Structure, Energetics, and Dynamics of Binding Coactivator Peptide to the Human Retinoid X Receptor α Ligand Binding Domain Complex with 9-cis-Retinoic Acid

Abstract

Retinoid X receptors (RXRs) are ligand-dependent nuclear receptors, which are activated by the potent agonist 9-cis retinoic acid (9cRA). 9cRA binds to the ligand binding domain (LBD) of RXRs, and recruits coactivator proteins for gene transcription. Using isothermal titration calorimetry, the binding of a 13-mer coactivator peptide, GRIP-1, to the hRXRα-LBD homodimer complex containing 9cRA (hRXRα-LBD:9cRA:GRIP-1) is reported between 20° and 37 °C. ΔG is temperature independent (−8.5 kcal/mol), and GRIP-1 binding is driven by ΔH (−9.2 kcal/mol) at 25 °C. ΔC p is large and negative (−401 cal/mol-K). The crystal structure of hRXRα-LBD: 9cRA:GRIP-1 is reported at 2.05 Å. When the structures of hRXRα-LBD:9cRA:GRIP-1 and hRXRα-LBD:9cRA (1FBY) homodimers are compared, E453 and E456 on helix 12 bury and form ionic interactions with GRIP-1. R302 on helix 4 realigns to form new salt bridges to both E453 and E456. F277 (helix 3), F437 (helix 11), and F450 (helix 12) move toward the hydrophobic interior. The changes in the near-UV spectrum at 260 nm of the hRXRα-LBD: 9cRA:GRIP-1 support this structural change. Helix 11 tilts toward helix 12 by ≈ 1 Å modifying the ring conformation of 9cRA. Hydrogen-deuterium exchange mass spectroscopy indicate GRIP-1 binding to hRXRα-LBD:9cRA significantly decreases the exchange rates for peptides containing helices 3 (F277), 4 (R302), 11 (F437) and 12 (E453; E456). The structural changes and loss of dynamics of the GRIP-1 bound structure are used to interpret the energetics of coactivator peptide binding to the agonist-bound hRXRα-LBD. -205-934-8285. Fax: 1-205-934-2543. muccio@uab.edu. # These authors contributed equally to this work.The atomic coordinates and structure factors (code 3OAP) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/). Supporting Information AvailableThe ITC measurements of GRIP-1 coactivator peptide binding to hRXRα-LBD:9cRA at 20, 25, 30, and 37 °C are presented in Figure S1, and the ITC measurement of -1 coactivator peptide binding to hRXRα-LBD:9cRA at 25 °C in HEPES Buffer is presented in Figure S2. The interaction between E456 of RXR and H688 of GRIP-1 observed in our crystal structure is presented in Figure S3. The deuterium incorporation measurements of all peptide fragments are provided in Figure S4. The contact measurements between hRXRα-LBD and GRIP-1 coactivator peptide or 9-cis-retinoic acid are provided in Table S1 and Table S2, respectively. The optimized extinction coefficients used in our near-UV-Vis difference simulation in Figure 4 are presented in Table S3. Table S4 provides the complete list of 38 HDX MS peptides and their observed differences in the various complexes. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2012 June 8. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptNuclear ...

Defining the Communication between Agonist and Coactivator Binding in the Retinoid X Receptor α Ligand Binding Domain

“…X-ray quality crystals of the hRXR␣-LBD⅐Targretin⅐GRIP-1 complex were obtained at 5-15% PEG 4000, 6 -12% glycerol, 0.1 M BisTris at pH 7.0. Both crystals had a P4 (3) 2 (1) 2 space group, which was the same as the crystals of the hRXR␣-LBD complex with 9cRA and GRIP-1 reported in our previous paper (12).…”

“…Crystallization of hRXR␣-LBD⅐9cUAB30⅐GRIP-1 and hRXR␣-LBD⅐9cRA⅐GRIP-1 homodimer complexes was similar to the method described previously (12) and those reported by Moras and co-workers (30). The crystals of hRXR␣-LBD homodimers with 9cUAB30 were obtained in a reservoir solution containing 5-10% PEG 4000, 2-5% glycerol, 0.1 M BisTris at pH 7.0.…”

“…These studies revealed the structural and dynamical rearrangements of H12 and other residues associated with coactivator binding to the holo-RXR␣-LBD (12). In this structure, we observed four tertiary structural changes that connected the retinoid in the LBP to H12 residues.…”

“…The structures and purity of the rexinoids and peptide were confirmed by NMR and LC-MS. The concentration of peptide was determined by 1 H NMR added to a known concentration of tryptophan as reported previously (12).…”

Structural basis for a molecular allosteric control mechanism of cofactor binding to nuclear receptors

“…In the LXRβ structure, different conformations of the ligand were also observed. A recent study of RXR complex to 9-cis retinoic acid and SRC-1 NR2 revealed a difference in the conformation of the 9-cis RA in the coactivator bound RXR (32).…”