Solution structure of the orphan nuclear receptor Rev-erb  response element by 1 H, 31 P NMR and molecular simulation* Claire Castagné a , Hernan Terenzi b **, Mario M. Zakin b , Muriel Delepierre a*** a Laboratoire de Résonance Magnétique Nucléaire, CNRS URA 1773, Institut Pasteur, 28, rue du Dr.-Roux, 75724 Paris cedex 15, France b Unité d’Expression des Gènes Eucaryotes, CNRS URA2185, Institut Pasteur, 28, rue du Dr.-Roux, 75724 Paris cedex 15, France (Received 10 February 2000; accepted 16 May 2000) Abstract — Rev-erb  is an orphan receptor that binds as a homodimer or as a monomer to DNA. The solution structure of the non-palindromic 15 bp DNA duplex d(TAGAATGTAGGTCAG), the response element of Rev-erb  for monomeric binding, was determined by 1 H and 31 P NMR, energy minimization with NMR-derived restraints for distances and NOE back-calculation methods. The refined final structures have the typical overall features of B-type DNA. However, titration of this 15 bp duplex with ReDBD, the DNA binding domain of Rev-erb , showed large shifts of imino protons and 31 P signals, suggesting major conformational changes. © 2000 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS Rev-erb  / homodimer / imino protons / DNA binding domain 1. Introduction The superfamily of hormone nuclear receptors includes more than 150 different proteins that play important roles in cellular regulation, providing a direct link between transcriptional control and physiological responses. This family contains nuclear receptors for hydrophobic ligands, such as steroid and thyroid hormones, retinoids and vitamin D and receptors for which only artificial ligands or no ligands have been identified: these are called ‘orphan receptors’. Target gene recognition is specified by the DNA binding domain (DBD) which, in the glucocor- ticoid (GR) and oestrogen (ER) receptors, consists of two type II zinc fingers containing all the elements required for interaction with their reactive DNA sites [1]. In contrast, the retinoic X receptor (RXR) DBD requires an additional helix extending from its second zinc finger for specific binding to its DNA target as homodimer [2]. For other receptors interacting with DNA as monomers, a region homologous to the additional helix of the RXR DBD, known as the T/A box, is thought to be involved in contact with DNA. Most response elements for nuclear receptors contain a six-nucleotide core for binding with the consen- sus sequence (A/G)GGTCA. Typically, two core elements are organized into either a palindrome or a direct repeat, to form a hormone responsive element (HRE). Generally, nuclear receptors bind their HRE as homodimers or heterodimers. However, in the recent years, many orphan receptors have been identified that are able to bind as monomers to a single 5’-extended consensus sequence of DNA [3] and the X-ray structure of one of them, the nuclear growth factor induced B (NGFI-B) was recently solved [4]. The Rev-erb family is a sub-group of orphan receptors, three members of which have been isolated from mam- malian genotypes: Rev-erbAα, also known as Ear-1 [3, 5, 6], Rev-erb also known as RVR or BD73 [7–9] and HZF-2 [10]. Mouse Rev-erb sequences map to chromosomes 19 and 14 [11] and their transcripts have a wide distribution, suggesting a broad range of biological activities. How- ever, their physiological effects are poorly understood. It has recently been reported that mouse Rev-erb is ex- pressed early in the development of the nervous system [12]. In contrast, the chicken Rev-erb seems to be involved in late stages of neuronal development, as part of the complex network of induction signals controlling neuron differentiation [7]. Rev-erb acts as a negative transcriptional regulator and competes with another or- phan receptor, RORα [13]. A consensus binding site for Rev-erb and RORα has been identified in the first intron of a nuclear phosphoprotein implicated in the genesis and progression of naturally occurring tumors [14]. A possible transcription repressing domain has been identified in * Coordinates for the final structure have been deposited in the Brookhaven Protein Data Bank under the file name 1bn9. Chemical shifts of the 15-mer duplex have been deposited in the BioMagResBank under accession number 4172. ** Present address: Departamento de Bioquimica-CCB-UFSC- 88040–900 Florianoplis-SC, Brazil. *** Correspondence and reprints: murield@pasteur.fr Biochimie 82 (2000) 739-748 © 2000 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS. All rights reserved. S0300908400011482/FLA