Impaired helix 12 dynamics due to proline 892 substitutions in the androgen receptor are associated with complete androgen insensitivity.
Structural studies of the ligand-binding domain (LBD) of several steroid receptors have revealed that the dynamic properties of the C-terminal helix 12 (H12) are the major determinant of the activation mode of these receptors. H12 exhibits high mobility and different conformations in the absence of ligand. Upon ligand binding, H12 is stabilized in a precise position to seal the ligand-binding pocket and finalize the assembly of the activation function (AF-2) domain. In this study, we investigated the role of the conserved proline 892 of the androgen receptor (AR) in directing the dynamic location and orientation of the AR-H12. We used a combined approach including kinetic and biochemical assays with molecular dynamic simulations to analyze two substitutions (P892A and P892L) identified in individuals with complete androgen insensitivity syndrome. Our analyses revealed distinct mechanisms by which these substitutions impair H12 function resulting in severely defective receptors. The AR-P892A receptor exhibited reduced ligand binding and transactivational potential because of an increased flexibility in H12. The AR-P892L substitution renders the receptor inactive due to a distorted, unstructured and misplaced H12. To confirm the mutants' inability to stabilize H12 in an active position, we have developed a novel in vivo assay to evaluate the accessibility of the H12-docking site on the AR-LBD surface. An extrinsic AR-H12 peptide was able to interact with wild-type and mutant LBDs in the absence of ligand. Ligand-induced proper positioning of the intrinsic H12 of wild-type AR prevented these interactions, whereas the misplacement of the mutants' H12 did not. Proline at this position may be critical for H12 dynamics not only in the AR, but also in other nuclear receptors where this proline is conserved.
Elhaji
Youssef A
YA
Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital, McGill University, National Research Council of Canada, Montreal, Que., Canada. youssef.elhaji@mail.mcgill.ca
Stoica
Ileana
I
Dennis
Sheldon
S
Purisima
Enrico O
EO
Lumbroso
Rose
R
Beitel
Lenore K
LK
Trifiro
Mark A
MA
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D013734:Androgen-Insensitivity Syndrome / Q000175:diagnosis / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D019556:COS Cells; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005260:Female; D005347:Fibroblasts / Q000378:metabolism / Q000473:pathology; D006801:Humans; D008024:Ligands; D008297:Male; D008969:Molecular Sequence Data; D009154:Mutation; D011392:Proline / Q000235:genetics*; D011485:Protein Binding / Q000235:genetics; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D011944:Receptors, Androgen / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D013816:Thermodynamics
D004268:DNA-Binding Proteins; D008024:Ligands; D011944:Receptors, Androgen; D011392:Proline