Journal of Molecular Biology
Regular articleStructural basis of RXR-DNA interactions1
Introduction
The nuclear receptors form a superfamily of transcription factors that use a conserved DNA-binding domain (DBD) to recognize highly related DNA target sequences Mangelsdorf and Evans 1995, Mangelsdorf et al 1995. The 9-cis retinoic acid receptor RXR is a central player in hormonal signaling because of its ability to form heterodimers with numerous other receptors Bugge et al 1992, Gearing et al 1993, Kliewer et al 1992a, Kliewer et al 1992b, Marks et al 1992, Yu et al 1991. The partners for RXR include the all-trans retinoic acid receptor (RAR), the vitamin D receptor (VDR), the thyroid hormone receptor (TR) and the peroxisome proliferator activated receptor (PPAR). In addition, RXR regulates gene expression in response to 9-cis retinoic acid through a homodimeric complex Heery et al 1994, Heyman et al 1992, Levin et al 1992, Zechel et al 1994. The homo- and heterodimeric complexes of RXR target specific DNA sequences known as hormone response elements. The correct receptor dimerization on the DNA is required to recruit the necessary coactivator or corepressor proteins to the transcription complex Glass et al 1997, Zamir et al 1997.
Most non-steroid nuclear receptors recognize the consensus sequence 5′-AGGTCA-3′ (see Figure 1(a)). RXR can bind as a homodimer with high affinity and specificity to response elements composed of tandem or direct repeats of the consensus hexamer, but not as a monomer to a single hexameric site Leblanc and Stunnenberg 1995, Umesono et al 1991, Lee et al 1993. The identity element in the direct DNA response elements that fosters highly cooperative specific binding is the size of the intervening spacer that separates the two half-sites (Umesono et al., 1991). Figure 1(a) illustrates the “one to five rule” describing how the homo- and heterodimeric forms of RXR recognize direct repeats (DR) with inter-half-site spacings ranging from one to five base-pairs, referred to as DR1 to DR5, respectively (Umesono et al., 1991).
Here, we describe the high-resolution crystal structure of the RXR-DNA-binding domain assembled as a homodimer on its direct repeat DNA target. The complex reveals the details of the protein-protein and protein-DNA contacts in this central member of the nuclear receptor superfamily. The inter-half-site spacing of the direct repeat provides the geometry needed for two subunits to interact effectively at their interface. A comparison with a previous NMR structure of the unbound RXR supports the idea that a switch in protein conformation occurs upon DNA binding Holmbeck et al 1998a, Holmbeck et al 1998b. The switch in RXR conformation facilitates dimerization and leads to a cooperative enhancement in DNA-affinity.
Section snippets
Structure determination and crystallographic arrangement
Figure 1(b) and (c) show the protein and DNA constructs used in co-crystallization. The RXR-DNA-binding region used in this study contains a carboxyl-terminal extension, known as the T-box, which was shown to be necessary for homo-cooperative assembly Lee et al 1993, Wilson et al 1992. The DR1 sequence used is a 15 base-pair sequence containing a tandem repeat of the consensus 5′-AGGTCA-3′ half-sites used by RXR, TR, VDR, RAR and other non-steroid nuclear receptors. The crystal structure was
Discussion
The ability of a DNA response element to act as an allosteric effector has been suggested in the nuclear receptor superfamily, where alternative DNA-binding sites are likely to lead to different receptor conformations and signaling pathways Lefstin and Yamamoto 1998, Forman et al 1995, Kurokawa et al 1994, Kurokawa et al 1995, La Vista-Picard et al 1996. The comparison of the bound RXR-DBD with the free protein provides compelling evidence that a key conformational change in the T-box DNA
Crystallization and data collection
The RXR-DNA-binding region and the DNA oligonucleotides were purified as described (Zhao et al., 1998). Initial crystals (type I) belonged to an orthorhombic space group (P212121) with unit cell dimensions a = 35.7 Å, b = 74.5 Å, c = 102.1 Å. These crystals were grown using a longer RXR construct (130-223) and the DNA shown in Figure 1(c). The crystals grew at 8 °C in hanging drops using 2 μl of macromolecules in 25 mM Tris buffer (pH 7.5) and 2 μl of the reservoir solution; 18-23 %
Acknowledgements
F.R. dedicates this paper to the memory of Professor Paul B. Sigler who provided inspiration and insight throughout this study. We are grateful to Drs Ronald Evans and Steve Kliewer for initiating this work, and Dr Michael L. Johnson for assistance with equilibrium binding data analysis. We thank the staff at the EMBL X31 beamline at the DORIS storage ring, DESY, Hamburg, Germany, and Dr Craig Ogata at Brookhaven National Laboratories beamline X-4A. S.A.C. was supported by an NIH training grant
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