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J. Biol. Chem., Vol. 278, Issue 49, 49145-49153, December 5, 2003

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The Interleukin 1 (IL-1) Receptor Accessory Protein Toll/IL-1 Receptor Domain

ANALYSIS OF PUTATIVE INTERACTION SITES BY IN VITRO MUTAGENESIS AND MOLECULAR MODELING^*

Jürgen Radons, Stefan Dove¶, Detlef Neumann||, Reinhold Altmann**, Alexander Botzki¶, Michael U. Martin||, and Werner Falk

From the Klinik und Poliklinik für Innere Medizin I, Universität Regensburg, D-93042 Regensburg, Germany, the ^¶Institut für Pharmazie, Abteilung Pharmazeutische Chemie II, Universität Regensburg, D-93053 Regensburg, Germany, and the ^||Institut für Pharmakologie, Medizinische Hochschule Hannover, D-30623 Hannover, Germany

Received for publication, June 10, 2003 , and in revised form, August 19, 2003.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The Toll/interleukin 1 (IL-1) receptor family plays an important role in both innate and adaptive immunity. These receptors are characterized by a C-terminal homology motif called the Toll/IL-1 receptor (TIR) domain. A principal function of the TIR domain is mediating homotypic protein-protein interactions in the signal transduction pathway. To suggest interaction sites of TIR domains in the IL-1 receptor complex, we modeled the putative three-dimensional structure of the TIR domain within the co-receptor chain, IL-1 receptor accessory protein. The model was based on homology with the crystal structures of human TLR1 and TLR2. The final structure of the IL-1 receptor accessory protein TIR domain suggests the conserved regions box 1 and 2, including Pro-446, as well as box 3 within the C-terminal -helix as possible protein-protein interaction sites due to their exposure and their electrostatic potential. Pro-446, corresponding to the Pro/His mutation in dominant negative TLR4, is located in the third loop at the outmost edge of the TIR domain and does not play any structural role. Inhibition of IL-1 responsiveness seen after substitution of Pro-446 by charged amino acids is due to the loss of an interaction site for other TIR domains. Amino acids 527–534 as part of the loop close to the conserved box 3 are critical for recruitment of myeloid differentiation factor 88 and to a lesser extent for IL-1 responsiveness. Modeling suggests that native folding of the TIR domain may be approached by the responsive deletion mutants 528–534 and 527–533, whereas the C-terminal -strand and/or -helix is displaced in the nonresponsive mutant 527–534.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The pleiotropic cytokine interleukin 1 (IL-1)¹ plays an important role in inflammation and regulation of immune responses including B cell growth and differentiation, thymocyte proliferation, and production of several cytokines (1, 2). The effects of IL-1 are mediated via the type I IL-1 receptor (IL-1RI) (3). Upon binding to its receptor, IL-1 induces multiple physiological responses and the expression of a large number of proinflammatory enzymes such as acute phase proteins, adhesion molecules, tissue-degrading enzymes, and inducible cyclooxygenase (for a review, see Ref. 4). Three ligands are known to bind to IL-1RI: IL-1, IL-1, and IL-1Ra, the latter acting as a true receptor antagonist. The IL-1RI complex consists of two type I integral membrane proteins: IL-1RI and IL-1RAcP (5, 6). It has been shown previously that co-expression of IL-1RAcP is essential for complete IL-1 responsiveness (7–10). Several intracellular signaling molecules have been reported to associate with the receptor complex. Four homologs of the Drosophila melanogaster molecule pelle have been found in the murine system (mPLK, mIRAK-2, mIRAK-4, and mIRAK-M) (11–16) and in other mammals (IRAK-1, IRAK-2, IRAK-M, and IRAK-4) (11, 12, 16–19). IRAK-1 associates with IL-1RAcP, and IRAK-2 associates with IL-1RI (12); both are preassociated with the regulatory protein Tollip (20), whereas IRAK-4 functions as a central element in the early signal transduction of Toll/IL-1 receptors upstream of IRAK-1 (11). It is assumed that the cytosolic adapter molecule MyD88 associates with the TIR domains of receptor and co-receptor (12, 21), possibly as a dimer (22), and thus introduces death domains into the active receptor complex. Upon activation, IRAK is phosphorylated weakening its affinity for Tollip in favor of interaction with MyD88 (23). After further phosphorylation, the MyD88·IRAK complex is disrupted. The free cytosolic IRAK interacts with tumor necrosis factor- receptor-associated factor 6 (TRAF-6) (24). IL-1 stimulates the membrane-cytosol translocation of the TAK-1-binding protein TAB-2, which functionally links TAK-1 to TRAF-6 (25). TRAF-6 is the upstream regulator of TAK-1 (26) thereby coupling IL-1RI to activation of NF-B (27). However, a minor part of the IL-1-dependent NF-B activation is independent of IRAK and uses an alternative pathway involving the recruitment of phosphatidylinositol 3-kinase (28) to a distinct site within the cytoplasmic domain of the IL-1 receptor complex (29). Signal transduction is also mediated via additional signaling cascades such as the p38 mitogen-activated protein kinase and the c-Jun N-terminal kinase pathways (26, 30, 31).

