Molecular Structure of the IL-1R-Associated Kinase-4 Death Domain and Its Implications for TLR Signaling

Cutting Edge: Molecular Structure of the IL-1R-Associated Kinase-4 Death Domain and Its Implications for TLR Signaling

This information is current as Michael V. Lasker, Mark M. Gajjar and Satish K. Nair of September 27, 2021. J Immunol 2005; 175:4175-4179; ; doi: 10.4049/jimmunol.175.7.4175 http://www.jimmunol.org/content/175/7/4175 Downloaded from

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JOURNAL OF IMMUNOLOGY CUTTING EDGE

Cutting Edge: Molecular Structure of the IL-1R-Associated Kinase-4 Death Domain and Its Implications for TLR Signaling1 Michael V. Lasker,*† Mark M. Gajjar,* and Satish K. Nair2*‡

IL-1R-associated kinase (IRAK) 4 is an essential compo- shown to have kinase activity. IRAK-4 is a unique member of nent of innate immunity. IRAK-4 deficiency in mice and the IRAK family in that it is the only member to have true ki- humans results in severe impairment of IL-1 and TLR sig- nase activity and is the most homologous to the Drosophila naling. We have solved the crystal structure for the death IRAK-like kinase, Pelle (3). The central role IRAK-4 plays in TLR/IL-1R signaling and subsequent immunological protec-

domain of Mus musculus IRAK-4 to 1.7 Å resolution. Downloaded from This is the first glimpse of the structural details of a mam- tion was demonstrated in mouse models (10) and in humans malian IRAK family member. The crystal structure re- with inherited IRAK-4 deficiencies, who suffer from recurrent pyogenic bacterial infections (11, 12). Modulation of this key veals a six-helical bundle with a prominent loop, which mediator in TLR/IL-1R signaling could prove useful in treating among IRAKs and Pelle, a Drosophila homologue, is disease states of inflammation, septic shock, and in the manage- unique to IRAK-4. This highly structured loop contained ment of autoimmune disorders, making IRAK-4 an attractive between helices two and three, comprises an 11-aa stretch. drug target (3, 13). To mediate TLR/IL-1R signaling, IRAK-4 http://www.jimmunol.org/ Although innate immune domain recognition is thought associates with the intermediate domain of the adaptor MyD88 to be very similar between Drosophila and mammals, this presumably via its own DD (3, 5). Failure to associate with structural component points to a drastic difference. This MyD88 disrupts phosphorylation of IRAK-1 (5) by IRAK-4 structure can be used as a framework for future mutation and and subsequent downstream signaling. The DD of IRAK-4 is deletion studies and potential drug design. The Journal of Im- also necessary in facilitating heterodimerization with other munology, 2005, 175: 4175–4179. IRAKs (9). To begin to understand the molecular structure of IRAK-4 and its implications for TLR/IL-1R signaling, we solved the crystal structure of the IRAK-4 DD to 1.7 Å resolu- by guest on September 27, 2021 he mammalian TLRs and the IL-1R are known to play tion. Using the crystal structure as a guide, we describe some important roles in innate immunity and are character- important structural components of the IRAK-4 DD, which T ized by a homologous cytoplasmic Toll/IL-1R domain have implications for its function, molecular recognition, and (1). Disruption of this domain, as in the P712H mutation of subsequent TLR/IL-1R signaling. Tlr4 (2), where mice are completely resistant to endotoxic shock, perturbs downstream signaling and the production of proinflammatory cytokines. In addition, elimination of down- Materials and Methods Generation of IRAK-4 DD stream signaling molecules can either severely hamper or totally disturb an effective innate immune response (3). These down- A cDNA construct encoding Mus musculus IRAK-4 DD (residues 1–113) flanked by NdeI and BamHI restriction sites was created by PCR amplification stream signaling molecules include a set of Toll/IL-1R-domain and cloned into a pGEX6p-1 vector (Amersham Biosciences) in frame with an containing adaptor proteins: MyD88, TIRAP/Mal, Trif/Ti- N-terminal GST tag. The construct was transformed into BL21 (DE3) cells. cam, and TRAM (4). MyD88 has a modular construction with Transformed cells were grown in Luria broth to an A600 of 0.6–0.8 at 37°C, ␤ an N-terminal death domain (DD)3 (5). This domain serves to where overexpression was induced with 0.5 mM isopropyl -D-thiogalactoside and the temperature was adjusted to 18°C for overnight expression. Purifica- recruit a family of kinases known as IL-1R- associated kinases tion of GST-IRAK-4 DD from the soluble fraction was conducted by affinity (IRAKs). The IRAK family has four known members: IRAK-1 chromatography. Cleavage and removal of the GST tag from IRAK-4 DD was (6), IRAK-2 (7), IRAK-M (8), and IRAK-4 (9). All four IRAKs performed by the addition of rhinovirus 3C protease and cation exchange chromatography. Buffer exchange into 20 mM HEPES (pH 7.5) and 125 mM KCl, contain a DD at the N terminus and a kinase domain at the C and subsequent concentration were conducted using a 5-kDa molecular mass terminus; however, only IRAK-1 (6) and IRAK-4 (9) have been limit centrifugal filter device (Millipore).

