RESUMO
The human nuclear receptor liver receptor homolog 1 (hLRH-1) plays an important role in the development of breast carcinomas. This orphan receptor is efficiently downregulated by the unusual co-repressor SHP and has been thought to be ligand-independent. We present the crystal structure at a resolution of 1.9 A of the ligand-binding domain of hLRH-1 in complex with the NR box 1 motif of human SHP, which we find contacts the AF-2 region of hLRH-1 using selective structural motifs. Electron density indicates phospholipid bound within the ligand-binding pocket, which we confirm using mass spectrometry of solvent-extracted samples. We further show that pocket mutations reduce phospholipid binding and receptor activity in vivo. Our results indicate that hLRH-1's control of gene expression is mediated by phospholipid binding, and establish hLRH-1 as a novel target for compounds designed to slow breast cancer development.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Fosfolipídeos/farmacologia , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Células HeLa , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Receptores Citoplasmáticos e Nucleares/genética , Alinhamento de Sequência , Espectrometria de Massas por Ionização por Electrospray , Fatores de TranscriçãoRESUMO
The DNA-binding and ligand-binding functions of nuclear receptors are localized to independent domains separated by a flexible hinge. The DNA-binding domain (DBD) of the human liver receptor homologue-1 (hLRH-1), which controls genes central to development and metabolic homeostasis, interacts with monomeric DNA response elements and contains an Ftz-F1 motif that is unique to the NR5A nuclear receptor subfamily. Here, we present the 2.2A resolution crystal structure of the hLRH-1 DBD in complex with duplex DNA, and elucidate the sequence-specific DNA contacts essential for the ability of LRH-1 to bind to DNA as a monomer. We show that the unique Ftz-F1 domain folds into a novel helix that packs against the DBD but does not contact DNA. Mutations expected to disrupt the positioning of the Ftz-F1 helix do not eliminate DNA binding but reduce the transcriptional activity of full-length LRH-1 significantly. Moreover, we find that altering the Ftz-F1 helix positioning eliminates the enhancement of LRH-1-mediated transcription by the coactivator GRIP1, an action that is associated primarily with the distantly located ligand-binding domain (LBD). Taken together, these results indicate that subtle structural changes in a nuclear receptor DBD can exert long-range functional effects on the LBD of a receptor, and significantly impact transcriptional regulation.