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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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