*Department of Biochemistry, †Medical Scholars Program, and ‡Center for Biophysics National Institutes of Health National Research Service Award 1 F30 NS 048779-01 from and Computational Biology, University of Illinois, Urbana-Champaign, Urbana, IL the National Institute of Neurological Disorders and Stroke. 61801 2 Address correspondence and reprint requests to Dr. Satish K. Nair, Department of Bio- Received for publication June 20, 2005. Accepted for publication July 18, 2005. chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Ur- bana, IL 61801. E-mail address: [email protected] 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. 3 Abbreviations used in this paper: DD, death domain; IRAK, IL-1R-associated kinase; Section 1734 solely to indicate this fact. MAD, multiwavelength anomalous dispersion; FOM, figure of merit. 1 This work was supported by start-up funds and the Institute for Genomic Biology, Uni- versity of Illinois, Urbana-Champaign (to S.K.N.). M.V.L. was supported by an individual

Crystallization factor of 17.7% (free R factor of 22.3%) against data to a resolution of 1.7 Å (Table I). Purified and concentrated (10 mg/ml) IRAK-4 DD was crystallized using the hanging drop method. Crystals were grown at 8°C in 2.5-␮l drops in 100 mM Data deposition HEPES (pH 7.5), 25% polyethylene glycol 3350, and using 10 mM MnCl2 as an additive. Crystals took ϳ5 days to form. Atomic coordinates for IRAK-4 DD have been deposited within the Protein Data Bank and can be downloaded using the accession code 2A9I. Data collection, phase determination, and refinement IRAK-4 DD crystals were cryoprotected by sequential 30-min soaks at 4°C in Results and Discussion mother liquor containing 10, 20, and 30% glycerol and then plunged into liq- The core structure of the IRAK-4 DD lies within residues 4–106 and is uid nitrogen. For halide-soaked crystals, the above process was conducted in a hexahelical bundle addition to a brief soak in the last cryoprotectant solution containing1Mso- dium bromide. DDs typically consist of a hexahelical bundle (18). Because Native amplitudes were collected, under standard cryogenic conditions, at a there is no signature motif that indicates initiation or termina- high-flux insertion device synchrotron beam line (Argonne National Labora- tory, sector 14-ID), to a limiting resolution of 1.7 Å. A multiwavelength anom- tion of the DD, we performed a primary sequence alignment alous dispersion (MAD) data set was collected on bromide-soaked crystals (Ar- with other IRAK family members to find a region of conserva- gonne National Laboratory, sector 32-ID). The halide soak resulted in the tion in IRAK-4 that might be amenable to crystallization. This specific incorporation of four ordered bromide ions into the structured solvent primary sequence alignment suggested that the initial 113 aa of region and anomalous scattering from these bromide ions were exploited for phase determination. IRAK-4 represents the core structure of the DD. The resulting The substructure of bromide ions present in the asymmetric unit was deter- crystal structure revealed no electron density for the first three mined using Shake and Bake (͗www.hwi.buffalo.edu/SnB͘) (14). These bro- and last seven amino acids, indicating these regions are disor- Downloaded from mide positions were used for heavy atom refinement and to derive estimates of protein phases using SOLVE (͗www.solve.lanl.gov͘) (15). The overall figure of dered and unstructured. This suggests that amino acid residues merit (FOM) calculated to a resolution of 2.0 Å was 0.41 (Table I). Density 4–106 represent the true core structure of the IRAK-4 DD. modification and solvent flattening using RESOLVE (͗www.solve.lanl.gov͘) The structure of the IRAK-4 DD did indeed reveal a hexahelical (15) dramatically improved the quality of the phase estimates (mean FOM ϭ bundle with the first ␣ helix beginning with valine 16, desig- 0.67) as judged by the interpretability of the resultant electron density maps. nated ␣1-1 (Fig. 1A). This residue numbering system will be Model building with XtalView (16) was guided by prior knowledge that the http://www.jimmunol.org/ structure likely contained a six-helical bundle as seen in structures of other DDs. used throughout. A highly structured N-terminal tail contain- REFMAC (17) was used for maximum-likelihood structure refinement. Several ing two proline residues (P4 and P7) precedes this helix. The cycles of model building, interspersed with refinement, resulted in a final model N-terminal tail also makes several hydrogen bond contacts with containing residues 4–106 of IRAK-4, 145 well-ordered solvent molecules, and ␣ ␣ a single manganese ion. The geometry of all residues falls within the allowed the loop located between 4 and 5. A rigid tail was also ob- regions of the Ramachandran plot. The structure has been refined to a final R served at the C terminus with two proline residues (P102 and

