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Structure of the retinoid X receptor α–liver X receptor β (RXRα–LXRβ) heterodimer on DNA

Abstract

Nuclear receptors (NRs) are conditional transcription factors with common multidomain organization that bind diverse DNA elements. How DNA sequences influence NR conformation is poorly understood. Here we report the crystal structure of the human retinoid X receptor α–liver X receptor β (RXRα–LXRβ) heterodimer on its cognate element, an AGGTCA direct repeat spaced by 4 nt. The complex has an extended X-shaped arrangement, with DNA- and ligand-binding domains crossed, in contrast to the parallel domain arrangement of other NRs that bind an AGGTCA direct repeat spaced by 1 nt. The LXRβ core binds DNA via canonical contacts and auxiliary DNA contacts that enhance affinity for the response element. Comparisons of RXRα–LXRβs in the crystal asymmetric unit and with previous NR structures reveal flexibility in NR organization and suggest a role for RXRα in adaptation of heterodimeric complexes to DNA.

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Figure 1: Organization of the RXRα–LXRβ heterodimer.
Figure 2: Flexibility in the LXRβ–RXRα complex.
Figure 3: Differences between RXRα–LXRβ DBD positions in the two heterodimers.
Figure 4: RXRα–LXRβ DNA contacts.
Figure 5: Reorganization of RXRα in LXRβ and PPARγ heterodimers.

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Acknowledgements

This work was supported by the Welch Foundation chair E-0004 (J.-A.G.), Emerging Technology Fund of Texas 300-9-1958 (J.-A.G.), Swedish Science Council (J.-A.G.), US National Institutes of Health DK41482 (P.W.) and an European Molecular Biology Organization long-term fellowship (X.L.). We thank J.D. Baxter for the inspiration to pursue this project.

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Authors and Affiliations

Authors

Contributions

X.L. expressed, purified and crystallized the complex, collected diffraction data and solved the structure. G.T. took part in expression and purification of the proteins. C.B. and J.H.S. took part in functional analysis. K.J.P. assisted in supervising the project. P.W. and J.-A.G. wrote the manuscript and supervised the project.

Corresponding author

Correspondence to Jan-Ake Gustafsson.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Schematic diagram of expression constructs.

Domain organization and amino acid coordinates of full length LXRβ and RXRα with truncated versions of the proteins used to generate crystals. At the side is an image of an SDS-PAGE gel used to verify integrity and purity of RXRα/LXRβ proteins in the complex.

Supplementary Figure 2 Electron density maps for part structures.

(a) Close up of H1 interactions that bridge the two heterodimers in the asymmetric unit of the crystal. The two heterodimer pairs interact through nearly symmetric interactions between LXRβ LBD H1. Stereo view of 2Fo-Fc map for LXRβ H1 from the two heterodimers with key contact residues marked. (b-c) Localization of ligands in RXRα and LXRβ LBDs. Fo-Fc electron density maps (in orange) for ligands reveal (b), 9-cis Retinoic Acid in RXRα and (c), GW3965 in LXRβ.

Supplementary Figure 3 Spacing between RXRα and LXRβ domains.

Ribbon diagram representations of the RXRα-LXRβ complex with a, the overall shape of LXRβ highlighted in blue and structural elements marked and b, RXRα shape highlighted in red. Note that there is clear separation between LBDs and DBDs in both receptors.

Supplementary Figure 4 Influences of the LXRβ CTE helix upon DNA-element selection.

(a) Overlapping CTE positions in LXRβ and TR DBDs on DR-4 elements. Comparison of RXRα-LXRβ and RXRα-TRβ heterodimers created by superposing two helices of the helix-turn-helix motifs of LXRβ (blue) and TRβ (cyan) DBDs. The RXRα DBD (red) occupies a similar position and the CTE helices from the two proteins partly overlap at the N-terminus and diverge over the length of the extended TRβ CTE helix. (b-d), Steric Clashes in RXRα-LXRβ DBD heterodimer on DR elements with shortened spacing. Representation of models of the RXRα-LXRβ DBD heterodimer on DR elements with shortened spacers. RXRα DBD is in red; LXRβ DBD is in marine. RXRα/LXRβ DBDs on (b), DR-3, with the clashing regions are shown by blue arrows. (c), DR-2. (d), DR-1.

Supplementary Figure 5 RXRα-LXRβ interactions in paired heterodimers.

(a) Close contact between RXRα LBD and LXRβ DBD in the heterodimer represented in Figure 1 of the main text. The figure shows stereo view with RXRα in red and the LXRβ in blue with key contact residues marked. Residues from the loop preceding RXRα LBD H1 and the N-terminus of H1 contact the LXR DBD; side chains of Asn227 and Glu228 point to a small dimple upon the LXRβ DBD formed by residues 115-122, 143 and 146-147 and 156. Additionally, RXRα His288 and Glu291 side chains, located in H3 and the H3-H4 loop, contact LXRβ DBD Gln143 and side chains of Glu233 and Arg234 in RXRα H1 also lie close to LXR DBD surface. (b) Contacts appear different and weaker in the second heterodimer. Stereo view of contacts from the other heterodimer reveal interactions are different from those seen in (a) with few close contacts. (c) Canonical LXRβ DBD/DNA contacts. The figure is a stereo view of the bases on AGGTCA half-site recognized by side chains of the LXRβ helix that merges in the major groove. Contacts resemble those of other receptors with DNA.

Supplementary Figure 6 LXRβ LBD–DNA crystal-packing contacts.

(a, b) Two views of a stick model of one of the RXRα-LXRβ heterodimer on its cognate DNA element and positions of nearby DNA elements in the crystal packing arrangement reveals close contacts between the LBD and DNA. The ligands are represented by sticks, LXRβ H12 helix is represented in cyan and cofactor peptide in red. The two LXRβ LBD DNA contact surfaces are marked “1” and “2”. (c) Stereo close up view of the contact surface of LXRβ LBD with DNA “1”. (d) Stereo close up view of the contact surface of LXRβ LBD with DNA “2”. (e) Gel shift assay to check LXRβ LBD binding on DNA. Note that the LXRβ LBD exhibits binding to the DR-4 element that is unaffected by heterodimer formation and the RXRα LBD shows no evidence for DNA binding.

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Lou, X., Toresson, G., Benod, C. et al. Structure of the retinoid X receptor α–liver X receptor β (RXRα–LXRβ) heterodimer on DNA. Nat Struct Mol Biol 21, 277–281 (2014). https://doi.org/10.1038/nsmb.2778

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