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 and three-dimensional NMR data using an ensemble iterative relaxation matrix approach followed by direct NOE refinement with DINOSAUR. A set of 47 structures of the rat GR fragment Cys440-Arg510 was generated with distance geometry and further refined with a combination of restrained energy minimization and restrained molecular dynamics in a parallel refinement protocol. Distance constraints were obtained from an extensive set of NOE build-up curves in H2O and 2H2O via relaxation matrix calculations (1186 distance constraints from NOE intensities, 10 phi and 22 chi 1 dihedral angle constraints from J- coupling data were used for the calculations). The root-mean-square deviation values of the 11 best structures on the well-determined part of the protein (Cys440 to Ser448, His451 to Glu469 and Pro493 to Glu508) are 0.60 A and 1.20 A from the average for backbone and all heavy atoms, respectively. The final structures have R-factors around 0.40 and good stereochemical qualities. The first zinc-coordinating domain of the GR DBD is very similar to the crystal structure with a root-mean-square difference of 1.4 A. 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 studies on the estrogen receptor DBD-DNA and GR DBD-DNA complexes, part of this region will form a distorted helix and the D-box will undergo a conformational change upon cooperative binding to DNA.
van Tilborg
M A
MA
Bijvoet Center for Biomolecular Research Utrecht University, The Netherlands.
Bonvin
A M
AM
Hård
K
K
Davis
A L
AL
Maler
B
B
Boelens
R
R
Yamamoto
K R
KR
Kaptein
R
R
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008961:Models, Structural; D008969:Molecular Sequence Data; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000737:chemistry*; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D011965:Receptors, Glucocorticoid