Table I. Statistics for native data collection and reﬁnement, MAD data collection, and phasing by guest on September 27, 2021

Cell parameters Space group C2 Unit cell dimensions a ϭ 76.1 Å, b ϭ 38.0 Å, c ϭ 51.6 Å

Data collection statisticsa Native ␭1 ␭2 ␭3 Wavelength (Å) 0.97888 0.91947 0.91919 0.91270 Resolution range (Å) 50–1.7 35–2.0 50–2.0 50–2.0 Unique reﬂections 11,988 8,336 8,382 8,441 Redundancy 6.1 7.4 7.5 7.5 Completeness (%) 100 (100) 95.0 (95.4) 95.0 (95.5) 95.4 (94.8) I/␴ 50.6 (8.9) 39.0 (23.8) 45.7 (23.5) 51.2 (21.4) b Rmerge (%) 3.2 (19.6) 3.6 (8.6) 3.2 (8.8) 2.9 (9.8) Mosaicity 0.4–0.5 Bromide MAD phases Number of bromide sites 4 FOM 0.41 FOM following density modiﬁcations 0.67 Model statistics Resolution 50–1.7 c Rwork/Rfree 0.1767/0.2226 rmsd bond lengths (Å) 0.017 rmsd bond angles (°) 1.719 Ramachandran plot Most favored ␾-␺ (%) 92.1 Additionally allowed 7.9 Generously allowed 0.0 Disallowed 0.0 Average B factor (Å2) 28.9 Number of nonhydrogen protein atoms per asymmetric unit 823 Mn2ϩ ions per asymmetric unit 1 Water molecules per asymmetric unit 145

a Numbers in parentheses represent values for the highest resolution shell. b ϭ⌺ ͉͗ ͘Ϫ ͉ ⌺ ͉ ͉ Rmerge hkl I I / hkl I . c ϭ⌺ ͉ Ϫ ͉ ⌺ ͉ ͉ Rwork hkl Fo Fc / hkl Fo , and Rfree is equivalent to Rwork but is calculated for a randomly chosen 7% of reﬂections excluded from model reﬁnement. The Journal of Immunology 4177

FIGURE 1. Schematic representation of the hexahelical bundle formed by IRAK-4 DD and a primary sequence alignment. A, A ribbon diagram of IRAK-4 DD with the tail regions and interconnecting loops denoted in gray. N and C termini are represented by the letters N and C, respectively. This ribbon diagram was generated with Py- MOL (DeLano Scientific, ͗www.pymol.org͘). B, Protein sequences of murine IRAK-4, murine IRAK-1, murine IRAK-2, murine IRAK-M, and Drosophila melanogaster

