ABSTRACT
Co-repressor proteins mediate transcriptional repression by nuclear receptors in the absence of ligand. The identification of a co-repressor-receptor interaction motif, and the finding that co-repressors and co-activators compete for the same site on the receptor, suggests a simple mechanism for the switch from repression to activation upon ligand binding. Defects in this mechanism result in dominant-negative receptors that repress transcription. Such receptors have been implicated in several clinically important diseases, including thyroid hormone resistance and diabetes mellitus.
Subject(s)
Cell Nucleus/metabolism , Gene Expression Regulation , Transcription, Genetic , Amino Acid Sequence , Binding Sites , Diabetes Mellitus/metabolism , Humans , Leukemia, Erythroblastic, Acute/metabolism , Leukemia, Promyelocytic, Acute/metabolism , Ligands , Models, Biological , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Structure, Tertiary , Thyroid Hormone Resistance Syndrome/metabolismABSTRACT
The association of transcription corepressors SMRT and N-CoR with retinoid and thyroid receptors results in suppression of basal transcriptional activity. A key event in nuclear receptor signaling is the hormone-dependent release of corepressor and the recruitment of coactivator. Biochemical and structural studies have identified a universal motif in coactivator proteins that mediates association with receptor LBDs. We report here the identity of complementary acting signature motifs in SMRT and N-CoR that are sufficient for receptor binding and ligand-induced release. Interestingly, the motif contains a hydrophobic core (PhixxPhiPhi) similar to that found in NR coactivators. Surprisingly, mutations in the amino acids that directly participate in coactivator binding disrupt the corepressor association. These results indicate a direct mechanistic link between activation and repression via competition for a common or at least partially overlapping binding site.
Subject(s)
Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Receptors, Cytoplasmic and Nuclear/chemistry , Receptors, Cytoplasmic and Nuclear/metabolism , Repressor Proteins/chemistry , Repressor Proteins/metabolism , Saccharomyces cerevisiae Proteins , Amino Acid Sequence , Animals , Binding Sites , Cell Line , Cloning, Molecular , DNA Mutational Analysis , DNA-Binding Proteins , Fungal Proteins/metabolism , Molecular Sequence Data , Nuclear Receptor Co-Repressor 1 , Protein Structure, Secondary , Receptors, Retinoic Acid/chemistry , Receptors, Retinoic Acid/metabolism , Receptors, Thyroid Hormone/chemistry , Receptors, Thyroid Hormone/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Signal Transduction , Transcription Factors/metabolism , beta-Galactosidase/metabolismABSTRACT
Gelsolin is a calcium-regulated actin severing and capping protein that binds two calcium ions and has three sites for actin; two recognize monomeric actin and one attaches to the sides of filaments. It contains six repeating sequence segments (G1-6). Here, we have analyzed the effects of calcium ions on (i) limited proteolysis of bacterially expressed human gelsolin by plasmin and (ii) dynamic light scattering and circular dichroism of gelsolin and various of its subdomains. Following cleavage of gelsolin in the absence of calcium between Lys150 and His151 (the junction between G1 and G2), the molecule does not fall apart, nor does it bind actin without added calcium. This same molecule can be reconstituted by mixing an excess of G1 with G2-6 in EGTA. The noncovalently linked form of gelsolin shows three actin binding sites in calcium and requires 3 microM calcium for 50% activation of actin binding. Measurements of light scattering and circular dichroism revealed structural changes in response to calcium for intact gelsolin and a number of its actin-binding subdomains. Many of these changes occurred at calcium concentrations below 100 nM. These results are discussed in relation to the calcium control of gelsolin function and its three-dimensional structure (Burtnick et al.(1997) Cell 90, 661-670). Nanomolar concentrations of calcium initiate the unlatching of structural constraints that maintain the inaccessibility of the actin binding sites, but actin binding occurs only after additional micromolar calcium sites in both the N-terminal and C-terminal halves of the molecule are occupied.
Subject(s)
Calcium/pharmacology , Gelsolin/chemistry , Actins/metabolism , Binding Sites , Chromatography, Ion Exchange , Circular Dichroism , Egtazic Acid/pharmacology , Electrophoresis, Polyacrylamide Gel , Escherichia coli/genetics , Ethylmaleimide/metabolism , Fibrinolysin/metabolism , Fluorescence , Gelsolin/metabolism , Humans , Light , Molecular Weight , Peptide Fragments/chemistry , Peptide Fragments/isolation & purification , Peptide Fragments/metabolism , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Scattering, RadiationABSTRACT
The structure of the segment 1 domain of gelsolin, a protein that fragments actin filaments in cells, is reported in complex with actin. Segment 1 binds monomer using an apolar patch rimmed by hydrogen bonds in a cleft between actin domains. On the actin filament model it binds tangentially, disrupting only those contacts between adjacent subunits in one helical strand. The segment 1 fold is general for all segments of the gelsolin family because the conserved residues form the core of the structure. It also provides a basis for understanding the origin of an amyloidosis caused by a gelsolin variant.
