DNA-induced conformational changes are the basis for cooperative dimerization by the DNA binding domain of the retinoid X receptor.

Dimerization of the DNA-binding domains of nuclear hormone receptors occurs in a manner that is highly cooperative with DNA binding. We have investigated the molecular basis for this cooperativity through an NMR study of the interaction between the monomeric DNA-binding domain (DBD) of the retinoid-X-receptor (RXR) and a single DNA half-site. Major changes were observed in the chemical shifts of the backbone resonances and in the pattern of medium-range nuclear Overhauser enhancement connectivities of the RXR upon binding to DNA, indicating that the DNA induces conformational changes in the monomer. Binding to DNA induces and stabilizes the structure in a region of the second zinc binding domain that forms the dimerization interface when RXR binds as a dimer to a direct repeat recognition element. These studies provide direct experimental evidence that DNA-induced protein conformational changes constitute the molecular basis for cooperative enhancement of dimer formation and DNA binding by the nuclear hormone receptor DBDs. In contrast to the localized folding induced in the dimerization interface, DNA binding leads to unfolding of the C-terminal helix found in the free RXR DBD. Unwinding of this helix may facilitate homodimer formation by maximizing interactions between the two DNA-bound RXR domains.

High-resolution solution structure of the retinoid X receptor DNA-binding domain.

The retinoid X receptor (RXR) is a member of the nuclear hormone receptor superfamily of transcriptional regulators and plays a central role in the retinoid and, through its ability to heterodimerize with other nuclear hormone receptors, non-steroid signaling pathways. The DNA-binding and recognition functions of RXR are located in a conserved 83 amino acid residue domain that recognizes the consensus sequence AGGTCA. In order to provide a detailed picture of its structure, we have calculated a high-resolution solution structure of the C195A RXRalpha DNA-binding domain. Structures were calculated using 1131 distance and dihedral angle constraints derived from 1H, 13C and 15N NMR spectra. The structures reveal a perpendicularly packed, "loop-helix" fold similar to other nuclear hormone receptor DNA-binding domains and confirm the existence of the C-terminal helix, which was first observed in the low-resolution NMR structure. The C-terminal helix is well formed and is stabilized by packing interactions with residues in the hydrophobic core. The solution structure of RXR is very similar to that determined by X-ray crystallographic studies of the RXR-TR heterodimer complex with DNA, except that in the latter case no electron density was observed for residues corresponding to the C-terminal helix. Other differences between the X-ray and NMR structures occur in the second zinc-binding loop, which is disordered in solution. Heteronuclear 15N NOE measurements suggest that this loop has enhanced flexibility in the free protein.

The solution conformation of hyaluronan: a combined NMR and molecular dynamics study.

Hyaluronan (HA) is a negatively charged glycosaminoglycan that exhibits a wide variety of biological effects mediated by binding to cell-surface and extracellular matrix proteins (hyaladherins). Short HA oligosaccharides have been shown to retain the specific interactions and biological effects of high molecular weight HA. Although it has a simple disaccharide repeating unit, the aqueous solution conformation of HA has been very difficult to determine because of strong coupling and overlapping resonances. In this study, we propose aqueous solution conformations for an octasaccharide of HA, derived from proton-proton NOE data and restrained molecular dynamics. To overcome spectral overlap and strong coupling, alternate methods for extracting distance restraints were employed. Restrained molecular dynamics calculations yielded one set of interglycosidic angle values for the beta (1,3) linkage (phi 13 = 46 degrees, psi 13 = 24 degrees). In contrast, two sets of values for the beta (1,4) linkage were consistent with the NOE restraints (phi 14 = 24 degrees, psi 14 = -53 degrees or phi 14 = 48 degrees, psi 14 = 8 degrees). The potential difference in flexibility for the two linkages is consistent with unrestrained as well as the restrained molecular dynamics trajectories described here. The conformational parameters obtained from restrained molecular dynamics are used to predict helical parameters of high molecular weight HA and will provide a basis for studies of HA binding to proteins.