Diagram of the structural elements of Tube and Pelle homologs.

<p><i>D. melanogaster</i> Tube and <i>N. vitripennis</i> Tube-like proteins lack their individual kinase domains. Repeat motifs are observed in both insect Tube and Tube-like proteins and <i>Sp</i>Pelle.</p

Phylogenetic analysis of the retrieved Tube and Pelle homologs from insects, crustaceans, and humans.

<p>NJ trees were generated using the MEGA 4.0 software. The corresponding GenBank accession numbers are listed in this figure. <i>Sp</i>Tube and <i>Sp</i>Pelle are marked with red and blue frames, respectively. <i>Dm, Drosophila melanogaster; Cp, Culex pipiens; Aa, Aedes aegypti; Ag, Anopheles gambiae; Nv, Nasonia vitripennis; Dp, Daphnia pulex; Am, Apis mellifera; Cf, Camponotus floridanus; Tc, Tribolium castaneum; Lv, Litopenaeus vannamei; Hs, Homo sapiens; Sp, Scylla paramamosain</i>.</p

Pull-down assays to test the binding activity of <i>Sp</i>Tube death domain (TubeDD) with <i>Sp</i>MyD88 death domain (MyDD).

<p>(A) Recombinant MyDD was bound to His Bind resin, to which purified GST-TubeDD protein was added. MyDD and TubeDD were eluted simultaneously with elution buffer but not with wash buffer. (B) Recombinant MyDD was added to His Bind resin, to which GST alone was added as a control. Only MyDD was eluted by elution buffer. M, protein markers; Lane 1, recombinant MyDD; Lane 2, GST-TubeDD; Lane 3, collection of wash buffer; Lane 4, collection of elution buffer; Lane 5, recombinant MyDD; Lane 6, GST; Lane 7, collection of wash buffer; Lane 8, collection of elution buffer.</p

Complete cDNA and translated amino acid sequences of <i>Sp</i>Pelle.

<p>The death domain (first domain), two conserved repeat motifs (two middle sequences), and kinase domain (last domain) are shadowed orderly. The stop codon is indicated by an asterisk (*). GD dipeptide rather than RD dipeptide is shown in blue frame.</p

(A) SDS-PAGE analysis of recombinant <i>Sp</i>Tube-DD expressed in <i>Escherichia coli</i>.

<p>Lane M, protein marker; Lane 1, total protein obtained from <i>E. coli</i> expressing pGEX4T1-<i>Sp</i>Tube-DD; Lane 2, total protein obtained from <i>E. coli</i> expressing GST<i>-Sp</i>Tube-DD after IPTG induction; Lane 3, recombinant GST<i>-Sp</i>Tube-DD purified by glutathione Sepharose 4B chromatography. (B) SDS-PAGE analysis of recombinant <i>Sp</i>MyD88-DD expressed in <i>E</i>. <i>coli</i>. Lane 1, total protein obtained from <i>E. coli</i> expressing pET30a-<i>Sp</i>MyD88-DD; Lane 2, total protein obtained from <i>E. coli</i> expressing <i>Sp</i>MyD88-DD after IPTG induction; Lane 3, recombinant <i>Sp</i>MyD88-DD purified by His-tag resin chromatography. (C) SDS-PAGE analysis of recombinant <i>Sp</i>Pelle-DD expressed in <i>E</i>. <i>coli</i>. Lane 1, total protein obtained from <i>E. coli</i> expressing pET30a-<i>Sp</i>Pelle-DD; Lane 2, total protein obtained from <i>E. coli</i> expressing <i>Sp</i>Pelle-DD after IPTG induction; Lane 3, recombinant <i>Sp</i>Pelle-DD purified by His-tag resin chromatography.</p

Full-length cDNA and translated amino acid sequences of <i>Sp</i>Tube.

<p>The death domain (the first domain) and kinase domain (the second domain) are shadowed. The stop codon is indicated by an asterisk (*). The initiation codon and the polyadenylation signal are shown in bold and red letters, respectively. The RD dipeptide existing only in Tube-like kinases but not in Pelle or Pelle-like protein sequences is marked with red box.</p

Comparisons of the predicted 3D structure of the <i>Sp</i>Tube death domain (DD).

<p>(14 aa to 144 aa, yellow) and the crystal structure of DmTube DD (PDB ID: 1d2z, chain B) (red) (A); the predicted 3D structure of <i>Sp</i>Pelle DD (13 aa to 109 aa, yellow) and the solution structure of DmPelle DD (PDB ID: 1d2z, chain A) (red) (B); the predicted 3D structure of <i>Sp</i>Tube kinase domain (251 aa to 488 aa, yellow) and the crystal structure of human IRAK4 kinase domain (PDB ID: 2nru, chain B) (red) (C); and the predicted 3D structure of <i>Sp</i>Pelle kinase domain (540 aa to 825 aa, yellow) and the crystal structure of human IRAK4 kinase domain (PDB ID: 2nru, chain B) (red) (D). Residues required for binding to MyD88 (blue) and Pelle (red) are indicated (A). The position of R (ARG) residue in RD dipeptide, which is the hallmark of Tube-like kinases, is also shown (C). These four predicted structures merge well with their corresponding templates.</p

Sequences of the primers used in this study.

<p>Underlined nucleotides indicate the locations of the restricted endonucleases.</p

Alignment of the death domains of the Tube and Pelle homologs from insects and crustaceans.

<p>The death domains of <i>Sp</i>Tube and <i>Drosophila</i> Tube share three conserved core residues (R, K, and R, indicated by blue arrows) required for binding to dMyD88. The death domains of Insect Tube homologs contain several common conserved core residues (E, A, GPXA, indicated by red arrows) required for binding to Pelle. The death domains of <i>Sp</i>Tube and <i>Drosophila</i> Tube share two core residues (A, A).</p

Pull-down assays to test the binding activity of <i>Sp</i>Tube death domain (TubeDD) with <i>Sp</i>Pelle death domain (PelleDD).

<p>(A) Recombinant PelleDD was bound to His Bind resin, to which purified GST-TubeDD protein was added. PelleDD and TubeDD were eluted simultaneously with elution buffer but not with wash buffer. (B) Recombinant PelleDD was added to His Bind resin, to which GST alone was added as a control. Only PelleDD was eluted by elution buffer. M, protein markers; Lane 1, recombinant PelleDD; Lane 2, GST-TubeDD; Lane 3, collection of wash buffer; Lane 4, collection of elution buffer; Lane 5, recombinant PelleDD; Lane 6, GST; Lane 7, collection of wash buffer; Lane 8, collection of elution buffer.</p