IL-1RI has been identified as part of the Toll/IL-1 receptor (TIR) superfamily whose members encode various receptors involved in inflammation and host defense (23, 32, 33). These receptors share a conserved cytoplasmic motif consisting of about 150–180 amino acids, the TIR domain, named due to the sequence similarity between IL-1RI and the D. melanogaster protein Toll (34). Within the TIR domain three typical regions of conservation can be found called box 1, 2, and 3 (35). A principal function of TIR domains is thought to be mediating homotypic protein-protein interactions in the signal transduction pathway (36). To elucidate the molecular basis of TIR domain signaling, we derived a homology model of the three-dimensional structure of the IL-1RAcP TIR domain based on crystal structures of the human Toll-like receptors TLR1 and TLR2 (37). The model suggests at least two conserved regions, 1) box 1 and 2, including Pro-446 in place of the Pro/His mutation in dominant negative TLR4 and 2) box 3 within the C-terminal -helix, as putative interaction sites for downstream adapter molecules due to their exposure and their electrostatic potential. Structural considerations and functional analyses of mutants suggested aa 527–534, which form the loop preceding box 3, as being critical for MyD88 recruitment and, to a lesser extent, for IL-1 responsiveness.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cell Culture and Biological Reagents—Mouse thymoma EL4D6/76 cells were cultured in RPMI 1640 medium (PAN Biotech GmbH, Aidenbach, Germany) containing 2 mM glutamine, 10% fetal calf serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 30 µM 2-mercapthoethanol (culture medium) at 37 °C in humidified air with 5% CO₂. For stimulation, 2 x 10⁵ cells were seeded in 96-well plates at a density of 1 x 10⁶ cells/ml. Human (h) recombinant (r) IL-1 (rhIL-1) and mouse (m) recombinant (r) IL-18 (rmIL-18) were purchased from PAN Biotech GmbH, whereas PMA was purchased from Sigma. Human embryonic kidney cells 293IL-1RI stably expressing human IL-1RI (18) were cultured and stimulated in Dulbecco's modified essential medium high glucose (PAA Laboratories, Linz, Austria) containing 10% fetal calf serum, 100 units/ml penicillin, and 100 µg/ml streptomycin under the conditions indicated above.

Construction of Mutated Forms of IL-1RAcP—Mutated forms of IL-1RAcP were constructed by PCR technique. Deletions in the IL-1RAcP sequence were performed with the ExSite^TM PCR-based site-directed mutagenesis kit (Stratagene Europe, Amsterdam, The Netherlands), whereas point mutations and multiple amino acid exchanges were generated by site-directed mutagenesis using the QuikChange^TM site-directed mutagenesis kit (Stratagene) according to the manufacturer's recommendations. To generate multiple mutations within IL-1RAcP, random mutagenesis was performed using degenerated primers as follows: 5'-CAC GGT CAT TAA ATG GA(C/T) AA(A/G) A(C/T)A GA(C/T) A(A/G)(A/T) CA(C/T) A(G/C)A T(G/C)(A/G) TCA GGG CAG GTT CTG G-3' (forward primer) and 5'-CCA GAA CCT GCC CTG A(C/T)(G/C) AT(G/C) TAT G(A/T)(C/T) T(A/G)T CT(A/G) T(C/T)T T(A/G)T CCT TTT AAT GAC CGT G-3' (reverse primer). The vector pFLAG-IL-1RAcP, a derivative of pFLAG-CMV-1 (Sigma) was used as a template in all mutagenesis experiments. All mutations were verified by sequence analysis.

Transient Transfection of EL4D6/76 and 293IL-1RI Cells— EL4D6/76 cells, lacking IL-1RAcP expression (9), at a density of 5 x 10⁵/ml were transiently transfected by the DEAE-dextran method. Briefly, the transfection reagent was freshly prepared by mixing 300 µl of 0.5 mg/ml DEAE-dextran in TBS (25 mM Tris-HCl, pH 7.4, 123 mM NaCl, 5 mM KCl, 0.7 mM CaCl₂, 0.5 mM MgCl₂, 0.6 mM Na₂HPO₄) and 300 µl of chloroquine (80 µg/ml in TBS) containing 500 ng of IL-1RAcP expression plasmid encoding the various mutated forms of the co-receptor chain together with 1 µg of a luciferase reporter plasmid containing the IL-1-inducible proximal region of the murine IL-2 promoter (pGL3-IL-2(-303)) (38, 39). 5 x 10⁶ cells (in logarithmic growth phase) were washed in phosphate-buffered saline, pH 7.4 and resuspended in TBS. After centrifugation the supernatant was decanted, and the cells were resuspended in the residual liquid. Subsequently the DEAE-dextran-DNA transfection mixture was added, and the cells were incubated for 30 min at room temperature. Afterward cells were washed twice with phenol red-free RPMI 1640 medium (PAN Biotech GmbH) plus supplements and cultured for 24 h in 6-well plates (Costar) at a density of 10⁶ cells/ml. 293IL-1RI cells were co-transfected by the calcium phosphate precipitation method with different amounts of pFLAG-IL-1RAcP encoding N-terminally FLAG-tagged mouse wt or mutant co-receptor chain in combination with either pTollip-noTag encoding untagged human Tollip or pRK7-Myc/MyD88 (a kind gift from H. Wesche, Tularik, South San Francisco, CA) encoding human MyD88 fused to an N-terminal Myc tag. The total amount of DNA in all transfections was kept constant by adding empty vector DNA.

