Functional probing of the human glucocorticoid receptor steroid-interacting surface by site-directed mutagenesis. Gln-642 plays an important role in steroid recognition and binding.
To elucidate which amino acids in the glucocorticoid receptor ligand-binding domain might be involved in determining steroid binding specificity by interaction with the D-ring of glucocorticoids, we have performed site-directed mutagenesis of the four amino acids Met-560, Met-639, Gln-642, and Thr-739 based on their proximity to the steroid in a model structure. Mutations of these residues affected steroid binding affinity, specificity, and/or steroid-dependent transactivation. The results indicate that these residues are located in close proximity to the ligand and appear to play a role in steroid recognition and/or transactivating sensitivity, possibly by changes in the steroid-dependent conformational change of this region, resulting in the formation of the AF-2 site. Mutation of Gln-642 resulted in a marked decrease in affinity for steroids containing a 17alpha-OH group. This effect was alleviated by the presence of a 16alpha-CH(3) group to a varying degree. Thr-739 appears to form a hydrogen bond with the 21-OH group of the steroid, as well as possibly forming hydrophobic interactions with the steroid. Met-560 and Met-639 appear to form hydrophobic interactions with the D-ring of the steroid, although the nature of these interactions cannot be characterized in more detail at this point.
Lind
U
U
Department of Medical Nutrition, Karolinska Institutet, Huddinge Hospital, Novum, S-141 86 Huddinge, Sweden.
Greenidge
P
P
Gillner
M
M
Koehler
K F
KF
Wright
A
A
Carlstedt-Duke
J
J
D000818:Animals; D019556:COS Cells; D005998:Glycine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D015335:Molecular Probes; D016297:Mutagenesis, Site-Directed; D011965:Receptors, Glucocorticoid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013256:Steroids / Q000378:metabolism*
D015335:Molecular Probes; D011965:Receptors, Glucocorticoid; D013256:Steroids; D005998:Glycine