STRUCTURE AND DYNAMICS OF THE GLUCOCORTICOID RECEPTOR DNA-BINDING DOMAIN - COMPARISON OF WILD-TYPE AND A MUTANT WITH ALTERED SPECIFICITY

Nuclear magnetic resonance was used to compare parameters reflecting s olution structure and dynamics of the glucocorticoid receptor DNA-bind ing domain (GRDBD), which binds specifically to a GRE binding site on DNA, and a triple mutant (GRDBD(EGA)), which binds to an ERE sits. The studies were prompted by an earlier observation that the cooperativit y for dimeric DNA-binding is 10 times higher for the GRDBD(EGA)-ERE as sociation than for the GRDBD-GRE association (Lundback et al., 1994). The higher binding cooperativity of the mutant was unexpected since th e triple mutation (G458E, S459G, and V462A) is made in the recognition helix and distant from the dimerization surface which is formed by re sidues in the fragment A477-N491. Sequential and long-range NOE connec tivities and measured (3)J(HNH alpha) coupling constants indicate that the overall structures of the two proteins are very similar, possibly with a less well-defined structure of the fragment K486-N491 in GRDBD (EGA). However, chemical shift changes, line broadening, and increased amide proton exchange rates are observed for several residues at. or close to, the dimerization surface of the mutant. These observations a re interpreted as a lower stability and/or several slowly interconvert ing folded conformations of this region of GRDBD(EGA) The effects are likely to be due to the loss of a hydrogen bond which links S459 to th e dimerization region in GRDBD. Different mechanisms for the increased binding cooperativity of the mutant are discussed, and it is noted th at the properties of the GRDBD(EGA) dimerization region are reminiscen t of those reported for the estrogen receptor DBD, which also binds to an ERE site.