Crystal Structure of the Toll/Interleukin-1 Receptor Domain of Human IL-1RAPL

The Toll/interleukin-1 receptor (TIR) domain is conserved in the intracellular regions of Toll-like receptors (TLRs) and interleukin-1 receptors (IL-1Rs) as well as in several cytoplasmic adapter molecules. This domain has crucial roles in signal transduction by these receptors for host immune response. Here we report the crystal structure at 2.3-Å resolution of the TIR domain of human IL-1RAPL, the first structure of a TIR domain of the IL-1R superfamily. There are large structural differences between this TIR domain and that of TLR1 and TLR2. Helix αD in IL-1RAPL is almost perpendicular to its equivalent in TLR1 or TLR2. The BB loop contains a hydrogen bond unique to IL-1RAPL between Thr residues at the 8th and 10th positions. The structural and sequence diversity among these domains may be important for specificity in the signal transduction by these receptors. A dimer of the TIR domain of IL-1RAPL is observed in the crystal, although this domain is monomeric in solution. Residues in the dimer interface are mostly unique to IL-1RAPL, which is consistent with the distinct functional roles of this receptor. Our functional studies show IL-1RAPL can activate JNK but not the ERK or the p38 MAP kinases, whereas its close homolog, TIGIRR, cannot activate JNK. Deletion mutagenesis studies show that the activation of JNK by IL-1RAPL does not depend on the integrity of its TIR domain, suggesting a distinct mechanism of signaling through this receptor. The Toll/interleukin-1 receptor (TIR) domain is conserved in the intracellular regions of Toll-like receptors (TLRs) and interleukin-1 receptors (IL-1Rs) as well as in several cytoplasmic adapter molecules. This domain has crucial roles in signal transduction by these receptors for host immune response. Here we report the crystal structure at 2.3-Å resolution of the TIR domain of human IL-1RAPL, the first structure of a TIR domain of the IL-1R superfamily. There are large structural differences between this TIR domain and that of TLR1 and TLR2. Helix αD in IL-1RAPL is almost perpendicular to its equivalent in TLR1 or TLR2. The BB loop contains a hydrogen bond unique to IL-1RAPL between Thr residues at the 8th and 10th positions. The structural and sequence diversity among these domains may be important for specificity in the signal transduction by these receptors. A dimer of the TIR domain of IL-1RAPL is observed in the crystal, although this domain is monomeric in solution. Residues in the dimer interface are mostly unique to IL-1RAPL, which is consistent with the distinct functional roles of this receptor. Our functional studies show IL-1RAPL can activate JNK but not the ERK or the p38 MAP kinases, whereas its close homolog, TIGIRR, cannot activate JNK. Deletion mutagenesis studies show that the activation of JNK by IL-1RAPL does not depend on the integrity of its TIR domain, suggesting a distinct mechanism of signaling through this receptor. Toll-like receptors (TLRs) 1The abbreviations used are: TLR, Toll-like receptors; TIR, Toll/interleukin-1 receptor; IL-1R, interleukin-1 receptor; JNK, c-Jun NH2-terminal kinase; ERK, extracellular signal-regulated kinase; MAP, mitogen-activated protein; TIGIRR, three immunoglobulin domain-containing IL-1 receptor-related; IL-1RAPL, IL-1R accessory protein-like; r.m.s., root mean square; MAD, multi-wavelength anomalous diffraction. 1The abbreviations used are: TLR, Toll-like receptors; TIR, Toll/interleukin-1 receptor; IL-1R, interleukin-1 receptor; JNK, c-Jun NH2-terminal kinase; ERK, extracellular signal-regulated kinase; MAP, mitogen-activated protein; TIGIRR, three immunoglobulin domain-containing IL-1 receptor-related; IL-1RAPL, IL-1R accessory protein-like; r.m.s., root mean square; MAD, multi-wavelength anomalous diffraction. and interleukin-1 receptors (IL-1Rs) have crucial roles in host immune and inflammatory responses (1Dunne A. O'Neill L.A.J. Science's STKE. 2003; (http://stke.sciencemag.org/cgi/content/full/sigtrans;2003/171/re3)Google Scholar). A total of 10 TLRs have been identified from the human genome. They recognize conserved molecular patterns in pathogenic microbial organisms, and this recognition is indispensable for the activation of the innate immune systems as well as the adaptive immune systems in vertebrates (2Medzhitov R. Janeway Jr., C.A. Cell. 1997; 91: 295-298Google Scholar, 3Anderson K.V. Curr. Opin. Immunol. 