1)(§), Tracey Q. Devine (2), Barbara J. Brandhuber (3), Stephen R. Sprang (1) (2)(¶)

From the (1) Department of Biochemistry and (2) Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9050 and (3) Synergen, Boulder, Colorado 80301-2546

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 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.


INTRODUCTION

Tumor necrosis factors (TNF, 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) .

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 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.

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 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-R1TNF 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.


MATERIALS AND METHODS

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 P422 (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-R1TNF 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 > 3F 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.




RESULTS

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 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.

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-R1TNF 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-R1TNF 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.




DISCUSSION

Comparison of the structure of the unliganded sTNF-R1 with that crystallized as a complex with TNF 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.

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)() 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.

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-R1TNF 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.

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.

  
Table: 543582574p4in Chloromercurynitrophenol.

On-line formulae not verified for accuracy

(119)

  
Table: Dimer interactions



FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Theatomic coordinates and structure factors (code 1NCF) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.

§
A Biotechnology and Biochemical Sciences Research Council/NATO fellow. Present address: Institute of Biomolecular Sciences, Chemistry Dept., Purdie Bldg., University of St. Andrews, St. Andrews, Scotland KY16 9ST, United Kingdom.

To whom all correspondence should be addressed: Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9050. Tel.: 214-648-5008; Fax: 214-648-6336; E-mail: sprang@howie.swmed.edu.

The abbreviations used are: TNF, tumor necrosis factor; sTNF-R1, extracellular domain of the type I TNF receptor.

T. Q. Devine, unpublished data.


ACKNOWLEDGEMENTS

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-R1TNF complex prior to their general release.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.