Structural and Functional Insights into the Ligand-binding Domain of a Nonduplicated Retinoid X Nuclear Receptor from the Invertebrate Chordate Amphioxus

Abstract

Retinoid X nuclear receptors (RXRs), as well as their insect orthologue, ultraspiracle protein (USP), play an important role in the transcription regulation mediated by the nuclear receptors as the common partner of many other nuclear receptors. Phylogenetic and structural studies have shown that the several evolutionary shifts have modified the ligand binding ability of RXRs. To understand the vertebrate-specific character of RXRs, we have studied the RXR ligand-binding domain of the cephalochordate amphioxus (Branchiostoma floridae), an invertebrate chordate that predates the genome duplication that produced the three vertebrates RXRs (␣, ␤, and ␥). Here we report the crystal structure of a novel apotetramer conformation of the AmphiRXR ligand-binding domain, which shows some similarity with the structures of the arthropods RXR/USPs. AmphiRXR adopts an apo antagonist conformation with a peculiar conformation of helix H11 filling the binding pocket. In contrast to the arthropods RXR/USPs, which cannot be activated by any RXR ligands, our functional data show that AmphiRXR, like the vertebrates/mollusk RXRs, is able to bind and be activated by RXR ligands but less efficiently than vertebrate RXRs. Our data suggest that amphioxus RXR is, functionally, an intermediate between arthropods RXR/USPs and vertebrate RXRs. The nuclear receptor (NR)6 superfamily is specific to animals and performs an abundance of functions, from embryonic development to metamorphosis and from homeostasis of various physiological functions to the control of metabolism (for a review, see Ref. 1). Nuclear receptors are ligand-activated transcription factors. Many members of the superfamily thus bind major hormones, such as steroids, thyroid hormones, or retinoids. These occupy a special position in gene regulation by providing a direct link between the ligand, which they bind, and the target gene, whose expression they regulate. There are also many nuclear receptors that are not known to bind any ligand and are thus called "orphan nuclear receptors" (2-4). Some nuclear receptors which were originally discovered as orphans have since been shown to bind small hydrophobic molecules, such as fatty acids, but others appear to be true orphans (4).Nuclear receptors are composed of several functional domains. The N-terminal A/B domain is highly variable in length and sequence and contains a constitutively active transactivation function AF-1. Most conserved are domains C and E, corresponding grossly to the DNA-binding domain and the ligand-binding domain (LBD), respectively. The LBD contains the ligand-dependent transactivation function AF-2. The DNA-binding domain and LBD are connected via a flexible hinge (domain D). NRs act in vivo and in vitro as ligand-dependent transcriptional transregulators through binding, most often as heterodimers, to cis-acting response elements present in cognate target genes. Activation of gene transcription occurs after binding of ligand, which results in a conformational change of the LBD, leading to release of corep...

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