2000; 12: 13-19Google Scholar). Ten different IL-1Rs are present in the human genome. IL-1R type I (IL-1RI) and IL-1R accessory protein (IL-1RAcP) mediate the pleiotropic effects of the proinflammatory cytokine IL-1, whereas IL-18R and AcPL function as receptors for the IL-18 cytokine (4Sims J.E. Curr. Opin. Immunol. 2002; 14: 117-122Google Scholar). As their names imply, IL-1RI and IL-18R have high affinities for their ligands, whereas the accessory proteins (IL-1RAcP and AcPL) cannot bind the ligand directly. The TLRs and IL-1Rs have entirely different extracellular domains. The TLRs contain many copies of the leucine-rich repeat (LRR), whereas the IL-1Rs contain three copies of the Ig-like domains. However, these two families of receptors share a conserved intracellular domain, known as the Toll/interleu-kin-1 receptor (TIR) domain. This domain is also present in several cytoplasmic molecules, such as MyD88, TIRAP/MAL, TRIF/TICAM, and TIRP/TRAM, which function as signal adapters for these receptors (5O'Neill L.A.J. Fitzgerald K.A. Bowie A.G. Trends Immunol. 2003; 24: 286-289Google Scholar). TIR domains contain 150–200 amino acid residues, and their sequences are weakly conserved among the TLRs, IL-1Rs, and the adapter molecules (Fig. 1). The amino acid sequence identity between any pair of TIR domains is generally about 25%. The authenticity and integrity of this domain is crucial for the functions of these receptors. For example, a single-site mutation (Pro-712 → His, also known as the Lpsd mutation) in the TIR domain of TLR4 renders mice unresponsive to lipopolysac-charide, the cell wall component of Gram-negative bacteria (6Poltorak 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. Beutler B. Science. 1998; 282: 2085-2088Google Scholar). This Pro residue is conserved in many of the TIR domains (Fig. 1), and the mutation of this residue to His in these other proteins generally produces dominant negative molecules. IL-1RAPL (IL-1R accessory protein-like) was identified from patients suffering from nonspecific X-linked mental retardation (MRX) (7Carrie A. Jun L. Bienvenu T. Vinet M.-C. McDonell N. Couvert P. Zemni R. Cardona A. Buggenhout G.V. Frints S. Hamel B. Moraine C. Ropers H.H. Strom T. Howell G.R. Whittaker A. Ross M.T. Kahn A. Fryns J.-P. Beldjord C. Marynen P. Chelly J. Nat. Genet. 1999; 23: 25-31Google Scholar). Non-overlapping deletions as well as a nonsense mutation in the intracellular domain of this receptor are found in MRX patients. The exact function of this receptor is currently not known. It is highly expressed in postnatal brain structures that are important for memory and learning. IL-1RAPL was independently characterized from homology searches in the Expressed Sequence Tag (EST) data base, which also found TIGIRR (three immunoglobulin domain-containing IL-1 receptor-related) as its close homolog (8Born T.L. Smith D.E. Garka K.E. Renshaw B.R. Bertles J.S. Sims J.E. J. Biol. Chem. 2000; 275: 29946-29954Google Scholar). The TIR domains of IL-RAPL and TIGIRR share 72% amino acid sequence identity (Fig. 1). Like the other IL-1Rs, these two receptors contain a TIR domain in their intracellular region. However, they also contain a 130-residue segment C-terminal to the TIR domain, which is absent in most of the other TLRs and IL-1Rs. Recent studies suggest that residues in this C-terminal segment of IL-1RAPL may interact with neuronal calcium sensor-1 (NCS-1), and IL-1RAPL may have a role in regulating exocytosis of secretory and neurotransmitter substances (9Bahi N. Friocourt G. Carrie A. Graham M.E. Weiss J.L. Chafey P. Fauchereau F. Burgoyne R.D. Chelly J. Hum. Mol. Genet. 2003; 12: 1415-1425Google Scholar). NCS-1 is up-regulated in schizophrenic and bipolar patients (10Koh P.O. Undie A.S. Kabbani N. Levenson R. Goldman-Rakic P.S. Lidow M.S. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 313-317Google Scholar). We have recently reported the crystal structures of the TIR domains of human TLR1 and TLR2 (11Xu Y. Tao X. Shen B. Horng T. Medzhitov R. Manley J.L. Tong L. Nature. 2000; 408: 111-115Google Scholar, 12Tao X. Xu Y. Zheng Y. Beg A. Tong L. Biochem. Biophys. Res. Commun. 