ABSTRACT
AMPA receptors mediate fast excitatory synaptic transmission and are essential for synaptic plasticity. ANQX, a photoreactive AMPA receptor antagonist, is an important biological probe used to irreversibly inactivate AMPA receptors. Here, using X-ray crystallography and mass spectroscopy, we report that ANQX forms two major products in the presence of the GluR2 AMPAR ligand-binding core (S1S2J). Upon photostimulation, ANQX reacts intramolecularly to form FQX or intermolecularly to form a covalent adduct with Glu705.
Subject(s)
Excitatory Amino Acid Antagonists/pharmacology , Nitro Compounds/pharmacology , Quinolines/pharmacology , Receptors, AMPA/antagonists & inhibitors , Binding Sites , Crystallography, X-Ray , Excitatory Amino Acid Antagonists/chemistry , Mass Spectrometry , Models, Molecular , Molecular Structure , Nitro Compounds/chemistry , Quinolines/chemistry , Receptors, AMPA/chemistryABSTRACT
Nuclear receptors (NRs) are ligand-regulated transcription factors important in human physiology and disease. In certain NRs, including the androgen receptor (AR), ligand binding to the carboxy-terminal domain (LBD) regulates transcriptional activation functions in the LBD and amino-terminal domain (NTD). The basis for NTD-LBD communication is unknown but may involve NTD-LBD interactions either within a single receptor or between different members of an AR dimer. Here, measurement of FRET between fluorophores attached to the NTD and LBD of the AR established that agonist binding initiated an intramolecular NTD-LBD interaction in the nucleus and cytoplasm. This intramolecular folding was followed by AR self-association, which occurred preferentially in the nucleus. Rapid, ligand-induced intramolecular folding and delayed association also were observed for estrogen receptor-alpha but not for peroxisome proliferator activated receptor-gamma2. An antagonist ligand, hydroxyflutamide, blocked the NTD-LBD association within AR. NTD-LBD association also closely correlated with the transcriptional activation by heterologous ligands of AR mutants isolated from hormone-refractory prostate tumors. Intramolecular folding, but not AR-AR affinity, was disrupted by mutation of an alpha-helical ((23)FQNLF(27)) motif in the AR NTD previously described to interact with the AR LBD in vitro. This work establishes an intramolecular NTD-LBD conformational change as an initial component of ligand-regulated NR function.
Subject(s)
Estrogen Receptor alpha/metabolism , PPAR gamma/metabolism , Protein Folding , Receptors, Androgen/metabolism , Transcriptional Activation/physiology , Amino Acid Motifs/genetics , Blotting, Western , Fluorescence Resonance Energy Transfer , Flutamide/analogs & derivatives , Flutamide/metabolism , Genetic Vectors , HeLa Cells , Humans , Ligands , Luciferases , Luminescent Proteins , Mammary Tumor Virus, Mouse , Mutation/genetics , Protein Structure, Tertiary/physiology , Receptors, Androgen/geneticsABSTRACT
Ligands occupy the core of nuclear receptor (NR) ligand binding domains (LBDs) and modulate NR function. X-ray structures of NR LBDs reveal most NR agonists fill the enclosed pocket and promote packing of C-terminal helix 12 (H12), whereas the pockets of unliganded NR LBDs differ. Here, we review evidence that NR pockets rearrange to accommodate different agonists. Some thyroid hormone receptor (TR) ligands with 5' extensions designed to perturb H12 act as antagonists, but many are agonists. One mode of adaptation is seen in a TR/thyroxine complex; the pocket expands to accommodate a 5' iodine extension. Crystals of other NR LBDs reveal that the pocket can expand or contract and some agonists do not fill the pocket. A TRbeta structure in complex with an isoform selective drug (GC-24) reveals another mode of adaptation; the LBD hydrophobic interior opens to accommodate a bulky 3' benzyl extension. We suggest that placement of extensions on NR agonists will highlight unexpected areas of flexibility within LBDs that could accommodate extensions; thereby enhancing the selectivity of agonist binding to particular NRs. Finally, agonists that induce similar LBD structures differ in their activities and we discuss strategies to reveal subtle structural differences responsible for these effects.
Subject(s)
Receptors, Cytoplasmic and Nuclear/agonists , Receptors, Cytoplasmic and Nuclear/chemistry , Acetates/chemistry , Acetates/metabolism , Amino Acid Sequence , Benzhydryl Compounds/chemistry , Benzhydryl Compounds/metabolism , Binding Sites/genetics , Conserved Sequence , Crystallography, X-Ray , Drug Design , Humans , In Vitro Techniques , Ligands , Models, Molecular , Molecular Sequence Data , Protein Conformation , Protein Structure, Tertiary , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Thyroid Hormone/agonists , Receptors, Thyroid Hormone/chemistry , Receptors, Thyroid Hormone/genetics , Receptors, Thyroid Hormone/metabolism , Sequence Homology, Amino Acid , Thyroxine/chemistry , Thyroxine/metabolismABSTRACT
Selective therapeutics for nuclear receptors would revolutionize treatment for endocrine disease. Specific control of nuclear receptor activity is challenging because the internal cavities that bind hormones can be virtually identical. Only one highly selective hormone analog is known for the thyroid receptor, GC-24, an agonist for human thyroid hormone receptor beta. The compound differs from natural hormone in benzyl, substituting for an iodine atom in the 3' position. The benzyl is too large to fit into the enclosed pocket of the receptor. The crystal structure of human thyroid hormone receptor beta at 2.8-A resolution with GC-24 bound explains its agonist activity and unique isoform specificity. The benzyl of GC-24 is accommodated through shifts of 3-4 A in two helices. These helices are required for binding hormone and positioning the critical helix 12 at the C terminus. Despite these changes, the complex associates with coactivator as tightly as human thyroid hormone receptor bound to thyroid hormone and is fully active. Our data suggest that increased specificity of ligand recognition derives from creating a new hydrophobic cluster with ligand and protein components.
