Supporting InformationCharles et al. 10.1073/pnas.1116123109SI MethodsDextran-Coated Charcoal (DCC) Assays.DCC assays were performedessentially as described (1) with some modifications. An appro-priate volume of a [3H]juvenile hormone (JH) III solution inhexane was evaporated in narrow glass tubes previously coatedwith 1% polyethylene glycol 20,000. [3H]JH III was then dis-solved in 100 μL of a buffer [20 mM Tris·HCl (pH 7.9), 5 mMmagnesium acetate, 1 mM EDTA, 1 mM DTT, and 1 μM of 3-octylthio-1,1,1-trifluoro-2-propanone (OTFP)]. OTFP is a potentesterase inhibitor added to protect JH III from degradation (2,3). Proteins were transcribed/translated for 90 min at 30 °C in T7Quick Coupled (Promega) TnT reactions. Each 50-μL reactionwas divided by 15 μL into the tubes with the dissolved hormone,mixed by gentle vortexing, and incubated for 60 min at 22 °C.The remaining 5 μL of the TnT reaction was saved for immu-noblot analysis of the protein integrity. Per each assay, 20 μL ofthe DCC suspension [10 mM Tris·HCl (pH 7.5), 1.5 mM EDTA,1% dextran, 5% Norit A] was added and gently mixed by vor-texing. After 10 min, the tube was centrifuged for 3 min at 3,000 ×g, and a 50-μL aliquot of the supernatant was collected andcounted by scintillation. The samples were not decolorized be-cause we found that color quenching of radioactivity by the rabbitreticulocyte lysate was insignificant after dilution with the scin-tillation fluid. Under the described conditions, no significantdegradation of the radiolabeled ligand was detectable at the endof the incubation, as judged by TLC analysis (Fig. S8).

Immunoprecipitation. Per each assay, the HEK293 human em-bryonic kidney cells were grown in DMEM/10% FBS (Invitrogen)on 10-cm plates to half confluence. The cells were transientlytransfected with polyethyleneimine with 5 μg of each of twoprotein expression vectors: pEGFP-C2 (Clontech) and pK-Myc-C2, providing N-terminal EGFP and Myc epitope tags, re-spectively. At 40 h posttransfection, cells were treated with 1 μMmethoprene in 10 μL of ethanol (or ethanol only) and harvested1 h later by centrifugation in cold PBS. The cell pellet was re-suspended in 250 μL of lysis buffer [50 mM Tris·HCl (pH 7.4),150 mM NaCl, 1 mM EDTA (pH 8), 50 mM NaF, 1 mM Na3-VO4, 1% Nonidet P-40, and the Complete protease inhibitors(Roche)], and cells were lysed for 30 min on ice with mixing.Where appropriate, the lysis buffer was supplemented with 1 μMmethoprene. After clearing by centrifugation, 180 μL of the ly-sates was added to tubes with 50 μL of the Dynabeads Protein G(Invitrogen) magnetic beads, previously bound with a rabbit anti-EGFP antiserum, and incubated for 45 min at room temperatureon a rotator. After washing five times with 500 μL of PBS atroom temperature, the bound proteins were eluted from thebeads with 20 μL of SDS sample buffer for 10 min at 70 °C. Input(10%) and bound proteins were loaded on a 12% SDS/PAGE,transferred onto the Protran nitrocellulose membrane, and de-tected with mouse anti-GFP (Sigma-Aldrich) and anti-Myc 9E10(Roche) antibodies on immunoblots by using HRP-conjugatedanti-mouse IgG (all antibodies diluted 1:2,000).

1. Miura K, Oda M, Makita S, Chinzei Y (2005) Characterization of the DrosophilaMethoprene-tolerant gene product. Juvenile hormone binding and ligand-dependentgene regulation. FEBS J 272:1169e1178.

2. Prestwich GD, Eng WS, Roe RM, Hammock BD (1984) Synthesis and bioassay ofisoprenoid 3-alkylthio-1,1,1-trifluoro-2-propanones: Potent, selective inhibitors ofjuvenile hormone esterase. Arch Biochem Biophys 228:639e645.

3. Renou M, Lucas P, Malo E, Quero C, Guerrero A (1997) Effects of trifluoromethylketones and related compounds on the EAG and behavioural responses topheromones in male moths. Chem Senses 22:407e416.

Fig. S1. Expression of full-length Tribolium Methoprene-tolerant (Met)(1–516) and truncated versions of the Met and Taiman proteins used in JH III-bindingassays. Codon-optimized Met DNA sequences encoding the indicated amino acid residues and cDNA encoding Tribolium Taiman [accession no. XP_967666.1;residues 141–510 encompassing the basic helix–loop–helix (bHLH) and Per-Arnt-Sim (PAS)-A and PAS-B domains] were cloned into the pK-Myc-C2 plasmid.Standard 50-μL transcription/translation reactions with the T7 TnT Quick Coupled system (Promega) contained 400 ng of template plasmid DNA. Expression andstability of all proteins after every assay was checked in 2-μL aliquots of the TnT reaction on immunoblots with mouse anti-Myc monoclonal 9E10 diluted1:4,000 (Roche) and followed by detection with HRP-conjugated anti-mouse IgG (1:2,000) and the ECL Western Blotting Substrate (Pierce) for chem-iluminescence with the Fujifilm LAS-3000 imager. Removal of the native Met protein C terminus (constructs 1–386 and 240–386) reduced the protein yield.Arrow points to the weakly expressed truncated PAS-B domain of Met (construct 240–386).