2002; 299: 216-221Google Scholar). The structures contain a central five-stranded fully parallel β-sheet, which is surrounded by α-helices on both faces. A loop in the structure, known as the BB loop, forms a prominent feature on the surface of the domain and corresponds to a region of significant sequence conservation among the TIR domains (Fig. 1). The Lpsd mutation of TLR4 is located at the very tip of this loop, confirming the functional importance of the BB loop. The TIR domains of TLR1 and TLR2 share 50% sequence identity, but there are clear structural differences between them. The sequences of the TIR domains of the IL-1Rs are significantly more divergent from those of TLR1 and TLR2 (Fig. 1). We report here the crystal structure of the TIR domain of human IL-1RAPL at 2.3-Å resolution, the first structure from the IL-1R superfamily. The TIR domain has the same overall fold, but there are significant differences to the structures of the TIR domain of the TLRs. The crystal structure also reveals a dimeric association of TIR domains, which may be important for the functions of this receptor. Protein Expression, Purification, and Crystallization—The TIR domain of human IL-1RAPL (residues 403–563) was subcloned into the pET26b vector (Novagen) and overexpressed in Escherichia coli at 20 °C. The recombinant protein contains a hexahistidine tag at the C terminus. After cell lysis, the soluble protein was purified by nickel-agarose affinity chromatography and anion exchange and gel filtration chromatography. The protein was concentrated to 10 mg/ml in a buffer containing 25 mm Tris (pH 7.5), 200 mm NaCl, 5 mm dithiothreitol, and 5% (v/v) glycerol and stored at -80 °C. The C-terminal His tag was not removed for crystallization. For the production of selenomethionyl proteins, the expression construct was transformed into the methionine auxotroph E. coli DL41(DE3) cells. Bacterial growth was carried out in defined LeMaster media (13Hendrickson W.A. Horton J.R. LeMaster D.M. EMBO J. 1990; 9: 1665-1672Google Scholar), and the protein was purified using the same protocol as for the wild-type protein. Protein Crystallization—Crystals of the TIR domain were prepared by the vapor diffusion method at 4 °C. Initial crystallization conditions for the protein were identified by sparse matrix screening with commercial kits (Hampton Research). The reservoir solution contained 100 mm sodium acetate (pH 4.6), 8% (w/v) polyethylene glycol 4000, 5% (v/v) ethylene glycol, and 5 mm dithiothreitol. The crystals were cryo-protected with the reservoir solution supplemented with 35% (v/v) ethylene glycol and flash-frozen in liquid propane for data collection at 100 K. Data Collection and Processing—A selenomethionyl multiwave-length anomalous diffraction (MAD) data set to 2.3-Å resolution was collected on an ADSC CCD at the X4A beamline at Brookhaven National Laboratory. Three wavelengths were used: 0.9793 Å (edge), 0.9792 Å (peak), and 0.9200 Å (high energy remote). The diffraction images were processed and scaled with the HKL package (14Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Google Scholar). The crystal belongs to the space group P212121, with unit cell parameters of a = 52.0 Å, b = 53.3 Å, and c = 183.2 Å. There are two molecules in the asymmetric unit, giving a Vmax of 3.1 Å3/dalton. The data processing statistics are summarized in Table I.Table ISummary of crystallographic informationMaximum resolution (Å)2.3Number of observations150,158Rmerge (%)aRmerge = Σh Σi |Ihi - 〈Ih〉|/Σh Σi Ihi. The numbers in parentheses are for the highest resolution shell5.4 (19.7)Figure of merit from MAD phasing0.60Resolution range for refinement29-2.3Number of reflections41,323Completeness (%)95 (82)R factorbR = Σh|Fho - Fhc|/ΣhFho in bond in bond = Σh Σi |Ihi - 〈Ih〉|/Σh Σi Ihi. The numbers in parentheses are for the highest resolution = Σh|Fho - Fhc|/ΣhFho in a and of were with the MAD data using the J. Biol. 1999; Scholar), which also the to 2.3-Å After by located of the residues in the two molecules. residues were into the with the M. A. Scholar). The structure was carried out with the P. J. M. T. Biol. 1998; Scholar). The statistics on the structure are summarized in Table I. were with of of of and the of or using to the The of was by the of of the vector were for at with of buffer After 50% of the was used to and an equivalent was used for and was using of expression were as a for and expressed as the vector The of IL-1RAPL were by at in the with the The crystal structure of the Toll/interleukin-1 receptor (TIR) domain of human IL-1RAPL has been at 2.3-Å resolution by the selenomethionyl MAD method W.A. Science. Scholar). The of the as well as the of observed were with the MAD data J. Biol. 1999; Scholar), which the of of the residues of the two molecules in the asymmetric The has with the observed diffraction with an of The root mean in bond is Å, and that in bond is A total of of the residues is in the most and the of the residues is in the regions of the The expression construct residues of IL-1RAPL, which the first 20 residues of the intracellular region of the receptor as well as the 130-residue segment at the C that is present in this receptor and The contains residues residues for and residues and for the other in the crystallographic asymmetric The have been at the Protein Data to the of TIR is the first structure of a TIR domain from the IL-1R of receptors. The structure of the TIR domain of human IL-1RAPL contains a central five-stranded fully parallel β-sheet, which is surrounded by on both (Fig. The overall of this structure is the same as that of the TIR domain of human TLR1 and TLR2 that we reported (11Xu Y. Tao X. Shen B. Horng T. Medzhitov R. Manley J.L. Tong L. Nature. 