Unlike most chaperones, heat-shock protein 90 (Hsp90) interacts with a select group of ''client proteins'' that regulate essential biological processes. Little is known about how Hsp90 recognizes and binds these proteins. The glucocorticoid receptor (GR) is a well characterized Hsp90 client protein, whose hormone binding, nuclear-cytoplasmic trafficking, and transcriptional activity are regulated by Hsp90. Here, we provide evidence that unliganded and hormone-bound GR interact with two distinct, solventexposed hydrophobic sites in the Hsp90 C-terminal domain that contain the sequences ''MxxIM'' (HM10) and ''L/MxxIL'' (HM9). Our results indicate that binding of Hsp90 HM10 to unliganded GR stabilizes the unliganded ligand-binding pocket of GR indirectly by promoting an intramolecular interaction between the C-terminal ␣-helix (H12) and a solvent-exposed hydrophobic groove in the GR ligand binding domain. In the presence of hormone, Hsp90 appears to bind the hydrophobic groove of GR directly by mimicking the interactions of GR with transcriptional coactivators. The identified interactions provide insights into the mechanisms that enable Hsp90 to regulate the activity of both unliganded and hormonebound GR and to sharpen the cellular response to hormone.binding sites ͉ heat-shock protein 90 ͉ steroid hormone receptors

Specific activation of serotonin (5-HT) 5-HT2C G protein-coupled receptors may be therapeutic for obesity and neuropsychiatric disorders. Mutagenesis coupled with computational and molecular modeling experiments based on the human β2 adrenergic receptor structure were employed to delineate the interactions of different ligands at human 5-HT2C residues D3.32, S3.36 and Y7.43. No binding of the tertiary amine radioligand ([3H]-mesulergine) could be detected when the 5-HT2C D3.32 residue was mutated to alanine (D3.32A). The S3.36A point-mutation greatly reduced affinity of primary amine ligands, modestly reduced affinity of a secondary amine, and except for the 5-HT2C-specific agonist N(CH3)2-PAT, affinity of tertiary amines was unaffected. Molecular modeling results indicated that the primary amines form hydrogen bonds with the S3.36 residue, whereas, with the exception of N(CH3)2-PAT, tertiary amines do not interact considerably with this residue. The Y7.43A point-mutation greatly reduced affinity of 5-HT, yet reduced to a lesser extent the affinity of tryptamine that lacks the 5-hydroxy moiety present in 5-HT; modeling results indicated that the 5-HT 5-hydroxy moiety hydrogen bonds with Y7.43 at the 5-HT2C receptor. Additional modeling results showed that 5-HT induced a hydrogen bond between Y7.43 and D3.32. Finally, modeling results revealed two low-energy binding modes for 5-HT in the 5-HT2C binding pocket, supporting the concept that multiple agonist binding modes may stabilize different receptor active conformations to influence signaling. Ligand potencies for modulating WT and point-mutated 5-HT2C receptor-mediated phospholipase C activity were in accordance with the affinity data. Ligand efficacies, however, were altered considerably by the S3.36A mutation only.

Steroid hormone receptors (SRs) are transcription factors that act as regulatory switches by altering gene expression in response to ligands. The highly conserved ligand-binding domain of SRs is a precise but versatile molecular switch that can adopt distinct conformations. Differential stabilization of these conformations by ligands, DNA response elements and transcriptional coregulators controls the activity of SRs in a gene-specific and cell-specific manner. In the case of the glucocorticoid receptor (GR), high-affinity ligand binding requires the interaction of the LBD with the heat shock protein 90 (Hsp90). Here, we show that the dependence of the ligand binding ability of GR on Hsp90 can be modified by the replacement of single amino acids within an allosteric network that connects the buried ligand-binding pocket and a solvent-exposed coregulator interaction surface. Each of the identified mutations altered the equilibrium between alternative GR conformations distinctively, indicating that the Hsp90 dependence of SRs may correlate with differences in the conformational dynamics of these receptors. Our results suggest that Hsp90 stabilizes the GR ligand-binding pocket indirectly by utilizing the allosteric network, while allowing the receptor to remain structurally uncommitted. Thus, in addition to ensuring the accessibility of the GR ligand-binding pocket to ligands, Hsp90 seems to enable hormones and coregulators to act as allosteric effectors, which forms the basis for gene-specific and cell-specific responses of GR to ligands.