1H, 13C, and 15N resonance assignment of the TIR domain of human MyD88 (original) (raw)

KURENAI : Kyoto University Research Information Repository

1H,13C{ }^{1} \mathrm{H},{ }^{13} \mathrm{C}, and 15 N{ }^{15} \mathrm{~N} resonance assignment of the TIR domain of human MyD88

Hidenori Ohnishi 1{ }^{1}, Hidehito Tochio 2{ }^{2}, Zenichiro Kato 1{ }^{1}, Takeshi Kimura 1{ }^{1}, Hidekazu Hiroaki 3{ }^{3}, Naomi Kondo 1{ }^{1}, and Masahiro Shirakawa 2{ }^{2}

1. Department of Pediatrics, Graduate School of Medicine, Gifu University, Gifu, Japan
2. Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Kyoto, Japan.
3. Division of Structural Biology, Graduate School of Medicine, Kobe University, 7-5-1, Kusunoki-cho, Chuo-ku, Kobe, Hyogo, 650-0017, Japan.

Corresponding author:

Hidenori Ohnishi

Department of Pediatrics, Graduate School of Medicine, Gifu University

Yanagido 1-1, Gifu 501-1194, Japan

Phone: +81-58-230-6386

Fax: +81-58-230-6387

E-mail: ohnishih@gifu-u.ac.jp

Hidehito Tochio

Department of Molecular Engineering, Graduate School of Engineering

Kyoto University, Kyoto 615-8510, Japan

Phone: +81-75-383-2536

Fax: +81-75-383-2541

E-mail: tochio@moleng.kyoto-u.ac.jp

Key words:

Innate immunity, Toll-like receptor, NMR, MyD88, TIR domain

Abstract

Myeloid differentiating factor 88 (MyD88) is one of a critical adaptor molecule in the Toll-like receptor (TLR) signaling pathway. The TIR domain of MyD88 serves

as a protein-protein interaction module and interacts with other TIR-containing proteins such as Mal (MyD88 adaptor-like) and Toll-like receptor 4 to form signal initiation complexes. Here we report the 15 N,13C{ }^{15} \mathrm{~N},{ }^{13} \mathrm{C}, and 1H{ }^{1} \mathrm{H} chemical shift assignments of the TIR domain of MyD88. The resonance assignments obtained in this work will contribute to the study of heteromeric TIR-TIR interactions between MyD88 and TIR-containing receptors or adaptors.

Biological context

MyD88 is an essential and universal cytosolic adaptor protein in the signal transduction pathways mediated by members of the IL-1 family, including IL-18, and the Toll-like receptors (TLRs). It consists of an N-terminal death domain, a C-terminal Toll/Interleukin-1 receptor (TIR) domain ( 150 amino acid residues), and a small linker segment between them. In innate immune responses, MyD88 plays pivotal roles in bridging between membraneous TLR and downstream kinases, such as the IL-1 receptor-associated kinases (IRAKs), in the cytosol (O’Neill and Bowie, 2007). For example, in the TLR4 pathway, the cytosolic TIR

domain of lipopolysaccharide (LPS)-stimulated TLR4 interacts with the TIR domain of MyD88 (MyD88-TIR). Concurrently, the death domain of MyD88 interacts with the death domain of IRAK4, activating the kinase. This initiates a phosphorylation cascade that eventually activates the transcription factors NF- κB\kappa \mathrm{B} (nuclear factor κB\kappa B ) and AP-1 (activator protein 1) (Akira et al., 2006).