Reporter Gene Assay and Determination of IL-2 Secretion—The transfectants were seeded into 96-well plates at a density of 1 x 10⁶/ml and co-stimulated with either 20 units/ml rhIL-1 or 250 ng/ml rmIL-18 in the presence of 10 ng/ml PMA for 18 h or remained unstimulated. IL-2 promotor activation was determined by measuring the luciferase activity in transiently transfected cells using the LucLite Plus luciferase reporter gene assay kit (PerkinElmer Life Sciences) as described previously (39). Luciferase activity was normalized to the IL-1R complex-independent IL-18 response obtained after co-incubation of the transfectants in the presence of PMA + IL-18. IL-2 secretion into the culture supernatants was quantified by sandwich enzyme-linked immunosorbent assay as described elsewhere (39).

Immunoprecipitation and Immunoblotting—40 h after transfection, 293IL-1RI cells were incubated in the absence or presence of 10 ng/ml rhIL-1 (a kind gift from D. Boraschi, Dompé SpA, L'Aquila, Italy) for 5 min and lysed in 1 ml of lysis buffer (0.5% Nonidet P-40, 10% glycerol, 250 mM NaCl, 1 mM EDTA, 20 mM -glycerophosphate, 1 mM Na₃VO₄, 5 mM p-nitrophenyl phosphate, 5 mM dithiothreitol, 50 mM HEPES, pH 7.9 supplemented with protease inhibitor mixture (Roche Diagnostics). The lysates were cleared by centrifugation (20 min, 15,000 x g, 4 °C) and incubated under agitation with anti-FLAG M2-Sepharose (Sigma) for 4 h at 4 °C. The precipitates were thoroughly washed with lysis buffer and heated for 10 min at 95 °C in Laemmli buffer (40). Solubilized proteins were separated by 7.5% SDS-PAGE and blotted onto polyvinylidene difluoride membranes (Macherey-Nagel, Düren, Germany). Specific proteins were detected by chemiluminescence (Pierce) using anti-FLAG (Sigma), anti-Myc (Santa Cruz Biotechnology, Santa Cruz, CA), and anti-Tollip (Alexis Biochemicals, Lausen, Swizerland) antibodies.

Computational Methods—An initial computer model of the mouse IL-1RAcP TIR domain was generated by the program COMPOSER (41) as part of the molecular modeling package SYBYL 6.9 (Tripos Inc., St. Louis, MO). Three crystal structures from the Brookhaven Protein Data Bank were selected as templates by overall sequence identity: human TLR1 (1fyv [PDB] ), TLR2 (1fyw [PDB] ), and the TLR2 P681H mutant (1fyx [PDB] ) (37). Based on optimal sequence alignments, the structurally conserved regions (SCRs, Fig. 1) and an average C framework structure of the template SCRs were determined by an iterative approach, improving both the multiple alignment and the subsequent SCR framework by pairwise Needleman and Wunsch dynamic programming procedures with a similarity matrix constructed from inter-C distances. The backbone of each SCR of the IL-1RAcP TIR domain was then built by fitting the corresponding SCR from one of the known homologs (namely that with the highest block sequence identity, mostly TLR1) to the appropriate region of the framework. The least-squares fits are inversely weighted by the variation of the residue positions across the known structures. This approach provides a sufficient degree of diversity on constructing the SCRs of the model and avoids an arbitrary focus on one of the templates.

View larger version (28K): [in this window] [in a new window]   FIG. 1. Sequence alignment of the TIR domains of mouse IL-1RAcP and of human Toll-like receptors TLR1 and TLR2 as base of homology modeling with COMPOSER. -Helices and -strands are differently shaded. Additionally boxes 1–3 and SCRs (bold type) as used for model generation in COMPOSER are marked.

After adding hydrogens, the models were roughly energy-minimized usingthe Kollman force field (42) with Kollman all-atom charges (distance-dependent dielectricity constant, 4; 500 cycles; first 50 cycles with constrained backbone; steepest descent method) and finally optimized up to a root mean square (r.m.s.) gradient of 0.05 kcal/mol x Å (Powell conjugate gradient). Surfaces and electrostatic potentials of the model were calculated and visualized by the program MOLCAD implied in SYBYL 6.9.

