Identification of a Novel Co-regulator Interaction Surface on the Ligand Binding Domain of Nurr1 Using NMR Footprinting

Anna Codina,Gerard Benoit,John T Gooch,David Neuhaus,Thomas Perlmann,John W.R Schwabe

doi:10.1074/jbc.m409096200

Abstract

The nuclear receptor Nurr1 is a transcription factor essential for the development of midbrain dopaminergic neurons in vertebrates. Recent crystal structures of the Nurr1 ligand binding domain (LBD) and the Drosophila orthologue dHR38 revealed that, although these receptors share the classical LBD architecture, they lack a ligand binding cavity. This volume is instead filled with bulky hydrophobic side chains. Furthermore the "canonical" non-polar co-regulator binding groove is filled with polar side chains; thus, the regulation of transcription by this sub-family of nuclear receptor LBDs may be mediated by some other interaction surface on the LBD. We report here the identification of a novel co-regulator interface on the LBD of Nurr1. We used an NMR footprinting strategy that facilitates the identification of an interaction surface without the need of a full assignment. We found that non-polar peptides derived from the co-repressors SMRT and NCoR bind to a hydrophobic patch on the LBD of Nurr1. This binding surface involves a groove between helices 11 and 12. Mutations in this site abolish activation by the Nurr1 LBD. These findings give insight into the unique mechanism of action of this class of nuclear receptors.

Highlights

The nuclear receptor Nurr1 is a transcription factor essential for the development of midbrain dopaminergic neurons in vertebrates
Peptide Interaction with the Nurr1 ligand binding domain (LBD)—To identify potential interaction surfaces on the Nurr1 LBD, we first sought to identify, using a pull-down assay, short peptides that are able to interact with the Nurr1 LBD
Candidate peptides of a diverse character were derived from the nuclear receptor corepressors SMRT and NCoR and the N-terminal AF1 domain of Nurr1 (Fig. 1), which have previously been reported to interact with the LBD of this class of receptors [21, 23]

Summary

Introduction

The nuclear receptor Nurr is a transcription factor essential for the development of midbrain dopaminergic neurons in vertebrates. In particular, focused on Nurr because it plays an important role in the development of dopaminergic neurons in the midbrain and may represent a therapeutic target to treat Parkinson’s disease [20] We know that this group of receptors lacks a ligand binding cavity, because crystal structures of the ligand binding domain from Nurr1 [17] and its orthologue in Drosophila, dHR38 [18], revealed that the ligand binding cavity is almost entirely filled with bulky hydrophobic side chains. These structures revealed that the canonical co-regulator binding groove observed in other receptors is filled with polar side chains and is not likely to support conventional non-polar interactions with coregulators This would appear to suggest that the LBD of Nurr and related receptors might, at first sight, be inert and play no direct role in the regulation of transcription. The simplest answer to this is that there is another, as yet unidentified co-regulator interaction surface

Methods

Results

Conclusion