Millisecond to Microsecond Time Scale Dynamics of the Retinoid X and Retinoic Acid Receptor DNA-Binding Domains and Dimeric Complex Formation

Abstract

The all-trans retinoic acid and 9-cis retinoic acid receptors (RAR and RXR, respectively) belong to a family of ligand inducible transcription factors, which exert their effect via binding to hormone response elements. Both are members of the class II sub-family of nuclear receptors, which bind DNA as dimers, on tandem repeats of a hexamer motif separated by a variable spacer. The variability in spacer length and the head-to-tail organization of the hormone response elements result in different protein-protein interactions in each of the complexes. We show that the zinc-coordinating loop regions of RXR and RAR DNA-binding domains exhibit dynamics on the millisecond to microsecond time scale. The highly dynamic second zinc finger of RXR constitutes the primary protein-protein interface in many nuclear receptor assemblies on DNA. Dynamics is also observed in the first and second zinc fingers of RAR, which are implicated in dimeric interactions with RXR on response elements with spacers of 5 base pairs and 1 base pair, respectively. The striking correspondence between the regions that exhibit conformational exchange and the dimer interfaces of the proteins complexed with DNA suggests a functional role for the dynamics. The observed flexibility may allow the proteins to adapt to various partners and with different orientations upon assembly on DNA. Furthermore, the more extensive dynamics observed for RXR may reflect the greater ability of this protein to modulate its interaction surface since it participates in a wide variety of receptor complexes.

Insight into the Role of Dynamics in the Conformational Switch of the Small GTP-binding Protein Arf1

“…S1B). This indicates that GTP␥S does not induce notable conformational changes, in agreement with 31 P NMR spectroscopy comparison of Ras bound to GTP and GTP analogs, which identified GTP␥S as the analog that is most similar to physiological GTP (33).…”

“…Peak intensities were converted to relaxation rates, and uncertainties in relaxation rates were calculated from repeated experiments as described in Ref. 31. The dispersion curves at the two fields were fitted simultaneously to a global two-state fast exchange (Meiboom equation) using house written MATLAB procedures.…”

“…Surprisingly, the bound 15 N-labeled GDP was however replaced by unlabeled GTP␥S in the presence of ARNO, as shown by the disappearance of the 15 N doublet and the appearance of a 14 N singlet. Contamination of the GTP␥S sample was ruled out by 31 P spectroscopy. The fact that the additional phosphate of GTP␥S is accommodated in the GDP conformation of Arf1 without yielding protein CSVs in the nucleotide binding site suggests that the additional group is expelled from the nucleotide binding site.…”

Synthetic Retinoids and Their Nuclear Receptors

“…The RARβ DBD functional domain contains an N-terminal β-sheet, then two zinc-finger regions, which bind to the retinoid βRARE. The zinc fingers are separated by a short α-helix and a loop and are followed by another α-helical region [191]. These studies indicate that the flexibility of the second zinc finger and the proximate helical region (T-box) have a role in the ability of RXRα to function as a heterodimer with many different NRs and bind to a variety of REs [191].…”

“…High-resolution nuclear magnetic resonance (NMR) spectrometry has been used to determine the structures of the RARβ [190,191] and RXRα [191][192][193] DBDs. The RARβ DBD functional domain contains an N-terminal β-sheet, then two zinc-finger regions, which bind to the retinoid βRARE.…”