Pelle were aligned using ClustalW (24). Amino acid resi- Downloaded from dues strictly conserved throughout all five sequences are highlighted in magenta. Identical residues present in at least three of the aligned sequences are represented in yellow. Similar residues are tinted in blue and gaps are denoted by dashes. A schematic representation of the structural components of IRAK-4 DD is shown above the alignment. Dashed lines denote unstructured regions of IRAK-4 DD, http://www.jimmunol.org/ whereas solid lines demonstrate structured tails or loops. The cylinders correspond to the helices with the same color scheme given in the IRAK-4 DD ribbon diagram. by guest on September 27, 2021

P106). These C-terminal proline residues are strictly conserved Given the profound functional consequences of these in all mouse IRAKs, Pelle (Fig. 1B), and human IRAK-4 and IRAK-1 mutations, we examined whether corresponding resi- IRAK-1. The rigid nature of proline residues is potentially re- dues in IRAK-4 DD represent critical structural components. quired for orienting the C-terminal kinase domain so that Residue W73 in IRAK-1 corresponds to W74, ␣4-8, in the proper molecular recognition can take place. IRAK-4 DD and serves as a central residue in the hydrophobic Functionally important residues of IRAK-1 are critical structural core. This hydrophobic core is made up of I11, L19, L22, L35, components of the IRAK-4 DD I39, L70, L71, L84, and L87. Among these residues, L71 and L84 are strictly conserved throughout the IRAK family and in Previous studies of IRAK-1 have shown residues T66 and W73, Pelle kinase. The other residues are homologous aliphatic resi- both of which are strictly conserved in the IRAK family (Fig. dues (Fig. 1B). Disruption of this hydrophobic core by a W74- 1B), to serve important functional roles (19). The overexpres- directed point mutation would easily perturb the association of sion of wild-type IRAK-1 has been shown to spontaneously ac- helix 4 with helices 2 and 5. W74 acts as a central residue me- tivate NF-␬B (8), whereas IRAK-1 point mutants T66A, T66E, and W73A all show reduced spontaneous activation diating interactions between many helices in the IRAK-4 DD, (19). In addition, the T66A and T66E point mutations were whereas T67, corresponding to residue T66 of IRAK-1 (19), shown to change the intracellular localization of IRAK-1 (19). has a very different role in maintaining structural integrity. ␣ ␣ Confocal microscopy demonstrated wild-type IRAK-1 to be T67, 4-2, instead forms a hydrogen bond to D27, 1-12, ef- present in punctate cytoplasmic complexes, whereas the T66A fectively linking the end of the first helix to the beginning of and T66E IRAK-1 mutants were found to be diffuse in the cy- the fourth helix. This aspartate residue is strictly conserved toplasm (19). These results indicate that perturbation of T66 in the IRAK family and Pelle. The conservation of a nega- abrogates the ability of IRAK-1 to form high molecular mass tively charged residue in this position is a critical structural complexes. Mutation of T66 also prevents the normal autophos- component that maintains hydrogen bonds with the hydroxy- phorylation activity of IRAK-1 (19). lated side chain and the backbone of the fourth helix. However, 4178 CUTTING EDGE: STRUCTURE OF THE IRAK-4 DEATH DOMAIN introduction of a negatively charged side chain at T67 (as in a When the hexahelical bundles of the two structures are super- T67E/D mutation) would not only disrupt this hydrogen imposed on each other, the core root-mean-square deviation is bond, but also cause a charge-charge repulsion with the D27 calculated to be just 1.97 Å. However, the similarity between residue and thus destabilize the fourth helix. An electrostatic these two structures is met with one striking exception, that of repulsion such as this could be initiated by a negatively charged a loop between helices two and three. This sizeable loop in the posttranslational modification such as phosphorylation. In fact, IRAK-4 DD structure, designated ␣2-␣3, appears quite small IRAK-1-derived peptides containing the T66 residue have been in the Pelle-DD structure (Fig. 2). The loop encompasses an shown to be phosphorylated by ␫ protein kinase C (20) using an 11-aa stretch, residues 39–49, and is highly structured by in vitro kinase assay. Considering the functional roles the cor- maintaining five pairs of polar contacts within the loop itself. responding residue T66 assumes in IRAK-1 and the critical Upon further analysis using a sequence alignment of IRAK-4 structural role T67 plays in the IRAK-4 DD, this amino acid with other IRAK family members, it became evident that this position is a putative site of posttranslational modification. loop is unique to IRAK-4 (Fig. 1B). Structural comparison of IRAK-4 with Pelle reveals a loop unique to IRAK-4 Molecular recognition of cognate adaptors differ between IRAK-4 and All four IRAKs are homologues of the Drosophila protein kinase Pelle Pelle. Similar to the IRAK family, Pelle has been shown to play Because the IRAK-4 DD three-dimensional structure is nearly an important role in host defense. Pelle also is a bipartite mol- identical with the Pelle-DD structure, excluding the sizeable Downloaded from ecule with an N-terminal DD and a C-terminal serine-threo- ␣2-␣3 loop, we decided to analyze the Pelle-DD/Tube-DD nine kinase domain. The crystal structure of the Pelle DD has structure in more detail. Tube is an adaptor molecule in Dro- been solved previously (21) and forms a six-helical bundle in- sophila, which is known to bind to Pelle, and plays a critical role dicative of a member of the DD super family. Although the DD in mediating an innate immune response toward fungal infec- of Pelle and IRAK-4 have only a 13% sequence identity, their tion (22). From the Pelle-DD/Tube-DD complex crystal struc- three-dimensional structures are highly homologous (Fig. 2). ture, solved previously (21), it was shown that the C-terminal http://www.jimmunol.org/ tail of Tube fits within a groove of Pelle (Fig. 2). This groove is formed by a cavity between the Pelle ␣4-␣5 and ␣2-␣3 loops. Both loops are rather small and lack any secondary structure or polar contact pairs within the loops. In comparison, the ␣2-␣3 loop of IRAK-4 DD has a twist and five polar contact pairs within the loop. The twist within the ␣2-␣3 loop of IRAK-4 DD precludes the presence of any analogous groove or cavity as

