Molecular evolution of ultraspiracle protein (USP/RXR) in insects

Abstract

Ultraspiracle protein/retinoid X receptor (USP/RXR) is a nuclear receptor and transcription factor which is an essential component of a heterodimeric receptor complex with the ecdysone receptor (EcR). In insects this complex binds ecdysteroids and plays an important role in the regulation of growth, development, metamorphosis and reproduction. In some holometabolous insects, including Lepidoptera and Diptera, USP/RXR is thought to have experienced several important shifts in function. These include the acquisition of novel ligand-binding properties and an expanded dimerization interface with EcR. In light of these recent hypotheses, we implemented codon-based likelihood methods to investigate if the proposed shifts in function are reflected in changes in site-specific evolutionary rates across functional and structural motifs in insect USP/RXR sequences, and if there is any evidence for positive selection at functionally important sites. Our results reveal evidence of positive selection acting on sites within the loop connecting helices H1 and H3, the ligand-binding pocket, and the dimer interface in the holometabolous lineage leading to the Lepidoptera/Diptera/Trichoptera. Similar analyses conducted using EcR sequences did not indicate positive selection. However, analyses allowing for variation across sites demonstrated elevated non-synonymous/synonymous rate ratios (d(N)/d(S)), suggesting relaxed constraint, within the dimerization interface of both USP/RXR and EcR as well as within the coactivator binding groove and helix H12 of USP/RXR. Since the above methods are based on the assumption that d(S) is constant among sites, we also used more recent models which relax this assumption and obtained results consistent with traditional random-sites models. Overall our findings support the evolution of novel function in USP/RXR of more derived holometabolous insects, and are consistent with shifts in structure and function which may have increased USP/RXR reliance on EcR for cofactor recruitment. Moreover, these findings raise important questions regarding hypotheses which suggest the independent activation of USP/RXR by its own ligand.

Figure 1. Phylogeny of insect species used in our analysis.

The topology of the trees used for the USP/RXR (left) and EcR (right) datasets are based on known taxonomic relationships. Species contained in the Diptera (Dip.) are shaded green, Lepidoptera (Lep.) blue, Trichoptera (Tri.) yellow, Hymenoptera (Hym.) orange, Coleoptera (Col.) red, Hemiptera (Hem.) purple, Dictyoptera (Dic.) and Orthoptera (Ort.) grey. The origin of holometabolous insects is indicated by an encircled bold H. The highlighted branch is the foreground lineage for detecting positive selection, where a separate d _N/d _S ratio was estimated for the Mecopterida (ω_m).

Figure 2. Location of putative positively selected sites in Mecopterida USP/RXR.

(A) The distribution of sites inferred by PAML to be under positive selection (P>0.95) along the Mecopterida branch (red spheres) across the crystal structure of H. virescens USP (green) with the ligand shown in grey. (B) Positively selected sites (red spheres) located near ligand binding sites (white sidechains). Only L230 directly interacts with the phospholipid ligand (grey) via van der Waals (red dashes). (C) The involvement of sites L203 and R231 in a hydrogen bond network between H3, loop L1–3 and H12. Hydrogen bonds are indicated by dotted blue lines and waters by small red dots (adapted from [24]). The backbone and sidechains of residues not under positive selection are shown in white, with blue and red indicating nitrogen and oxygen respectively. (D) Polar interactions (dashed black lines) between the positively selected site E411 in USP loop L9–10 (green) and two arginine residues in H9 of the EcR (cyan). The images were created using PDB 1G2N and 1R1K, site numbering according to H. virescens USP (AX383958) and EcR (Y09009).

Figure 3. Posterior mean ω at each amino acid site across the USP/RXR gene.

The values of ω as estimated by M8 in PAML (black line) and the Dual model of HyPhy (shaded blue) are shown for each codon site across the gene for the Mecopterida (A) and Non-Mecopterida (B). All sites inferred to be under positive selection by branch-site analysis the along the Mecopterida branch are shown as red boxes on the x-axis in of the Mecopterida plot. A schematic of USP/RXR secondary structure is shown above both plots to illustrate the position of each functional domain (A/B, C, and D) as well as the helices (H1–H12) and β sheets (S1–S2) of the ligand-binding domain. The schematic for the Mecopterida and Non-Mecopterida genes are based of the crystal structures of H. virescens and B. tabaci USP/RXR, respectively. Site numbering is based on the USP/RXR alignment given in supporting figure S1.

Figure 4. Posterior mean ω at each amino acid site across the EcR gene.

The values of ω as estimated by M8 in PAML (black line) and the Dual model of HyPhy (shaded blue) are shown for each codon site across the gene for the Mecopterida (A) and Non-Mecopterida (B). A schematic of EcR secondary structure is shown above both plots to illustrate the position of each functional domain (A/B, C, and D) as well as the helices (H1–H12) and β sheets (S1–S3) of the ligand-binding domain. The schematic for the Mecopterida and Non-Mecopterida genes are based of the crystal structures of H. virescens and B. tabaci EcR, respectively. Site numbering is based on the EcR alignment given in supporting figure S2.