Statistical Analysis—Statistical differences between mean values were analyzed using the two-sided Student's t test.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
A homology model of the putative three-dimensional structure of the TIR domain within IL-1RAcP was generated starting from three Protein Data Bank crystal structures of human TLR1 (1fyv [PDB] ), TLR2 (1fyw [PDB] ), and the TLR2 P681H mutant (1fyx [PDB] ) (37) and from multiple alignments of the primary and secondary structure elements of mouse IL-1RAcP and several members of the Toll/IL-1 receptor family. The alignment of IL-1RAcP with TLR1 and TLR2 generated by the program COMPOSER as base for model construction is presented in Fig. 1. In general, the structurally conserved regions SCR1–SCR8 contain the six -helices A, B, C, C', D, and E and the five -strands A, B, C, D, and E but additionally parts of the loops AA, BB, and DD and the turn BC. The identity scores of IL-1RAcP with TLR1 (28.2%) and TLR2 (25%) are at the lower limit for application of homology modeling approaches, but within the SCRs identical and conservatively mutated aa together amount to 50.5% (IL-1RAcP versus TLR1) and 49.5% (IL-1RAcP versus TLR2). The long loops BB and CD were selected and inserted by COMPOSER from one of the TIR domain templates (1fyv [PDB] ). The energy-minimized model of IL-1RAcP is shown in Fig. 2A. Due to the homology modeling approach, its domain structure closely resembles that of the templates. The r.m.s. distances of the C atoms of the SCRs amount to 1.11, 1.26, and 1.25 Å comparing the IL-1RAcP model with 1fyv [PDB] , 1fyw [PDB] , and 1fyx [PDB] , respectively. The TIR domain structure of IL-1RAcP is predicted to contain a central multiple -sheet that is surrounded by six -helices. The disulfide bridge between the cysteines 667 and 686 in TLR1 is neither present in TLR2 nor in IL-1RAcP. The conserved region box 1, mainly consisting of A, and box 2, including B and nearly the complete loop BB, are adjacent in space and present most of their side chains for interaction with adapter molecules (37) (see surface representation, Fig. 2B, and electrostatic potential, Fig. 2C). Box 2 contains a highly conserved proline residue at position 446 farthest away from the rest of the TIR domain. The identical course of the BB loop in the crystal structures of TLR2 and its P681H mutant (r.m.s. of C atoms, 0.27 Å) suggests that this proline does not play any structural role. The BB loops of TLR1 and TLR2 are similar too as indicated by an r.m.s. deviation of 0.97 Å, which is low compared with the overall r.m.s. distance of both TIR domains (2.34 Å). The C-terminal homology motif box 3 as part of E, possibly together with loop EE, represents a second region for potential recruitment of downstream adapter molecules (Fig. 2, A–C). The eight-amino acid stretch between Lys-527 and Pro-534 (nearly the complete loop EE) was recently shown to be critical for IL-1 responsiveness (39). In particular, the correct position of helix E including box 3 seems to be of importance since deletion mutants like 527–533 and 528–534, enabling the same packing of E as in the wild type, are fully responsive (Fig. 2D, for more details see "Discussion").

View larger version (103K): [in this window] [in a new window]   FIG. 2. Models of the IL-1RAcP TIR domain. A, ribbon and tube representation of the tertiary TIR domain structure derived from the energy-minimized COMPOSER model. Secondary structure elements are labeled and colored (-helices, magenta; -strands, yellow; loops, cyan). Additionally the backbone and side chains of aa in conserved regions (box 1–3) and loop EE are drawn. B, MOLCAD surface of the model calculated without hydrogens. The position and the colors are the same as in A. C, electrostatic potential (based on Kollman all-atom charges) of the wild type model in A represented on a MOLCAD surface (hydrogens included). The position corresponds to A and B. The color scale (listed from positive to negative) is as follows: brown (most positive), orange, yellow, gray, cyan (about neutral), blue, and violet (most negative). White arrows, negatively charged pockets as possible binding sites for positively charged aa such as those indicated by golden arrows. Green arrow, neutral area near Pro-446. D, comparison of the E-loop EE-E motifs of the wild type model (ribbon and tube representation; C atoms of displayed aa, white) and of energy-minimized models of the deletion mutants 528–534 (orange) and 527–533 (green). Alignments of all C atoms except aa 527–534 indicate close similarity of the structures (r.m.s. distance wt versus 528–534, 0.26 Å; r.m.s. distance wt versus 527–533, 0.31 Å). The C distances between Lys-525 and Gln-535 (marked as balls) amount to 9.9 Å (wt), 10.7 Å (528–534), and 10.1 Å (527–533).

View larger version (11K): [in this window] [in a new window]   FIG. 3. Effect of mutations of Pro-446 within IL-1RAcP on IL-1-induced IL-2 promoter activation and IL-2 secretion. EL4D6/76 cells were co-transfected with 1 µg of luciferase reporter and 500 ng of the indicated IL-1RAcP constructs as described under "Experimental Procedures." 24 h after transfection cells were stimulated with 10 units/ml rhIL-1 or 250 ng/ml rmIL-18 together with 10 ng/ml PMA. After incubation for an additional 18 h luciferase activity was determined in triplicates as a measure of IL-2 promoter activation. IL-2 secretion into the supernatants was detected by sandwich enzyme-linked immunosorbent assay. Data are expressed as ratio of the luciferase activity or the IL-2 secretion, respectively, of the IL-1 and IL-18 response and represent means ± S.E. of three separate experiments performed in at least triplicates (*, p < 0.001 compared with wt). Ctrl., control.

View larger version (11K): [in this window] [in a new window]   FIG. 4. Effect of mutations of Gly-447 within IL-1RAcP on IL-1-induced IL-2 promoter activation and IL-2 secretion. EL4D6/76 cells were transiently transfected and stimulated as described in the legend to Fig. 3. Afterward IL-2 production and activation of the IL-2 promoter were detected as described. Data are expressed as ratio of the luciferase activity or IL-2 secretion, respectively, of the IL-1 and IL-18 response and represent means ± S.E. of three separate experiments performed in at least triplicates (*, p < 0.001 compared with wt). Ctrl., control.

View larger version (16K): [in this window] [in a new window]   FIG. 5. Effect of different mutant variants of IL-1RAcP on IL-1 responsiveness. EL4D6/76 cells were transiently transfected with pGL3-IL-2(-303) together with the indicated IL-1RAcP constructs and stimulated as described elsewhere. After stimulation IL-2 promoter activation and IL-2 secretion were determined as described. Data are expressed as means ± S.E. of three separate experiments performed in at least duplicate (*, p < 0.01; **, p < 0.001 compared with wt).