The TIR domains serve as protein-protein interaction modules, which are conserved in the intracellular region of TLRs and also in cytosolic adaptor proteins such as MyD88, Mal (MyD88 adaptor-like), TRIF (TIR domain-containing adaptor inducing IFN- β\beta ), TRAM (TRIF-related adaptor molecule), and SARM (sterile α\alpha and HEAT Armadillo motifs). The crystal structures of TIR domains of some mammalian membranous receptors (TLR1, TLR2, TLR10, IL-1RAPL) and a bacterial protein (Paracoccus denitrificans TIR (PdTIR)) have been reported (Chan et al., 2009; Khan et al., 2004; Nyman et al., 2008; Tao et al., 2002; Xu et al., 2000), in some of which homomeric TIR interfaces were observed. However, the functional relevance of such interactions remains obscure, as the formation of

these homo-dimers of TIR domains had not been observed in solution (Khan et al., 2004; Nyman et al., 2008).

In our previous study, the isolated MyD88-TIR was shown to exist as a monomer, while full-length MyD88 forms a dimer in solution. The dimerization appears to be mediated via homomeric interactions within its death domain (Ohnishi et al., 2009). In that study, we performed NMR titration experiments using 15 N{ }^{15} \mathrm{~N} MyD88-TIR and characterized a functionally relevant heteromeric TIR-TIR interaction with another TIR domain derived from Mal, which had been shown to enhance LPS-stimulated TLR4 signaling. Interestingly, no interaction has been observed between TLR4-TIR and MyD88-TIR, while Mal has been shown to directly bind TLR4-TIR. Thus, Mal seems to bridge between TLR4 and MyD88. These results exemplified the binding specificity among TIRs and its importance in forming correct signal initiation complexes (Ohnishi et al., 2009).

Here, we describe the NMR assignments of the TIR domain of MyD88. In IL-1R

and TLR signaling, the heteromeric TIR interactions of MyD88 play a central role in the above-mentioned formation of the TLR signalosome. The resonance assignments form a substantial contribution to the study of the heteromeric TIR-TIR interactions between MyD88 and TIR containing receptors or adaptors.

Methods and Experiments

Sample preparation

The portion of the human MyD88 gene encoding the TIR domain (amino acid residues 148-296) was cloned into the vector pGEX-5X-3 digested with restriction enzymes, ECORI and NotI (GE Healthcare). This vector was transformed into E. coli BL-21 (DE3) (Novagen). The transformed bacterial cells were cultured at 37∘C37^{\circ} \mathrm{C} until the OD600\mathrm{OD}_{600} reached approximately 0.5 . The cells were then induced with 1.0 mM IPTG and cultured for 16 hours at 25∘C25^{\circ} \mathrm{C}. For 15 N{ }^{15} \mathrm{~N}-or 13C,15 N{ }^{13} \mathrm{C},{ }^{15} \mathrm{~N}-double-labeling, rich growth media containing stable isotope-enriched nutrients (Silantes) were used. Harvested bacterial cells were once frozen and thawed before the next lysis step. The cells were resuspended in lysis buffer

(20mM Tris, pH 8.0, 400mM KCl, 1mM EDTA, 10mM 2-mercaptoethanol and protease inhibitors) and disrupted by sonication on ice. The supernatant was loaded onto a glutathione Sepharose 4B FF (GE Healthcare) affinity chromatography column. Lysis buffer at 30x the bed volume of this resin was used for the washing step. GST fused MyD88-TIR protein was eluted with elution buffer ( 50 mM Tris, pH 8.0 , and 10 mM 2-mercaptoethanol, and 10 mM reduced glutathione). The GST-tag was cleaved by digestion with Factor Xa (1% of total protein volume) (Haematologic Technologies Inc.). Subsequently, the TIR domain was purified by gel filtration (Sephacryl S-100 HR 26/60 column, GE Healthcare) and cation-exchange chromatography (Mono-S column, GE Healthcare). The gel filtration running buffer was 20 mM potassium phosphate buffer ( pH 6.0 ) containing 100mMKCl,0.1mM100 \mathrm{mM} \mathrm{KCl}, 0.1 \mathrm{mM} EDTA and 10 mM DTT. Using this purification protocol, 15 N{ }^{15} \mathrm{~N}-labeled or 13C,15 N{ }^{13} \mathrm{C},{ }^{15} \mathrm{~N}-doubly-labeled TIR domain of MyD88 wild-type proteins was prepared. The yields of these proteins were approximately 1.0 mg from 1 L of rich growth media containing stable isotope-enriched nutrients. For preparing samples where selected amino acids