2000; 408: 111-115Google Scholar). The for equivalent between the TIR domains of IL-1RAPL and TLR1 is Å, with the residues in the the highest structural conservation (Fig. However, there are significant differences in the of the TIR domains of TLR1 and This the that the TIR domains have large of which may be important for specificity in signal transduction by these receptors (11Xu Y. Tao X. Shen B. Horng T. Medzhitov R. Manley J.L. Tong L. Nature. 2000; 408: 111-115Google Scholar). For the β-sheet, in IL-1RAPL is at the in TLR1 (Fig. The differences between the two structures are in of their of the β-sheet, the of αD in IL-1RAPL is almost to that of the αD in TLR1 and TLR2 (Fig. the other of the β-sheet, the of the has different in the two This may be to the that IL-1RAPL has an between and (Fig. 1). most of the residues in this segment are in the structure, they may have the of the of the differences are also observed for the and between the two TIR domains (Fig. structural between IL-1RAPL and TLR1 is in the from to the TIR domain of TLR1 and two and were by a highly region. IL-1RAPL, the two are by a well (Fig. The of the and are also different between IL-1RAPL and TLR1 (Fig. The observed structural differences may these TIR domains or signal transduction The TIR domains from the IL-1Rs contain a highly conserved at its (Fig. 1), with the residue to the first residue in expression construct for This residue is fully to the and has in the of this we IL-1RAPL the terminus. of these residues soluble which the of weakly diffraction data set to 2.3-Å resolution was and the that the at the is in this This construct also contains 20 residues at the C but they are in the structure as in the BB BB loop contains about 10 residues and and in the structures of TIR domains (11Xu Y. Tao X. Shen B. Horng T. Medzhitov R. Manley J.L. Tong L. Nature. 2000; 408: 111-115Google Scholar). residues in this loop are highly conserved among the TIR domains, an residue at the at Pro at Lpsd mutation and at residues are in a is generally a residue in the TIR domains. The structures of the TIR domains of TLR1 and TLR2 that the residue at the is in an pair with a conserved residue the of This is also observed in the TIR domain of IL-1RAPL, and the residue also with the residue (Fig. the BB loop, large differences are observed both in the of the and the of the between the TIR domains of IL-1RAPL and TLR1 (Fig. The of is also different between the two The of the residue is into the in the TLR1 TIR domain, whereas is in the IL-1RAPL The of the residues in the two structures are by a of about 5 Å. the IL-1RAPL has a Thr whereas most other TIR domains have a (Fig. 1). The of this Thr residue on the surface of the domain, to the of the Thr residue at the (Fig. which is unique to IL-1RAPL and TIGIRR (Fig. 1). A of the TIR of IL-1RAPL in the studies suggest that TIR domains may function through receptor activation the TIR domains of the receptors may a that can in the adapter molecules and through among their TIR domains. Our studies with purified TIR domains, that the affinity of the TIR domains for is generally of the TIR domains that we have purified as in at mg/ml filtration and studies that the TIR domain of IL-1RAPL is also monomeric in solution. A. and L. the crystal of the TIR domain of IL-1RAPL, there are two molecules in the asymmetric unit, by a (Fig. the two share an interface in this with about of surface The two are such that the on of the and in interact with their in the other whereas on the other and are not in this interface (Fig. 1). the of this interface is the among and in with their in the other (Fig. in is from the by this of surface The of this residue is the αD in the other with and residue numbers the other as well as in the loop between and Three residues, and are located to the of the dimer and total to the surface and are in pair with residues and from the same of the The BB of the two are located at the of the dimer with residues in they the αD of the other total to the surface This TIR to between the two in the dimer of IL-1RAPL