3MNE

Crystal structure of the agonist form of mouse glucocorticoid receptor stabilized by F608S mutation at 1.96A

Summary for 3MNE

• Entry DOI: 10.2210/pdb3mne/pdb
• Descriptor: Glucocorticoid receptor, Nuclear receptor coactivator 2 peptide, GLYCEROL, ... (5 entities in total)
• Functional Keywords: protein-ligand complex, steroid nuclear receptor, mouse, agonist, co-activator, hormone receptor
• Biological source: Mus musculus (mouse); More
• Cellular location: Cytoplasm: P06537; Nucleus: Q61026
• Total number of polymer chains: 2
• Total formula weight: 32266.58

Authors

Schoch, G.A.,Seitz, T.,Benz, J.,Banner, D.,Stihle, M.,D'Arcy, B.,Thoma, R.,Sterner, R.,Hennig, M.,Ruf, A. (deposition date: 2010-04-21, release date: 2010-09-15, Last modification date: 2023-09-06)

Primary citation

Seitz, T.,Thoma, R.,Schoch, G.A.,Stihle, M.,Benz, J.,D'Arcy, B.,Wiget, A.,Ruf, A.,Hennig, M.,Sterner, R.
Enhancing the stability and solubility of the glucocorticoid receptor ligand-binding domain by high-throughput library screening.
J.Mol.Biol., 403:562-577, 2010

PubMed Abstract: The human glucocorticoid receptor ligand-binding domain (hGR-LBD) is an important drug target for the treatment of various diseases. However, the low intrinsic stability and solubility of hGR-LBD have rendered its purification and biophysical characterization difficult. In order to overcome these problems, we have stabilized hGR-LBD by a combination of random mutagenesis and high-throughput screening using fluorescence-activated cell sorting (FACS) with enhanced green fluorescent protein (eGFP) as folding reporter. Two plasmid-encoded gene libraries of hGR-LBD fused to the egfp gene were expressed in Escherichia coli, followed by eight rounds of FACS screening, in each of which 10(8) cells were analyzed. The hgr-lbd mutants isolated by this approach contained numerous amino acid exchanges, and four beneficial ones (A605V, V702A, E705G, and M752T) were followed up in detail. Their characterization showed that the fluorescence of hGR-LBD-eGFP fusions is correlated linearly with the stability and solubility of hGR-LBD in the absence of eGFP. When combined, the four exchanges increased the thermal stability of hGR-LBD by more than 8 °C and enhanced its purification yield after expression in E. coli by about 26-fold. The introduction of three beneficial exchanges into the homologous ligand-binding domain of mouse enabled its X-ray structure determination at high resolution, which showed how the exchanges stabilize the protein and revealed atomic details that will guide future drug design. Our results demonstrate that large eGFP fusion libraries can be screened by FACS with extreme sensitivity and efficiency, yielding stabilized eukaryotic proteins suitable for biophysical characterization and structure determination.

PubMed: 20850457
DOI: 10.1016/j.jmb.2010.08.048

Experimental method

X-RAY DIFFRACTION (1.96 Å)