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Fig. S2. JH III-binding capacity of the PAS-B domain of Tribolium Met was verified in equilibrium dialysis assays. Equal amounts of [3H]JH III in 70 μL of buffer[20 mM Tris·HCl (pH 7.9), 5 mM magnesium acetate, 1 mM EDTA, 1 mM DTT, and 1 μM OTFP] was loaded into both compartments of an equilibrium mi-crodialyzer (part no. 742202; Harvard Apparatus). One of the two dialysis compartments contained 15 μL of either one of the indicated in vitro-translated Metversions; the other compartment contained the TnT reticulocyte lysate mix without DNA. After overnight incubation at 4 °C, aliquots from each compartmentwere measured by scintillation counting. Accumulation of labeled JH III in the compartment containing a Met protein relative to the entire input of radio-activity was taken as a measure of specific JH III binding. Values are mean ± SD from three independent experiments.

Fig. S3. Homology between the PAS-B domain of hypoxia-inducible factor 2α (HIF2α), for which the crystal structure (PDB ID 3F1P) has been resolved (1), wasused as a template for structure modeling of the PAS-B domain of Tribolium Met (residues Leu-240–Leu-358 are shown).

Fig. S4. Computational docking of JH III and pyriproxyfen into the structural model of Tribolium Met PAS-B domain. (A) Superposition of four best dockingsolutions for JH III in the ligand-binding pocket. The solutions correspond to −7.4 kcal/mol (green), −7.2 kcal/mol (yellow), and −6.9 kcal/mol (orange andmagenta), respectively. Solutions 1 (yellow) and 4 (magenta) involve a hydrogen bond (dotted lines) between the epoxide group and Tyr-252. (B) Docking ofpyriproxyfen produced a single best solution with a theoretical affinity of −9.2 kcal/mol and two potential hydrogen bonds between two of its ether groupsand Tyr-252 and Lys-311. Side chains of residues contributing to the ligand-binding pocket of both JH III and pyriproxyfen are indicated.

1. Scheuermann TH, et al. (2009) Artificial ligand binding within the HIF2α PAS-B domain of the HIF2 transcription factor. Proc Natl Acad Sci USA 106:450e455.

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Fig. S5. Mutagenesis of the evolutionarily conserved PAS-B domain of the Tribolium Met protein. Letters above the Tribolium sequence show amino acidsubstitutions used in this study, and numbers indicate their positions. Mutations shown in black squares substantially reduce JH III binding. The Met PAS-Bdomain from the beetle (Coleoptera) Tribolium castaneum (accession no. ABR25244.1) is aligned with its orthologs from the mosquitoes (Diptera) Anophelesgambiae (DQ303468.1) and Aedes aegypti (AY902310.1); the honey bee, the wasp, and the ant (all Hymenoptera) Apis melifera (XP_395005.3), Nasonia vit-ripennis (XM_001606725.2), and Camponotus floridanus (EFN68682.1); the fly (Diptera) Drosophila melanogaster (Dm) Gce (NP_511160.1) and Met(NP_511126.2); the silk moth (Lepidoptera) Bombyx mori (Bm) Met1 (BAJ05085.1) and Met2 (BAJ05086.1); the true bugs and the aphid (all Hemiptera) Pyr-rhocoris apterus (JN416984), Rhodnius prolixus (JN416985), and Acyrthosiphon pisum (XP_003246906.1); the louse (Phthiraptera) Pediculus humanus(XP_002430841.1); and the firebrat (Zygentoma) Thermobia domestica (JN416986). Dm_Met, Dm_Gce, and Bm_Met1, Bm_Met2 are products of paralogousMet genes that have duplicated in the Drosophila and Bombyx lineages, respectively.

Fig. S6. Example of expression and stability of WT and mutant versions of the truncated (240–516) and full-length (1–516) Tribolium Met proteins. Codon-optimized DNA sequences encoding the indicated Met variants were expressed from 400 ng of the pK-Myc-C2 vector in 50-μL transcription/translation reactions(Promega). Aliquots (2-μL) of each TnT reaction were examined on immunoblots with the mouse anti-Myc antibody 9E10 (Roche) diluted 1:4,000 and followedby detection with a HRP-conjugated anti-mouse IgG (1:2,000) and the ECL Western Blotting Substrate (Pierce) for chemiluminescence.

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Fig. S8. JH III remains undegraded during binding assays. DCC binding assays containing 20 μL of Quick Coupled TnT reticulocyte lysate (Promega) expressingthe full-length Met protein were set up under standard conditions in the presence of 2 pmol of [3H]JH III and with or without 1 μM esterase inhibitor OTFP.Samples were then subjected to TLC analysis on a Whatman LK6D TC plate, which was developed (hexane/ethyl acetate/acetic acid: 70/40/1) and exposed at−80 °C overnight after impregnation with EN3HANCE (Perkin-Elmer). Arrow indicates the direction of migration. Asterisk shows a position of a very faint bandvisible only in the samples without OTFP, probably corresponding to JH acid. The leftmost lane contains 2 pmol of the input [3H]JH III stock solution.

1. Pandini A, Denison MS, Song Y, Soshilov AA, Bonati L (2007) Structural and functional characterization of the aryl hydrocarbon receptor ligand binding domain by homology modelingand mutational analysis. Biochemistry 46:696e708.

2. Pandini A, et al. (2009) Detection of the TCDD binding-fingerprint within the Ah receptor ligand binding domain by structurally driven mutagenesis and functional analysis.Biochemistry 48:5972e5983.

Fig. S7. Comparison of individual point mutations within the PAS-B domains of Tribolium Met and mouse aryl hydrocarbon receptor (mAhR, accession no.NP_038492.1) that abolish or reduce binding of JH III and dioxin (TCDD) ligands toMet andmAhR, respectively. Data for mAhR are from Pandini and colleagues (1, 2).

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