Backbone dynamics of the glucocorticoid receptor DNA-binding domain.

The extent of rapid (picosecond) backbone motions within the glucocorticoid receptor DNA-binding domain (GR DBD) has been investigated using proton-detected heteronuclear NMR spectroscopy on uniformly 15N-labeled protein fragments containing the GR DBD. Sequence-specific 15N resonance assignments, based on two- and three-dimensional heteronuclear NMR spectra, are reported for 65 of 69 backbone amides within the segment C440-A509 of the rat GR in a protein fragment containing a total of 82 residues (MW = 9200). Individual backbone 15N spin-lattice relaxation times (T1), rotating-frame spin-lattice relaxation times (T1 rho), and steady-state (1H)-15N nuclear Overhauser effects (NOEs) have been measured at 11.74 T for a majority of the backbone amide nitrogens within the segment C440-N506. T1 relaxation times and NOEs are interpreted in terms of a generalized order parameter (S2) and an effective correlation time (tau e) characterizing internal motions in each backbone amide using an optimized value for the correlation time for isotropic rotational motions of the protein (tau R = 6.3 ns). Average S2 order parameters are found to be similar (approximately 0.86 +/- 0.07) for various functional domains of the DBD. Qualitative inspection as well as quantitative analysis of the relaxation and NOE data suggests that the picosecond flexibility of the DBD backbone is limited and uniform over the entire protein, with the possible exception of residues S448-H451 of the first zinc domain and a few residues for which relaxation and NOE parameters were not obtained. in particular, we find no evidence for extensive rapid backbone motions within the second zinc domain. Our results therefore suggest that the second zinc domain is not disordered in the uncomplexed state of DBD, although the possibility of slowly exchanging (ordered) conformational states cannot be excluded in the present analysis.