STRUCTURE REFINEMENT OF THE GLUCOCORTICOID RECEPTOR-DNA BINDING DOMAIN FROM NMR DATA BY RELAXATION MATRIX CALCULATIONS

The solution structure of the glucocorticoid receptor (GR) DNA-binding domain (DBD), consisting of 93 residues, has been refined from two an d three-dimensional NMR data using an ensemble iterative relaxation ma trix approach followed by direct NOE refinement with DINOSAUR. A set o f 47 structures of the rat GR fragment Cys440-Arg510 was generated wit h distance geometry and further refined with a combination of restrain ed energy minimization and restrained molecular dynamics in a parallel refinement protocol. Distance constraints were obtained from an exten sive set of NOE build-up curves in H2O and (H2O)-H-2 via relaxation ma trix calculations (1186 distance constraints from NOE intensities, 10 phi and 22 chi(1) dihedral angle constraints from J- coupling data wer e used for the calculations). The root-mean-square deviation values of the 11 best structures on the well-determined part of the protein (Cy s440 to Ser448, His451 to Glu469 and Pro493 to Glu508) are 0.60 Angstr om and 1.20 Angstrom from the average for backbone and all heavy atoms , respectively. The final structures have X-factors around 0.40 and go od stereochemical qualities. The first zinc-coordinating domain of the GR DBD is very similar to the crystal structure with a root-mean-squa re difference of 1.4 Angstrom. The second zinc-coordinating domain is still disordered in solution. No secondary structure element is found in this domain in the free state. As suggested by crystallographic stu dies on the estrogen receptor DBD-DNA and GR DBD-DNA complexes, part o f this region will form a distorted helix and the D-box will undergo a conformational change upon cooperative binding to DNA.