View larger version (46K): [in this window] [in a new window]   FIG. 6. Binding of Tollip and MyD88 to different mutants of IL-1RAcP. 293IL-1RI cells were transiently transfected with wt or mutant IL-1RAcP together with either Tollip or MyD88 as indicated and described under "Experimental Procedures." After incubation in the presence of IL-1, cell lysates were prepared followed by immunoprecipitation of IL-1RAcP using their FLAG epitope tags. Afterward proteins were separated by SDS-PAGE and visualized by Western blotting for MyD88 (A) and Tollip (B) (the asterisk indicates an unspecific band always found when blotting Tollip after immunoprecipitation using anti-FLAG reagents). IP, immunoprecipitation; WB, Western blot.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

To understand the structural basis for the elimination of receptor signaling caused by the LPS^d mutation (43, 46), Xu et al. (37) determined the crystal structure of the experimentally generated Pro-681 His mutant of the human TLR2 TIR domain. The results obtained reveal that the proline residue does not have a structural role. Our own observations on the structure of the TIR domain within IL-1RAcP are in good agreement with these findings. The defective IL-1 response seen after mutation of Pro-446 and Gly-447 by charged aa residues (and by His) is not due to a disruption of the TIR domain structure itself but rather to the polar modification of a nonpolar interaction site for other TIR domain-harboring molecules since we found that substitution with neutral aa like in Pro-446 Ala is without any effect on the IL-1 response. As expected, binding of MyD88 to the Pro-446 His mutant of IL-1RAcP was abolished. This is in line with previous results demonstrating that the Pro-681 His mutant of human TLR2 (37) as well as the Pro-712 His mutant of human TLR4 (48) do not recruit MyD88.

Interestingly in our study mutation of Pro-446 Ala also blocked binding of MyD88 to IL-1RAcP without altering IL-1 responsiveness and recruitment of Tollip. Human and mouse TLR3 contain an alanine in place of the conserved proline. Signaling mediated via TLR3 appears to use specific pathways (49) independent of IRAK in contrast to all other TLRs (50). Very recently, specific tyrosine residues within the cytoplasmic part of human TLR3 have been identified as being essential for the assembly of the signaling complex (51). TLR3 recognizes intracellular double-stranded RNA, a common product of viral infections, and triggers downstream signals leading to interferon production via activation of interferon and NF-B promoter independent of the adapter molecules MyD88 and TIR domain-containing adapter protein (TIRAP) (52). However, when activated by poly(I·C), TLR3 induces cytokine production through a signaling pathway dependent on MyD88 (49). It is therefore not possible to conclude that an Ala mutation of the conserved Pro is generally incompatible with MyD88 binding.

The second region of interest is represented by box 3 and the loop EE close to the C-terminal -helix corresponding to the analogous loop in human TLR1 and TLR2 (37). To characterize critical aa within this loop in more detail we performed an extensive mutagenesis analysis leading to the identification of several mutant variants of IL-1RAcP that differentially affect IL-1 responsiveness. Among them, mutants harboring double and triple aa substitutions at certain positions showed improved IL-1 responsiveness. In a previous study we could demonstrate that truncation of the C terminus of IL-1RAcP up to aa 544 leads to a significantly enhanced IL-1-induced activation of the IL-2 promoter and IL-2 production (39). These observations might be explained by the removal of interaction with a negative regulator such as the IL-1 receptor-interacting protein 1 (53). IL-1 receptor-interacting protein 1 is thought to associate with the IL-1 receptor and to maintain a G protein in its GDP-bound form thus suppressing IL-1 responses (53).

It may be suggested that full functionality of IL-1RAcP depends on the proper three-dimensional arrangement of box 3 with respect to boxes 1 and 2 and thus on the native folding of all secondary structure elements. Any translation or rotation of helix E (box 3) relative to the remainder of the TIR domain may interfere with either dimerization with IL-1RI or interaction with adapters or both. This hypothesis implies an essential structural role of the loop EE in enabling the native position of helix E. The sequence-response relationships of loop EE mutants (Table I) are rather nondistinctive and do not indicate specific interactions of side chains with adapter molecules necessary for functionality, but they might reflect how different courses of the loop affect IL-2 promoter activation and IL-2 secretion. Direct evidence of the importance of the helix E position results from deletion mutants (39): 527–534 was not responsive, whereas the deletion of only one aa less (527–533 and 528–534) led to nearly full responsiveness. This raises the question how many residues are at least needed to build a loop between the last aa of E, Lys-525, and the first aa of E, Gln-535, without rigid body movement of both secondary structure elements. In the IL-1RAcP model, the C atom distance of these aa amounts to 9.9 Å (Fig. 2D). It is not possible to bridge this distance by one aa (Trp-526), corresponding to 527–534. If, however, two aa (Trp-526 and Lys-527 or Pro-534) are inserted, the gap may be closed without distortion of E and E. Results of SYBYL loop searches connecting Lys-525 and Gln-535 with two aa by suitable templates from 1480 proteins of a binary data base have shown that the distance between these residues may be close to the wild type reference (Fig. 2D).

