Changes in dynamical behavior of the retinoid X receptor DNA-binding domain upon binding to a 14 base-pair DNA half site.

The retinoid X receptor (RXR) is a prominent member of the nuclear receptor family of ligand-inducible transcription factors. Many proteins of this family exert their function as heterodimers with RXR as a common upstream partner. Studies of the DNA-binding domains of several nuclear receptors reveal differences in structure and dynamics, both between the different proteins and between the free- and DNA-bound receptor DBDs. We investigated the differences in dynamics between RXR free in solution and in complex with a 14 base-pair oligonucleotide, using (1)H and (15)N relaxation studies. Nano- to picosecond dynamics were probed on (15)N, employing Lipari-Szabo analysis with an axially symmetric tumbling model to estimate the exchange contributions to the transverse relaxation rates. Furthermore, milli- to microsecond dynamics were estimated qualitatively for (1)H and (15)N, using CPMG-HSQC and CPMG-T(2) measurements with differential pulse spacing. RXR shows hardly any nano- to picosecond time-scale internal motion. Upon DNA binding, the order parameters show a tiny increase. Dynamics in the milli- to microsecond time scale is more prevalent. It is localized in the first and second zinc fingers of the free RXR. Upon DNA-binding, exchange associated with specific/aspecific DNA-binding of RXR is observed throughout the sequence, whereas conformational flexibility of the D-box and the second zinc finger of RXR is greatly reduced. Since this DNA-binding induced folding transition occurs remote from the DNA in a region which is involved in protein-protein interactions, it may very well be related to the cooperativity of dimeric DNA binding.

Microsecond time scale dynamics in the RXR DNA-binding domain from a combination of spin-echo and off-resonance rotating frame relaxation measurements.

Slow protein dynamics can be studied by 15N spin-echo (CPMG) and off-resonance rotating frame relaxation through the effective field dependence of the exchange-mediated relaxation contribution. It is shown that, by a combination of these complementary techniques, a more extended sampling of the microsecond time scale processes is achieved than by either method alone. 15N R2 and improved off-resonance R1 rho experiments [Mulder et al. (1998) J. Magn. Reson., 131, 351-357] were applied to the 9-cis-retinoic acid receptor DNA-binding domain and allowed the identification of 14 residues exhibiting microsecond time scale dynamics. Assuming exchange between two conformational substates, average lifetimes ranging from 37 to 416 microseconds, and chemical shift differences of up to 3 ppm were obtained. The largest perturbation of tertiary structure was observed for the second zinc finger region, which was found to be disordered in the solution structure [Holmbeck et al. (1998) J. Mol. Biol., 281, 271-284]. Since this zinc-coordinating domain comprises the principal dimerization interface for RXR in a wide repertoire of complexes with different hormone receptors to their cognate response elements, this finding has important implications for our understanding of nuclear receptor assembly on DNA direct repeats. The flexibility observed for the dimerization domain may explain how RXR, through the ability to adaptively interact with a wide variety of highly homologous partner molecules, demonstrates such a versatile DNA-binding repertoire.

Millisecond to microsecond time scale dynamics of the retinoid X and retinoic acid receptor DNA-binding domains and dimeric complex formation.

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.

Identification of the ribosome binding sites of translation initiation factor IF3 by multidimensional heteronuclear NMR spectroscopy.

Titrations of Escherichia coli translation initiation factor IF3, isotopically labeled with 15N, with 30S ribosomal subunits were followed by NMR by recording two-dimensional (15N,1H)-HSQC spectra. In the titrations, intensity changes are observed for cross peaks belonging to amides of individual amino acids. At low concentrations of ribosomal subunits, only resonances belonging to amino acids of the C-domain of IF3 are affected, whereas all those attributed to the N-domain are still visible. Upon addition of a larger amount of 30S subunits cross peaks belonging to residues of the N-terminal domain of the protein are also selectively affected. Our results demonstrate that the two domains of IF3 are functionally independent, each interacting with a different affinity with the ribosomal subunits, thus allowing the identification of the individual residues of the two domains involved in this interaction. Overall, the C-domain interacts with the 30S subunits primarily through some of its loops and alpha-helices and the residues involved in ribosome binding are distributed rather symmetrically over a fairly large surface of the domain, while the N-domain interacts mainly via a small number of residues distributed asymmetrically in this domain. The spatial organization of the active sites of IF3, emerging through the comparison of the present data with the previous chemical modification and mutagenesis data, is discussed in light of the ribosomal localization of IF3 and of the mechanism of action of this factor.

The structure of the translational initiation factor IF1 from E.coli contains an oligomer-binding motif.

The structure of the translational initiation factor IF1 from Escherichia coli has been determined with multidimensional NMR spectroscopy. Using 1041 distance and 78 dihedral constraints, 40 distance geometry structures were calculated, which were refined by restrained molecular dynamics. From this set, 19 structures were selected, having low constraint energy and few constraint violations. The ensemble of 19 structures displays a root-mean-square deviation versus the average of 0.49 A for the backbone atoms and 1.12 A for all atoms for residues 6-36 and 46-67. The structure of IF1 is characterized by a five-stranded beta-barrel. The loop connecting strands three and four contains a short 3(10) helix but this region shows considerably higher flexibility than the beta-barrel. The fold of IF1 is very similar to that found in the bacterial cold shock proteins CspA and CspB, the N-terminal domain of aspartyl-tRNA synthetase and the staphylococcal nuclease, and can be identified as the oligomer-binding motif. Several proteins of this family are nucleic acid-binding proteins. This suggests that IF1 plays its role in the initiation of protein synthesis by nucleic acid interactions. Specific changes of NMR signals of IF1 upon titration with 30S ribosomal subunit identifies several residues that are involved in the interaction with ribosomes.

Solution structure of the Oct-1 POU homeodomain determined by NMR and restrained molecular dynamics.

The POU homeodomain (POUhd), a divergent member of the well-studied class of homeodomain proteins, is the C-terminal part of the bipartite POU domain, the conserved DNA-binding domain of the POU proteins. In this paper we present the solution structure of POUhd of the human Oct-1 transcription factor. This fragment was overexpressed in Escherichia coli and studied by two- and three-dimensional homo- and heteronuclear NMR techniques, resulting in virtually complete 1H and 15N resonance assignments for residues 2-60. Using distance and dihedral constraints derived from the NMR data, 50 distance geometry structures were calculated, which were refined by means of restrained molecular dynamics. From this set a total of 31 refined structures were selected, having low constraint energy and few constraint violations. The ensemble of 31 structures displays a root-mean-square deviation of the coordinates of 0.59 A with respect to the average structure, calculated over the backbone atoms of residues 6 to 54. The fold of POUhd is very similar to that of the canonical homeodomains. Interestingly, the recognition helix of the free POUhd ends at residue 53, while in the cocrystal structure of the intact POU domain with the DNA octamer motif [Klemm, J.D., Rould, M.A., Aurora, R., Herr, W. and Pabo, C.O. (1994) Cell, 77, 21-32] this helix in the POUhd subdomain is extended as far as residue 60.