Multiple sequence alignment of the death domains (DDs) of representative IRAK family members along with the IRAK4 DD crystal structure.

<p>The amino acid numbers corresponding to DD regions for each representative sequence are shown beside the IRAK protein names. Green colored circles represent conservation within each IRAK subfamily. The potential type I sites are represented by purple circles, whereas conserved and type I potential sites are both depicted as dark blue circles. The red triangles represent conserved amino acids in all IRAK subfamilies from the entire species list.</p

Overview of the kinase domain amino acid changes in 26 predicted sites during type II functional divergence.

<p>Sites that correlated with substantial biochemical change were detected in the pairwise type II functional analysis of the vertebrate IRAKs (KD). The residue numbering is based on the human IRAK4 KD sequence. The amino acid changes between the subfamilies along with their property changes are provided. (“+” represents a positive amino acid whereas “−” represents a negative amino acid).</p

Phylogenetic analysis of IRAK genes compared with <i>D. melanogaster</i> homologs determined using the Bayesian inference.

<p>A total of 73 coding sequences were included in this analysis (22 representative vertebrate IRAK subfamily sequences based on taxonomy; 19 Pelle, 26 Tube/TLK, and 6 PIK-1 sequences). <i>A. queenslandica</i> was considered as an outgroup. The numerical values represent posterior probability. Each IRAK subfamily clade is represented by a unique color: Tube/TLK (hot pink), PIK-1 (cyan), and Pelle (khaki). Other IRAK colors are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049771#pone-0049771-g001" target="_blank">Figure 1</a>. Taxonomic convention: IRAK protein name followed by an abbreviated form of the species name.</p

Phylogenetic relationship between vertebrate IRAK subfamilies determined using the Bayesian inference.

<p>A total of 139 IRAK coding sequences were included in this analysis. <i>A. queenslandica</i> was considered as an outgroup. The numerical values represent posterior probability. As depicted, the tree clustered into 4 major monophyletic clades. Each clade is represented by a unique color: IRAK4 (purple), IRAKM (blue), IRAK2 (green), and IRAK1 (light red). The species closest to the outgroup invertebrates are shown in red. Taxonomical convention: IRAK protein name followed by an abbreviated form of the species name.</p

IRAK4 DD and KD structures.

<p>(A) The crystal structure of the IRAK4 DD and the Type I potential sites are shown using a ball-and-stick representation as a representative for all the DDs. (B) The crystal structure of the IRAK4 KD and the Type I potential sites are shown using a ball-and-stick representation as a representative for all the KDs. (C) Type II potential sites are shown using a ball-and-stick representation with reference to the IRAK4 KD crystal structure.</p

Multiple sequence alignment of the kinase domains (KDs) from representative IRAK family members along with the IRAK4 KD crystal structure.

<p>The amino acid numbers corresponding to the KD regions of each representative sequence are shown beside each IRAK protein name. Green colored circles represent the conservation observed within each subfamily in the species list. The potential type I sites are represented by purple circles, whereas conserved and type I potential sites are both depicted by dark-blue circles. The red triangles represent the conserved amino acids of all IRAK subfamilies from the entire species list. The green arrow represents the critical Asp residue. Orange arrows indicate potential type II sites. Gray triangles on the arrows represent conserved amino acids involved in type II divergence.</p

Type I evolutionary functional divergence between vertebrate IRAK subfamilies (death domain).

<p>Type I functional divergence coefficients (<i>θ_I</i>) and their respective standard errors are shown above the diagonal (upper right). Functional divergence values are represented by 1><i>θ_I</i>>0.5. Below the diagonal (lower left) are the likelihood ratio (LRT) values to test the null hypothesis of <i>θ_I</i> = 0. The LRT values in bold significantly rejected the null hypothesis (p≤0.05).</p

Molecular Evolution and Structural Features of IRAK Family Members

<div><p>The interleukin-1 receptor-associated kinase (IRAK) family comprises critical signaling mediators of the TLR/IL-1R signaling pathways. IRAKs are Ser/Thr kinases. There are 4 members in the vertebrate genome (IRAK1, IRAK2, IRAKM, and IRAK4) and an IRAK homolog, Pelle, in insects. IRAK family members are highly conserved in vertebrates, but the evolutionary relationship between IRAKs in vertebrates and insects is not clear. To investigate the evolutionary history and functional divergence of IRAK members, we performed extensive bioinformatics analysis. The phylogenetic relationship between IRAK sequences suggests that gene duplication events occurred in the evolutionary lineage, leading to early vertebrates. A comparative phylogenetic analysis with insect homologs of IRAKs suggests that the Tube protein is a homolog of IRAK4, unlike the anticipated protein, Pelle. Furthermore, the analysis supports that an IRAK4-like kinase is an ancestral protein in the metazoan lineage of the IRAK family. Through functional analysis, several potentially diverged sites were identified in the common death domain and kinase domain. These sites have been constrained during evolution by strong purifying selection, suggesting their functional importance within IRAKs. In summary, our study highlighted the molecular evolution of the IRAK family, predicted the amino acids that contributed to functional divergence, and identified structural variations among the IRAK paralogs that may provide a starting point for further experimental investigations.</p> </div

Superimposition of the IRAK DD final structures obtained from MD simulations with initial structures.

<p>Differences in the final structures of IRAK1, IRAK2, IRAKM, IRAK4, Pelle, Tube, and PIK-1 are shown. Their respective initial structures are in golden blue. The major structural variations are observed from helix 2 to helix 4.</p

Type I evolutionary functional divergence between vertebrate IRAK subfamilies (kinase domain).

<p>Type I functional divergence coefficients (<i>θ_I</i>) and their respective standard errors are shown above the diagonal (upper right). Functional divergence values are represented by 1><i>θ_I</i>>0.5. Below the diagonal (lower left) are the likelihood ratio (LRT) values to test the null hypothesis of <i>θ_I</i> = 0. LRT values in bold significantly rejected the null hypothesis (p≤0.05).</p