were not labeled, proteins were over-expressed by the same method described above with a slight modification. The bacterial cells were first cultured in LB media and collected 30 min before IPTG induction by centrifugation. The cells were resuspended and cultured in 15 N{ }^{15} \mathrm{~N}-enriched M9 media containing unlabelled Gly, Ser and Cys, or Arg alone to a final composition of 100mg/L100 \mathrm{mg} / \mathrm{L} for protein expression (The details of this protocol will be published elsewhere by Dr. H. Hiroaki). To improve the sample solubility and stability, the sample buffer was replaced by 20 mM potassium phosphate buffer ( pH 6.0 ) containing 0.1 mM EDTA, 10 mM DTT, 50 mM deuterated L-arginine, and 50 mM deuterated L-glutamic acid. The final concentrations of the protein samples for typical NMR experiments were about 0.3 mM (Golovanov et al., 2004).

NMR spectroscopy

All NMR spectra were recorded at 25∘C25^{\circ} \mathrm{C} on a Bruker DRX500 or Avance800 spectrometer equipped with a cryogenic probe. For assignment of backbone 1H{ }^{1} \mathrm{H}, 13C{ }^{13} \mathrm{C}, and 15 N{ }^{15} \mathrm{~N} resonances, HNCA, HN(CO)CA,HNCACB,CBCA(CO)NH\mathrm{HN}(\mathrm{CO}) \mathrm{CA}, \mathrm{HNCACB}, \mathrm{CBCA}(\mathrm{CO}) \mathrm{NH} and 3D

1H−15 N{ }^{1} \mathrm{H}-{ }^{15} \mathrm{~N} NOESY-HSQC spectra were recorded. For side chain resonance assignment, 2D constant-time 1H−13C{ }^{1} \mathrm{H}-{ }^{13} \mathrm{C} HSQC, 1H−13C{ }^{1} \mathrm{H}-{ }^{13} \mathrm{C} NOESY-HSQC, HCCH-TOCSY, CC(CO)NNH and HCC(CO)NNH were recorded. All NMR data were processed using NMRPipe (Delaglio et al., 1995), and analyzed using Sparky (Goddard and Kneller, 1999).

Assignments and data deposition

Employment of 50 mM L-arginine and L-glutamic acid in the sample buffer significantly improved the solubility of the sample by approximately three fold, resulting in significantly enhanced NMR signal intensities. Following a standard sequential assignment procedure, 88.6%88.6 \% of the 1HN,15 N{ }^{1} \mathrm{H}^{N},{ }^{15} \mathrm{~N} resonances of backbone amide groups (124 out of the 140 non-Pro residues) were assigned (Fig. 1). In addition, 91.3%91.3 \% of Hα\mathrm{H} \alpha (136 out of 149 residues), 93.3%93.3 \% (139 out of 149 residues) of 13C∘{ }^{13} \mathrm{C}^{\circ}, and 91.3%91.3 \% (136 out of 149 residues) of 13C∘{ }^{13} \mathrm{C}^{\circ} resonances were assigned. The sequential correlations of the backbone resonances of 195-200, 202, and 203 were missing. Those residues are involved in the BB-loop region in the

structure of MyD88-TIR. Relatively lower {1H}−15 N\left\{{ }^{1} \mathrm{H}\right\}^{-15} \mathrm{~N} hetero NOE values of amide groups at the neighboring residues suggested that the region did not adopt a fixed conformation, but had some flexibility (Ohnishi et al., 2009). This might have caused NMR signal broadening at the BB-loop residues. Besides those above-mentioned residues, Thr-185, Asp-186, Tyr-187, and Arg-188 in the turn region were also missing, which could also be due to NMR signal broadening caused by local motions. Nevertheless, substantial numbers of side chain 1H{ }^{1} \mathrm{H} and 13C{ }^{13} \mathrm{C} atoms of those residues have been assigned on the basis of 13C{ }^{13} \mathrm{C} edited NOESY and HCCH-TOCSY spectra. The assigned chemical shifts of the TIR domain of MyD88 have been deposited in the BMRB database under accession number 11078. The coordinates of the TIR domain of human MyD88 have been deposited at the Protein Data Bank under accession code 2z5v.