to be unique to this receptor and its close homolog TIGIRR, as many of the residues in this interface are conserved in these two receptors (Fig. 1). For example, of the three residues in the IL-1RAPL dimer interface is conserved in the other TIR domains (Fig. 1). the pair for is not This unique dimer interface is consistent with the distinct functional of these two receptors (8Born T.L. Smith D.E. Garka K.E. Renshaw B.R. Bertles J.S. Sims J.E. J. Biol. Chem. 2000; 275: 29946-29954Google Scholar). the of the dimer interface is the residue in (Fig. It is to the of the dimer such that forms a hydrogen bond with its in the other a close for the equivalent residue of in was observed in the crystal structure of that TIR domain (11Xu Y. Tao X. Shen B. Horng T. Medzhitov R. Manley J.L. Tong L. Nature. 2000; 408: 111-115Google Scholar). As a of forms a bond with in the other of a dimer (Fig. As a of this the observed dimer interface in the TIR domain of TLR1 was to be (11Xu Y. Tao X. Shen B. Horng T. Medzhitov R. Manley J.L. Tong L. Nature. 2000; 408: 111-115Google Scholar, 12Tao X. Xu Y. Zheng Y. Beg A. Tong L. Biochem. Biophys. Res. Commun. 2002; 299: 216-221Google Scholar). However, the structure of the IL-1RAPL TIR domain that close of this residue in the dimer interface be This is by that mutation of the equivalent residue in TLR2 receptor function X. Xu Y. Zheng Y. Beg A. Tong L. Biochem. Biophys. Res. Commun. 2002; 299: 216-221Google Scholar). The of the TIR domains of IL-1RAPL and TLR1 are This is in large to the significant in the of the αD between the two TIR domains. The dimer association observed for IL-1RAPL is for the TLR1 TIR domain as for the αD A surface about for is by the dimer of the TIR domain of TLR1 (Fig. with the of the structural we functional studies with the IL-1RAPL receptor. is the functional role of the dimer that is observed in the This can be using IL-1RAPL and its close homolog TIGIRR as the dimer may not be present for the other receptors. studies have that IL-1RAPL and TIGIRR cannot activate the (8Born T.L. Smith D.E. Garka K.E. Renshaw B.R. Bertles J.S. Sims J.E. J. Biol. Chem. 2000; 275: 29946-29954Google Scholar). Recent studies with IL-1R that can activate the MAP ERK, and JNK, cannot activate Fitzgerald K.A. Smith P. O'Neill L.A.J. J. Biol. Chem. 2002; Scholar). This to IL-1RAPL can also activate the MAP Our data that this receptor can activate JNK (Fig. but not the ERK and p38 MAP kinases, and data also that IL-1RAPL cannot activate the conditions used not It is that in the a not expressed in these may be for IL-1RAPL to activate studies of this activation by IL-1RAPL suggest that is of the TIR domain. of or the TIR domain were found to be to activate JNK to the same as the receptor (Fig. It is that this activation is an as TIGIRR cannot activate this the sequence the by IL-1RAPL is more that by (Fig. IL-1RAPL may be to activate JNK by other molecules through its extracellular domain the sequence homology with TIGIRR is the here contain a intracellular segment to the TIR domain (residues However, is for this segment to mediate the activation of JNK, as significant homology with studies are to the of this JNK activation by we have the crystal structure of the TIR domain of human IL-1RAPL at 2.3-Å As the first structure of the TIR domain from the IL-1R of significant differences to the structures of the TIR domains of TLR1 and TLR2 that we reported This is consistent with the that sequence and structural diversity among these domains is important for specificity in the signal transduction The unique structural of the TIR domain in IL-1RAPL with TLR1 and TLR2 may be the molecular for its to activate The structural the of a dimer with a interface in the crystals of this domain. The in this dimer to be unique to IL-1RAPL and its close homolog TIGIRR, and they cannot be using IL-1RI and The of the functional of the observed dimer have to the of a for these two receptors. of the TIR domains of IL-1RAPL and TLR1 suggest that residues in the be important for the function of TIR domains. This is by studies that mutation of the equivalent residue in to the function of the receptor X. Xu Y. Zheng Y. Beg A. Tong L. Biochem. Biophys. Res. Commun. 2002; 299: 216-221Google Scholar). Sequence among the TIR domains show that this residue is almost conserved as among the TLRs, but is not conserved among the IL-1Rs and the adapter TIR domains (Fig. 1). The molecular mechanism of the functional of a residue at this in the TLRs to be We and for the X4A and Tao for with data collection at the and Sims for the IL-1RAPL and TIGIRR and for