The possible role of the conserved regions box 1 and 2 on the one hand and box 3 on the other hand may be further elucidated by analysis of the electrostatic potential of the IL-1RAcP TIR domain model (Fig. 2C). Obviously positive charges predominate in box 3 and its close neighborhood on E and loop EE, whereas the exposed surface of boxes 1 and 2 is mainly negative or neutral (e.g. at Pro-446). Also the different shape of these regions suggests that box 1/2-box 3 interactions could play a role: negatively charged pockets in or close to box 1 and 2 may be possible binding sites for protruding positively charged side chains in box 3, E, and loop EE. In box 1 and 2 the correspondence of charged residues is very close among IL-1RAcP, IL-1RI, and MyD88, in box 3 there is correspondence only between IL-1RAcP and IL-1RI, and in all conserved regions there is lower correspondence among IL-1RAcP, TLR1, and TLR2. Therefore IL-1RAcP/IL-1RI dimerization and recruitment of MyD88 might rely on box 1/2-box 3 interactions. This hypothesis is, however, rather speculative since the homology models do not allow refined simulations of proteinprotein interactions without detailed studies of appropriate IL-1RAcP mutants and their binding to IL-1RI and adapters. Nevertheless the surface and the electrostatic potential in Fig. 2, B and C, provides suggestions about two topological regions that may both be important for the recruitment of different TIR domains and contribute to the formation of multiprotein complexes necessary for functionality.

Our results on MyD88 and Tollip (see Fig. 6) suggest that the IL-1RAcP wt loop EE is, in conjunction with box 3, necessary for MyD88 binding, whereas recruitment of Tollip can occur independently of box 3 (Fig. 6) thus confirming previous results obtained by Burns et al. (20). However, our findings are still limited and somewhat contradictory since the mutant W51 was fully and the 8 x Ala mutant was partially IL-1-responsive. The responsiveness of these mutants could be due to the MyD88-independent recruitment of IRAK-2 to the receptor complex as suggested by Boch et al. (54). Signaling pathways activated by IL-1 also include the activation of phosphatidylinositol 3-kinase. The regulatory subunit p85 has been reported to interact directly with IL-1RI (55, 56) or with IL-1RAcP (57). Whereas the phosphatidylinositol 3-kinase pathway is sufficient to completely activate the transcription factor AP-1, it only partially activates NF-B (28). Since the IL-2 promoter contains consensus binding sites for AP-1 (58), it could be speculated that IL-2 promoter activation is at least in part mediated by phosphatidylinositol 3-kinase. We do not know to what extent this pathway is affected by our mutants and thus how it might influence the partial responsiveness of the major subgroup of IL-1RAcP mutants analyzed so far.

Questions such as the following arise. Is, under selected conditions, MyD88 dispensable for IL-2 promoter activation and IL-2 secretion? Does MyD88 interact, possibly by its boxes 1 and 2, with a site comprising box 3 and loop EE of IL-1RAcP, e.g. via involvement of Lys-530 not present in all negatively tested mutants in Fig. 6? These questions are far from being answered and will be the subject of further investigations. Our recent model of a synergistic interaction between aa 527–534 and box 3 (39) must be modified in some respect since not Tollip as supposed but rather MyD88 should interact with loop EE according to the observed binding pattern of both adapters to mutants.

In summary, our structural and functional analyses identify two distinct regions within the TIR domain of IL-1RAcP that might function as putative adapter protein interaction sites in the IL-1 receptor complex. The identification of these regions enhances our understanding of the molecular mechanisms in IL-1 and TIR domain signaling crucial for acquired and innate immunity. The generation of a fibroblast-type cell line derived from IL-1RI-deficient mice will help us to examine the role of the aa under study in the interaction between IL-1RI and IL-1RAcP.

	FOOTNOTES

 
^* This work was supported by grants from the Deutsche Forschungsgemeinschaft and by European Community Grants BIO4-CT97-2107 and QLG1-CT-1999-00549 (to W. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** Present address: Pfizer GmbH, D-76139 Karlsruhe, Germany.

Supported by multimmune GmbH. To whom correspondence should be addressed: Abteilung Hämatologie und Onkologie, Universität Regensburg, D-93053 Regensburg, Germany. Tel.: 49-941-944-7124; Fax: 49-941-944-7123; E-mail: juergen.radons{at}klinik.uni-regensburg.de.

¹ The abbreviations used are: IL, interleukin; aa, amino acid(s); IL-1RI, IL-1 receptor type I; IL-1RAcP, IL-1 receptor accessory protein; IRAK, IL-1 receptor-associated kinase; MyD88, myeloid differentiation factor 88; NF-B, nuclear factor B; PLK, Pelle-like kinase; PMA, phorbol 12-myristate 13-acetate; r.m.s., root mean square; TBS, Tris-buffered saline; TAK, transforming growth factor--activated kinase; TIR, Toll/IL-1 receptor; Tollip, Toll-interacting protein; TLR, Toll-like receptor; TRAF-6, tumor necrosis factor- receptor-associated factor 6; wt, wild type; h, human; m, mouse; r, recombinant; SCR, structurally conserved region; LPS, lipopolysaccharide.

	ACKNOWLEDGMENTS

 
We thank H. Wesche for the kind gift of the pRK7-Myc/MyD88 expression vector.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

