Structural insights into the assembly and activation of

interleukin 1β with its receptors

Dongli Wang, Senyan Zhang, Liang Li, Xi Liu, Kunrong Mei, Xinquan Wang*

Center for Structural Biology, School of Life Sciences

MOE Key Laboratory of Bioinformatics

Tsinghua University

Medical Science Building C226

Tsinghua University, Beijing 100084, P. R. China

Correspondence should be addressed to X.W. (xinquanwang@mail.tsinghua.edu.cn)

Tel: 86-10-62789401

Fax: 86-10-62792736

Nature Immunology: doi:10.1038/ni.1925

Supplementary Table 1: Selected interactions (d ≤ 3.5 Å) in the

IL-1β−IL-1RII−IL-1RAcP ternary complex and IL-1β−IL-1RI binary complex.

IL-1β−IL-1RII−IL-1RAcP Site I IL-1β−IL-1RI Site I IL-1β IL-1RII IL-1β IL-1RI Arg11 Pro126 Arg11 Lys114 Ser13 Val136 Gln14 Val138, His181 Gln14 Val124, Arg163 Gln15 Gln127, Ile128, Val136 Gln15 Lys1114, Gly122 Met20 Tyr22 Ser21 Tyr22, Gln39

Glu25 Asn30 Lys27 Tyr22 Lys27 Glu11 His30 Lys23, Val138, Pro140 Gln32 Phe26, Ile123 Gln32 Ile14, Val16, Ile110, Phe111 Gly33 Ile123, Ser124 Gly33 Ile110, Lys112 Gln34 Arg24, Phe26 Gln34 Gln108 Asp35 Tyr22, Lys23, Arg24 Gln38 Tyr22

Asp108 Asn204 Glu128 Asp141, Leu142 Glu128 Tyr127, Glu129 Met130 Leu44

IL-1β−IL-1RII−IL-1RAcP Site II IL-1β−IL-1RI Site II

IL-1β IL-1RII IL-1β IL-1RI Ala1 Asp260, Tyr261 Pro2 Tyr261

Arg4 Met258, Arg282 Arg4 Leu237, Asp239, Ser263, Leu275

Leu6 Leu237 Phe46 Met258 Glu51 Trp260, Phe321 Glu51 Lys298 Ser52 His316

Asn53 Ile303 Ile56 His316 Lys93 Glu269 Lys93 Ile250, Glu252, Glu259

Lys94 Glu252 Glu105 Thr318 Glu105 Thr300

IL-1β−IL-1RII−IL-1RAcP Site III

IL-1β IL-1RAcP D54 Arg286

Lys109 Glu132

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Glu111 Ser185 Gly140 Phe167, Asn168 Gln141 Asn166 Asp145 Ser185

IL-1β−IL-1RII−IL-1RAcP Site IV

IL-1RII IL-1RAcP Ser134 Glu132, Gly134 Val173 Leu180 Leu180 Gly134 Ile223 His226 Val225 Asn229 Ile226 Val232 Phe248 His231

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Supplementary Figure 1: Electron density maps, contoured at 1.0 σ and the

molecular packing in the crystal lattice. (a) Stereo view of the overall map calculated

by combining SAD phases from a crystal with only IL-1β selenomethionine-labeled

with calculated phases from the molecular replacement solution; (b) Stereo view of

the overall map calculated by combining SAD phases from a crystal with all

components selenomethionine-labeled with calculated phases from the molecular

replacement solution; (c) Stereo view of the final overall 2mFo-DFc map. In (a, b, c),

the final model is shown as ribbon. (d) A representative 2mFo-DFc map of the

interface between IL-1β (green) and IL-1RAcP (cyan). (e) The molecular packing in

the crystal lattice. The left panel shows the packing around the D1 domain of

IL-1RAcP, and the right panel shows the packing around the D3 domain of IL-1RAcP.

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Supplementary Figure 2: Sequence alignments. (a) Sequence alignment of

IL-1RAcP-dependent agonists (IL-1β, IL-1α, IL-33, IL-1F6, IL-1F8 and IL-1F9) and

their respective antagonists (IL-1Ra and IL-1F5). The alignment of IL-1β, IL-1α

(PDB code: 2ILA), IL-1Ra (PDB code: 1ILT), IL-33 (PDB code: 2KLL), and IL-1F5

(PDB code: 1MD6) is structure-based, and IL-1F6, IL-1F8, and IL-1F9 are aligned

with IL-1β respectively by using ClustalW server (http://www.ebi.ac.uk/clustalw/).

Green highlights IL-1β residues involved in the interactions with IL-1RII at site I.

Yellow highlights IL-1β residues involved in the interactions with IL-1RII at site II.

Cyan highlights IL-1β residues involved in the interactions with IL-1RAcP at site III.

The D145 position in IL-1β is labeled with red star. (b) Sequence alignment of

ligand-binding primary receptors IL-1RII, IL-1RI, ST2, and IL-1Rrp2 by using

ClustalW. Green highlights IL-1RII residues involved in the interactions with IL-1β at

site I. Yellow highlights IL-1RII residues involved in the interactions with IL-1β at

site II. Orange highlights IL-1RII residues involved in the interactions with IL-1RAcP

at sites IV. The NxS/T amino acid motifs that could be glycosylated in IL-1RII are

boxed. (c) Sequence of IL-1RAcP. Cyan highlights IL-1RAcP residues involved in the

interactions with IL-1β at site III. Orange highlights IL-1RAcP residues involved in

the interactions with IL-1RII at site IV. The NxS/T amino acid motifs that could be

glycosylated in IL-1RAcP are boxed.

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Supplementary Figure 3: The N-linked glycans in the IL-1β−IL-1RII−IL-1RAcP

complex structure.

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Supplementary Figure 4: Three different models of the IL-1β−IL-1RI−IL-1RAcP

architecture.

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Supplementary Figure 5: Purification of IL-1β−IL-1RI, IL-1β−IL-1RII, and

IL-1Ra−IL-1RI complexes containing native or mutated cytokine. (a), IL-1β mutants

are: IL-1β-mut1, β4−β5 loop substitution; IL-1β-mut2, β11−β12 loop substitution;

IL-1β-mut2’, extended β11−β12 loop substitution including D145K (red star) ;

IL-1β-mut3, β5−β6 loop substitution. IL-1Ra mutant (IL-1Ra-mut) contains double

substitution of β4−β5 and extended β11−β12 loops. (b) Size exclusion

chromatography of the binary complex purification. Inset is the SDS-PAGE gel, the

left lane showing the binary complex, and the right lane showing the cytokine.

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Supplementary Figure 6: The IL-1β β5−β6 loop is on the right side of the complex

seen from the concave surface of IL-1RII. It has no interaction with IL-1RAcP.

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