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 ...

Background: Some retinoid X receptor (RXR) agonists have potential as cancer drugs. Results: Structures of RXR in complex with two different agonists show similar folds. Dynamics analysis reveals unique ligand-induced dynamics in helices 3, 11, and 12. Conclusion: Two networks of interactions that connect RXR agonists to coactivator binding are defined. Significance: Recognition of common conformational changes and distinguishing dynamics of RXR-selective agonists is necessary for advances in drug design.

The thermal denaturation of bacteriorhodopsin in the purple membrane of Halobacterium halobium has been studied by differential scanning calorimetry (DSC) and temperature-dependent spectroscopy in the pH range from 5 to 11. Monitoring of protein fluorescence and absorbance in the near-UV and visible regions indicates that changes primarily occur in tertiary structure with denaturation. Far-UV circular dichroism shows only small changes in the secondary structure, unlike most globular water-soluble proteins of comparable molecular weight. The DSC transition can best be described as a two-state denaturation of the trimer. Thermodynamic analysis of the calorimetric transition reveals some similarity between the unfolding of bacteriorhodopsin and water-soluble proteins. Specifically, a pH dependence of the midpoint temperature of denaturation is seen as well as a temperature-dependent enthalpy of denaturation. Proteolysis experiments on denatured purple membrane suggest that bacteriorhodopsin may be partially extruded from the membrane as it denatures. Exposure of buried hydrophobic residues to the aqueous environment upon denaturation is consistent with the observed temperature-dependent enthalpy.

A number of retinoid X receptor (RXR) agonists have proven to be highly effective in preventing methylnitrosourea (MNU) induced mammary cancers. However, these agonists have side effects; particularly causing an increase in serum triglyceride levels. A series of ligands for RXR were designed based on computer modeling to the ligand binding domain (LBD) of the RXR receptors and on structure-activity relationships. The chemopreventive effects of these retinoids were evaluated in the relatively long-term MNU model. As a short-term assay to predict their efficacy, the ability of the retinoids to modulate cell proliferation and apoptosis was also determined in mammary cancers after only 7 days of treatment. The five UAB retinoids evaluated included two Class I UAB retinoids (UAB20, UAB112) and three Class II UAB retinoids (UAB30, 4-methyl-UAB30 and the benzosuberone-analog of UAB30). The previously evaluated RXR agonist targretin and the pan-agonist 9-cis-retinoic acid (9-cis-RA), which interacts with both RAR and RXR receptors, were included as positive agonists known to prevent cancer in the MNU model. In the prevention studies, in which the agents were administered beginning 5 days after MNU until the end of the study, targretin (150 mg/kg diet) and 4-methyl-UAB30 (200 mg/kg diet) were highly effective in decreasing cancer numbers by 75-85%. UAB30 (200 mg/kg diet) and 9-cis-RA (60 mg/kg diet) gave intermediate inhibitions of 60 and 45%, respectively. Targretin (15 mg/kg diet), UAB20 (200 mg/kg diet) and the benzosuberone analog of UAB30 (200 mg/kg diet) showed limited activity by decreasing cancer multiplicity 25-30%, while UAB112 had no effect on mammary cancer multiplicity. A direct correlation was observed between the long-term chemopreventive efficacy of these agents and their ability to decrease cell proliferation in mammary cancers after short-term treatment. Furthermore, the highly effective agents (4-methyl-UAB30 and targretin at 150 mg/kg diet) increased apoptosis 3-5 times, while agents with moderate or limited preventive efficacy failed to significantly increase apoptosis. Although the more effective retinoid treatments increased serum triglycerides 2.5- to 4.0-fold, one moderately effective agent (UAB30) had no significant effect on lipid levels. In summary, a short-term in vivo method has been identified for screening newly synthesized retinoids both for chemopreventive efficacy and for their adverse effect on serum triglycerides.

(2E,4E,6Z,8Z)-8-(3′,4′-Dihydro-1′(2H)-naphthalen-1′-ylidene)-3,7-dimethyl-2,3,6-octatrienoinic acid (UAB30) is currently undergoing clinical evaluation as a novel cancer prevention agent. In efforts to develop even more highly potent rexinoids that prevent breast cancer without toxicity, we further explore here the structure-activity relationship of two separate classes of rexinoids. UAB30 belongs to the class II rexinoids and possesses a 9Z-tetraenoic acid chain bonded to a tetralone ring, whereas the class I rexinoids contain the same 9Z-tetraenoic acid chain bonded to a di-substituted cyclohexenyl ring. Among the twelve Class I and Class II rexinoids evaluated, the Class I rexinoid 11 is most effective in preventing breast cancers in an in vivo rat model alone or in combination with tamoxifen. Rexinoid 11 also reduces the size of established tumors and exhibits a therapeutic effect. However, 11 induces hypertriglyceridemia at its effective dose. On the other hand rexinoid 10 does not increase triglyceride levels while being effective in the in vivo chemoprevention assay. X-ray studies of four rexinoids bound to the ligand binding domain of the retinoid X receptor reveal key structural aspects that enhance potency as well as those that enhance the synthesis of lipids.