1. Dinarello, C. A. (1996) Blood 87, 2095–2147[Abstract/Free Full Text]
2. Dinarello, C. A. (1998) Int. Rev. Immunol. 16, 457–499[Medline] [Order article via Infotrieve]
3. Heguy, A., Baldari, C., Bush, K., Nagele, R., Newton, R. C., Robb, R. J., Horuk, R., Telford, J. L., and Melli, M. (1991) Cell Growth Differ. 2, 311–315[Abstract]
4. O'Neill, L. A. (2000) Sci. STKE http://stke.sciencemag.org/cgi/content/full/OC_sigtrans;2000/44/re1
5. Sims, J. E., March, C. J., Cosman, D., Widmer, M. B., MacDonald, H. R., McMahan, C. J., Grubin, C. E., Wignall, J. M., Jackson, J. L., Call, S. M., Friend, D., Alpert, A. R., Gillis, S. R., Urdal, D. L., and Dower, S. K. (1988) Science 241, 585–589[Abstract/Free Full Text]
6. Greenfeder, S. A., Nunes, P., Kwee, L., Labow, M., Chizzonite, R. A., and Ju, G. (1995) J. Biol. Chem. 270, 13757–13765[Abstract/Free Full Text]
7. Hofmeister, R., Wiegmann, K., Korherr, C., Bernardo, K., Krönke, M., and Falk, W. (1997) J. Biol. Chem. 272, 27730–27736[Abstract/Free Full Text]
8. Huang, J., Gao, X., Li, S., and Cao, Z. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 12829–12832[Abstract/Free Full Text]
9. Korherr, C., Hofmeister, R., Wesche, H., and Falk, W. (1997) Eur. J. Immunol. 27, 262–267[Medline] [Order article via Infotrieve]
10. Wesche, H., Korherr, C., Kracht, M., Falk, W., Resch, K., and Martin, M. U. (1997) J. Biol. Chem. 272, 7727–7731[Abstract/Free Full Text]
11. Li, S., Strelow, A., Fontana, E. J., and Wesche, H. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 5567–5572[Abstract/Free Full Text]
12. Muzio, M., Ni, J., Feng, P., and Dixit, V. M. (1997) Science 278, 1612–1615[Abstract/Free Full Text]
13. Rosati, G., and Martin, M. U. (2002) Biochem. Biophys. Res. Commun. 293, 1472–1477[CrossRef][Medline] [Order article via Infotrieve]
14. Rosati, G., and Martin, M. U. (2002) Biochem. Biophys. Res. Commun. 297, 52–58[CrossRef][Medline] [Order article via Infotrieve]
15. Trofimova, M., Sprenkle, A. B., Green, M., Sturgill, T. W., Goebl, M. G., and Harrington, M. A. (1996) J. Biol. Chem. 271, 17609–17612[Abstract/Free Full Text]
16. Wesche, H., Gao, X., Li, X., Kirschning, C. J., Stark, G. R., and Cao, Z. (1999) J. Biol. Chem. 274, 19403–19410[Abstract/Free Full Text]
17. Grosshans, J., Bergmann, A., Haffter, P., and Nusslein-Volhard, C. (1994) Nature 372, 563–566[CrossRef][Medline] [Order article via Infotrieve]
18. Cao, Z., Henzel, W. J., and Gao, X. (1996) Science 271, 1128–1131[Abstract]
19. Yamin, T. T., and Miller, D. K. (1997) J. Biol. Chem. 272, 21540–21547[Abstract/Free Full Text]
20. Burns, K., Clatworthy, J., Martin, L., Martinon, F., Plumpton, C., Maschera, B., Lewis, A., Ray, K., Tschopp, J., and Volpe, F. (2000) Nat. Cell Biol. 2, 346–351[CrossRef][Medline] [Order article via Infotrieve]
21. Wesche, H., Henzel, W. J., Shillinglaw, W., Li, S., and Cao, Z. (1997) Immunity 7, 837–847[CrossRef][Medline] [Order article via Infotrieve]
22. Burns, K., Martinon, F., Esslinger, C., Pahl, H., Schneider, P., Bodmer, J. L., Di Marco, F., French, L., and Tschopp, J. (1998) J. Biol. Chem. 273, 12203–12209[Abstract/Free Full Text]
23. Dunne, A., and O'Neill, L. A. (2003) Sci. STKE http://stke.sciencemag.org/cgi/content/full/OC_sigtrans;2003/171/re3
24. Cao, Z., Xiong, J., Takeuchi, M., Kurama, T., and Goeddel, D. V. (1996) Nature 383, 443–446[CrossRef][Medline] [Order article via Infotrieve]
25. Takaesu, G., Kishida, S., Hiyama, A., Yamaguchi, K., Shibuya, H., Irie, K., Ninomiya-Tsuji, J., and Matsumoto, K. (2000) Mol. Cell 5, 649–658[CrossRef][Medline] [Order article via Infotrieve]
26. Ninomiya-Tsuji, J., Kishimoto, K., Hiyama, A., Inoue, J., Cao, Z., and Matsumoto, K. (1999) Nature 398, 252–256[CrossRef][Medline] [Order article via Infotrieve]
27. Song, H. Y., Regnier, C. H., Kirschning, C. J., Goeddel, D. V., and Rothe, M. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 9792–9796[Abstract/Free Full Text]
28. Guo, F., and Wu, S. (2000) Inflammation 24, 305–316[CrossRef][Medline] [Order article via Infotrieve]
29. Auron, P. E. (1998) Cytokine Growth Factor Rev. 9, 221–237[CrossRef][Medline] [Order article via Infotrieve]
30. Ridley, S. H., Sarsfield, S. J., Lee, J. C., Bigg, H. F., Cawston, T. E., Taylor, D. J., DeWitt, D. L., and Saklatvala, J. (1997) J. Immunol. 158, 3165–3173[Abstract]
