ABSTRACT
Several crystal structures of human estrogen receptor alpha ligand-binding domain (hERalpha LBD) complexed with agonist or antagonist molecules have previously been solved. The proteins had been modified in cysteine residues (carboxymethylation) or renatured in urea to circumvent aggregation and denaturation problems. In this work, high-level protein expression and purification together with crystallization screening procedure yielded high amounts of soluble protein without renaturation or modifications steps. The native protein crystallizes in the space group P3(2) 21 with three molecules in the asymmetric unit. The overall structure is very similar to that previously reported for the hERalpha LBD with cysteine carboxymethylated residues thus validating the modification approach. The present strategy can be adapted to other cases where the solubility and the proper folding is a difficulty.
Subject(s)
Peptide Fragments/chemistry , Peptide Fragments/isolation & purification , Receptors, Estrogen/chemistry , Receptors, Estrogen/isolation & purification , Cloning, Molecular , Computer Simulation , Crystallization , Crystallography, X-Ray , Dimerization , Estrogen Receptor alpha , Humans , Ligands , Models, Molecular , Peptide Fragments/genetics , Peptide Fragments/metabolism , Protein Folding , Protein Structure, Secondary/genetics , Protein Structure, Tertiary/genetics , Receptors, Estrogen/genetics , Receptors, Estrogen/metabolism , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolismABSTRACT
The crystal structure of a triple cysteine to serine mutant ERalpha ligand-binding domain (LBD), complexed with estradiol, shows that despite the presence of a tightly bound agonist ligand, the protein exhibits an antagonist-like conformation, similar to that observed in raloxifen and 4-hydroxytamoxifen-bound structures. This mutated receptor binds estradiol with wild type affinity and displays transcriptional activity upon estradiol stimulation, but with limited potency (about 50%). This partial activity is efficiently repressed in antagonist competition assays. The comparison with available LBD structures reveals key features governing the positioning of helix H12 and highlights the importance of cysteine residues in promoting an active conformation. Furthermore the present study reveals a hydrogen bond network connecting ligand binding to protein trans conformation. These observations support a dynamic view of H12 positioning, where the control of the equilibrium between two stable locations determines the partial agonist character of a given ligand.
Subject(s)
Receptors, Estrogen/chemistry , Receptors, Estrogen/metabolism , Cloning, Molecular , Crystallography, X-Ray , Estrogen Antagonists/pharmacology , Estrogen Receptor alpha , Humans , Hydrogen Bonding , Ligands , Models, Molecular , Mutagenesis, Site-Directed , Protein Conformation , Receptors, Estrogen/agonists , Receptors, Estrogen/geneticsABSTRACT
Choline acetyltransferase (ChAT) is the key enzyme responsible for the synthesis of the neurotransmitter acetylcholine and is reduced in various central neurodegenerative diseases. From a previously selected 12.6 kb human choline acetyltransferase (hChAT) genomic clone, we have identified and characterized a promoter region of 895 bp. Sequence analysis revealed the presence of a TATA-like box, a CAAT box and several putative regulatory responsive elements. Three transcription initiation sites were determined by primer extension analysis. The Northern blot of poly(A)+ RNA, showed a single band of 2300 Nt in the human nucleus accumbens and facial nucleus. By using transient transfections into NE-1-115 and COS-1 cells of the 5' flanking region of the hChAT gene we identified a sequence of 66 bp upstream of the transcription start site which confers responsiveness to proto-oncogenes c-Fos/c-Jun. These data suggest that the hChAT gene may be a physiological target of c-Fos/c-Jun and therefore may play a role in neuronal responses to various stimuli.