in Pelle (Fig. 2). Thus, a steric clash with the ␣2-␣3 loop would by guest on September 27, 2021 prevent an interaction like Pelle-DD/Tube-DD taking place with the IRAK-4 DD. Although no mammalian counterpart has been found for Tube, Pelle is most homologous to IRAK-4 (9). IRAK-4 is known to bind to a number of different molecules including: MyD88, IRAK-1, TNF-related activation factor 6, and Pellino-1, a protein known to play a role in IL-1- mediated signaling (23). If indeed this cognate adaptor interface is evolutionarily conserved in IRAK-4 from Pelle, then there are two obvious possibilities for interaction to take place. One is that the adaptor molecule for IRAK-4 has an interface, which accommodates the sizeable ␣2-␣3 loop. An alternative is that the ␣2-␣3 loop could undergo a conformational change. However, this may require breaking many polar contacts and considerable rearrangement. The results of these structural studies show that the core structure of the IRAK-4 DD comprises amino acid residues 4–106 and forms a hexahelical bundle. The hexahelical bundle is followed by two conserved proline residues, P102 and P106, whereas the hydrophobic core is centralized around W74. The end of the first helix is tethered to the beginning of the fourth helix by means of a hydrogen bond between D27 and T67. A loop, ␣2-␣3, unique to IRAK-4 within the IRAK family, forms a twist and five pairs of polar contacts with itself. This twist FIGURE 2. Structural comparison of Pelle-DD and IRAK-4 DD. A ribbon reduces the possibility that a groove is formed as in the Pelle diagram of Pelle-DD, IRAK-4 DD, and Tube-DD is represented in orange, crystal structure. Given that humans with mutations in IRAK-4 cyan, and blue, respectively. The structural data file for Pelle and Tube was are susceptible to recurrent pyogenic infections, these results taken from the Protein Data Bank (1D2Z) and the figure was generated with PyMOL (DeLano Scientific, ͗www.pymol.org͘). The ␣2-␣3 loop in Pelle-DD have considerable implications on innate immune defense in and IRAK-4 DD is colored in magenta. A red dashed ellipse represents the area mammals and will hopefully serve as a guide for future bio- the IRAK-4 ␣2-␣3 loop would encompass if it were superimposed on Pelle. chemical and cellular studies. The Journal of Immunology 4179

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