Acknowledgments

This work was supported in part by the Research and Development Program for New Bio-industry Initiatives (2005-2009) of the Bio-oriented Technology Research Advancement Institution (BRAIN), Japan to NK. This work was also

supported in part by Grants-in-Aid for Scientific Research and the National
Project on Protein Structural and Functional Analyses from the Ministry of Education, Science, and Culture of Japan to HO and HT.

References

Akira, S., Uematsu, S., and Takeuchi, O. (2006). Pathogen recognition and innate immunity. Cell 124, 783-801.

Chan, S. L., Low, L. Y., Hsu, S., Li, S., Liu, T., Santelli, E., Le Negrate, G., Reed, J. C., Woods, V. L., Jr., and Pascual, J. (2009). Molecular mimicry in innate immunity: crystal structure of a bacterial TIR domain. J Biol Chem 284, 21386-21392.

Delaglio, F., Grzesiek, S., Vuister, G. W., Zhu, G., Pfeifer, J., and Bax, A. (1995).

NMRPipe: a multidimensional spectral processing system based on UNIX pipes.

J Biomol NMR 6, 277-293.

Goddard, T. D., and Kneller, D. G. (1999). SPARKY 3 ( San Francisco: University of California).

Golovanov, A. P., Hautbergue, G. M., Wilson, S. A., and Lian, L. Y. (2004). A simple method for improving protein solubility and long-term stability. J Am Chem Soc 126, 8933-8939.

Khan, J. A., Brint, E. K., O’Neill, L. A., and Tong, L. (2004). Crystal structure of the Toll/interleukin-1 receptor domain of human IL-1RAPL. J Biol Chem 279, 31664-31670.

Nyman, T., Stenmark, P., Flodin, S., Johansson, I., Hammarstrom, M., and Nordlund, P. (2008). The Crystal Structure of the Human Toll-like Receptor 10 Cytoplasmic Domain Reveals a Putative Signaling Dimer. J Biol Chem 283, 11861-11865.

O’Neill, L. A., and Bowie, A. G. (2007). The family of five: TIR-domain-containing adaptors in Toll-like receptor signalling. Nat Rev Immunol 7, 353-364.

Ohnishi, H., Tochio, H., Kato, Z., Orii, K., Li, A., Kimura, T., Hiroaki, H., Kondo, N., and Shirakawa, M. (2009). Structural basis for the multiple interactions of the MyD88 TIR domain in TLR4 signaling. Proc Natl Acad Sci U S A.

Tao, X., Xu, Y., Zheng, Y., Beg, A. A., and Tong, L. (2002). An extensively

associated dimer in the structure of the C713S mutant of the TIR domain of human TLR2. Biochem Biophys Res Commun 299, 216-221.

Xu, Y., Tao, X., Shen, B., Horng, T., Medzhitov, R., Manley, J. L., and Tong, L. (2000). Structural basis for signal transduction by the Toll/interleukin-1 receptor domains. Nature 408, 111-115.

Figure Legend

Fig. 11H,15 N1{ }^{1} \mathrm{H},{ }^{15} \mathrm{~N} HSQC spectrum of uniformly 15 N{ }^{15} \mathrm{~N}-labeled TIR domain of human MyD88 in 20 mM potassium phosphate buffer ( pH 6.0 ) containing 0.1 mM EDTA, 10 mM DTT, 50 mM deuterated L-arginine, and 50 mM deuterated L-glutamic acid recorded at 25∘C25^{\circ} \mathrm{C}.