31. Baud, V., Liu, Z. G., Bennett, B., Suzuki, N., Xia, Y., and Karin, M. (1999) Genes Dev. 13, 1297–1308[Abstract/Free Full Text]
32. Martin, M. U., and Wesche, H. (2002) Biochim. Biophys. Acta 1592, 265–280[Medline] [Order article via Infotrieve]
33. O'Neill, L. A., Fitzgerald, K. A., and Bowie, A. G. (2003) Trends Immunol. 24, 286–290[CrossRef][Medline] [Order article via Infotrieve]
34. Gay, N. J., and Keith, F. J. (1991) Nature 351, 355–356[Medline] [Order article via Infotrieve]
35. Bowie, A., and O'Neill, L. A. (2000) J. Leukoc. Biol. 67, 508–514[Abstract]
36. Kopp, E. B., and Medzhitov, R. (1999) Curr. Opin. Immunol. 11, 13–18[CrossRef][Medline] [Order article via Infotrieve]
37. Xu, Y., Tao, X., Shen, B., Horng, T., Medzhitov, R., Manley, J. L., and Tong, L. (2000) Nature 408, 111–115[CrossRef][Medline] [Order article via Infotrieve]
38. Stricker, K., Serfling, E., Krammer, P. H., and Falk, W. (1993) Eur. J. Immunol. 23, 1475–1480[Medline] [Order article via Infotrieve]
39. Radons, J., Gabler, S., Wesche, H., Korherr, C., Hofmeister, R., and Falk, W. (2002) J. Biol. Chem. 277, 16456–16463[Abstract/Free Full Text]
40. Laemmli, U. K. (1970) Nature 227, 680–685[CrossRef][Medline] [Order article via Infotrieve]
41. Blundell, T., Carney, D., Gardner, S., Hayes, F., Howlin, B., Hubbard, T., Overington, J., Singh, D. A., Sibanda, B. L., and Sutcliffe, M. (1988) Eur. J. Biochem. 172, 513–520[Medline] [Order article via Infotrieve]
42. Weiner, S. J., and Kollman, P. A. (1986) J. Comput. Chem. 7, 230–252[CrossRef]
43. Poltorak, A., He, X., Smirnova, I., Liu, M. Y., Huffel, C. V., Du, X., Birdwell, D., Alejos, E., Silva, M., Galanos, C., Freudenberg, M., Ricciardi-Castagnoli, P., Layton, B., and Beutler, B. (1998) Science 282, 2085–2088[Abstract/Free Full Text]
44. Qureshi, S. T., Lariviere, L., Leveque, G., Clermont, S., Moore, K. J., Gros, P., and Malo, D. (1999) J. Exp. Med. 189, 615–625[Abstract/Free Full Text]
45. Hoshino, K., Takeuchi, O., Kawai, T., Sanjo, H., Ogawa, T., Takeda, Y., Takeda, K., and Akira, S. (1999) J. Immunol. 162, 3749–3752[Abstract/Free Full Text]
46. Underhill, D. M., Ozinsky, A., Hajjar, A. M., Stevens, A., Wilson, C. B., Bassetti, M., and Aderem, A. (1999) Nature 401, 811–815[CrossRef][Medline] [Order article via Infotrieve]
47. Ozinsky, A., Underhill, D. M., Fontenot, J. D., Hajjar, A. M., Smith, K. D., Wilson, C. B., Schroeder, L., and Aderem, A. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 13766–13771[Abstract/Free Full Text]
48. Rhee, S. H., and Hwang, D. (2000) J. Biol. Chem. 275, 34035–34040[Abstract/Free Full Text]
49. Alexopoulou, L., Holt, A. C., Medzhitov, R., and Flavell, R. A. (2001) Nature 413, 732–738[CrossRef][Medline] [Order article via Infotrieve]
50. Jiang, Z., Zamanian-Daryoush, M., Nie, H., Silva, A. M., Williams, B. R. G., and Li, X. (2003) J. Biol. Chem. 278, 16713–16719[Abstract/Free Full Text]
51. Sarkar, S. N., Smith, H. L., Rowe, T. M., and Sen, G. C. (2003) J. Biol. Chem. 278, 4393–4396[Abstract/Free Full Text]
52. Matsumoto, M., Kikkawa, S., Kohase, M., Miyake, K., and Seya, T. (2002) Biochem. Biophys. Res. Commun. 293, 1364–1369[CrossRef][Medline] [Order article via Infotrieve]
53. Sims, J. E., Bird, T. A., and Mitcham, J. L. (1996) Eur. Cytokine Netw. 7, 480 (abstr.)
54. Boch, J. A., Yoshida, Y., Koyama, Y., Wara-Aswapati, N., Peng, H., Unlu, S., and Auron, P. E. (2003) Biochem. Biophys. Res. Commun. 303, 525–531[CrossRef][Medline] [Order article via Infotrieve]
55. Reddy, S. A., Huang, J. H., and Liao, W. S. (1997) J. Biol. Chem. 272, 29167–29173[Abstract/Free Full Text]
56. Marmiroli, S., Bavelloni, A., Faenza, I., Sirri, A., Ognibene, A., Cenni, V., Tsukada, J., Koyama, Y., Ruzzene, M., Ferri, A., Auron, P. E., Toker, A., and Maraldi, N. M. (1998) FEBS Lett. 438, 49–54[CrossRef][Medline] [Order article via Infotrieve]
57. Sizemore, N., Leung, S., and Stark, G. R. (1999) Mol. Cell. Biol. 19, 4798–4805[Abstract/Free Full Text]
58. Jain, J., Loh, C., and Rao, A. (1995) Curr. Opin. Immunol. 7, 333–342[CrossRef][Medline] [Order article via Infotrieve]

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