Biochemical and NMR Mapping of the Interface between CREB-binding Protein and Ligand Binding Domains of Nuclear Receptor: BEYOND THE LXXLL MOTIF

Fabrice A.C Klein,R Andrew Atkinson,Noelle Potier,Dino Moras,Jean Cavarelli

doi:10.1074/jbc.m411697200

Abstract

Biochemical and NMR Mapping of the Interface between CREB-binding Protein and Ligand Binding Domains of Nuclear Receptor: BEYOND THE LXXLL MOTIF

Highlights

The best characterized Nuclear receptors (NRs) coactivators belong to the p160 family and interact with ligand binding domains (LBDs) as well as with A/B domains of NRs
We show that distinct zones of that fragment are involved in the interactions: a 20-residue segment containing the LXXLL motif is implicated in the interaction with all three domains tested, whereas a second N-terminal well conserved block of around 25 residues centered on a consensus L40PDEL44 motif constitutes a secondary motif of interaction with peroxisome proliferator-activated receptor ␥-LBD
Cloning and Expression—cDNA corresponding to amino acids 228 – 467 of murine RXR␣-LBD was cloned in pET3a and pET28b bacterial expression vectors (Novagen), allowing production of native RXR␣LBD and a His6-RXR␣-LBD fusion protein. cDNA corresponding to amino acids 204 – 478 of human peroxisome proliferator-activated receptors (PPARs)␥-LBD was cloned in pET28b, for expression of a His6-PPAR␥-LBD fusion protein

Summary

BEYOND THE LXXLL MOTIF*

CBP plays an important role as a general cointegrator of various signaling pathways and interacts with a large number of transcription factors and cofactors, through numerous protein binding domains spread along its primary sequence (Fig. 1A) [12]. Both CBP and p160s possess a histone acetyltransferase domain [13, 14]. Functional studies performed in vitro as well as in vivo have highlighted the essential role of NR boxes in the interaction of coactivators with NR LBDs (18, 26 –32; for a recent review, see Ref. 33) Both functional and structural studies suggest a general mechanism for the assembly of NRs and zinc binding domain; TOCSY, total correlation spectroscopy; TOF, time-of-flight. The interaction with human ERR␥-LBD in the absence and presence of the antagonist, 4-hydroxytamoxifen, was investigated

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION