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The structural basis of direct glucocorticoid-mediated transrepression.

Hudson WH, Youn C, Ortlund EA - Nat. Struct. Mol. Biol. (2012)

Bottom Line: The nGRE differs dramatically from activating response elements, and the mechanism driving GR binding and transrepression is unknown.To unravel the mechanism of nGRE-mediated transrepression by the GR, we characterized the interaction between GR and an nGRE in the thymic stromal lymphopoietin (TSLP) promoter.We show using structural and mechanistic approaches that nGRE binding is a new mode of sequence recognition by human GR and that nGREs prevent receptor dimerization through a unique GR-binding orientation and strong negative cooperativity, ensuring the presence of monomeric GR at repressive elements.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia, USA.

ABSTRACT
A newly discovered negative glucocorticoid response element (nGRE) mediates DNA-dependent transrepression by the glucocorticoid receptor (GR) across the genome and has a major role in immunosuppressive therapy. The nGRE differs dramatically from activating response elements, and the mechanism driving GR binding and transrepression is unknown. To unravel the mechanism of nGRE-mediated transrepression by the GR, we characterized the interaction between GR and an nGRE in the thymic stromal lymphopoietin (TSLP) promoter. We show using structural and mechanistic approaches that nGRE binding is a new mode of sequence recognition by human GR and that nGREs prevent receptor dimerization through a unique GR-binding orientation and strong negative cooperativity, ensuring the presence of monomeric GR at repressive elements.

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GR employs unique interactions to recognize the high-affinity site within nGREs(a) Close up view of the high-affinity GR–TSLP nGRE interaction with side chains and nucleotide depicted as sticks (O, red; N, blue). Hydrogen bonds and van der Waals interactions are represented by red and black dashed lines, respectively. Three base-specific contacts are present between GR and the high-affinity nGRE binding site. Val443 makes two hydrophobic contacts, and Lys442 donates a hydrogen bond to guanine 849. (b) Arg447 makes unique non-specific interactions with DNA at the high-affinity nGRE binding site. In contrast, Arg447 makes base-specific contacts with a guanine base when bound to a (+)GRE (orange; PDB FYL)12. c) 2Fo-Fc electron density (blue mesh) contoured at 1σ showing the conformation of the lever arm residues in TSLP nGRE-bound GR alone and (d) superimposed on (+)GRE-bound GR (orange).
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Figure 2: GR employs unique interactions to recognize the high-affinity site within nGREs(a) Close up view of the high-affinity GR–TSLP nGRE interaction with side chains and nucleotide depicted as sticks (O, red; N, blue). Hydrogen bonds and van der Waals interactions are represented by red and black dashed lines, respectively. Three base-specific contacts are present between GR and the high-affinity nGRE binding site. Val443 makes two hydrophobic contacts, and Lys442 donates a hydrogen bond to guanine 849. (b) Arg447 makes unique non-specific interactions with DNA at the high-affinity nGRE binding site. In contrast, Arg447 makes base-specific contacts with a guanine base when bound to a (+)GRE (orange; PDB FYL)12. c) 2Fo-Fc electron density (blue mesh) contoured at 1σ showing the conformation of the lever arm residues in TSLP nGRE-bound GR alone and (d) superimposed on (+)GRE-bound GR (orange).

Mentions: This suspected high-affinity GR DBD–nGRE DNA interaction involves three base specific contacts within the major groove (Fig. 2a): Val443 makes hydrophobic contacts with cytosine 846 and thymine 847 and Lys442 donates a hydrogen bond to N7 of guanine 849. Mutation of this guanine to adenine increases the Kd of GR for the high affinity site, confirming the identity of the high-affinity GR binding site (Table 1). In a previous study, the identical mutation ablates the repressive ability of the mouse TSLP nGRE10. Likewise, mutation of Lys442 significantly diminishes nGRE binding (Table 1). Unlike its DNA-reading function in (+)GRE structures, the Arg447 side chain is prevented from making base-specific contacts due to a steric clash with thymine 845 (Fig. 2b). The repositioned Arg447 instead make hydrophobic interactions with this base and ionic interactions with the cytosine 844 backbone phosphate. Mutation of thymine 845 to guanine, which would permit the “active” conformation of Arg447, abrogates transrepression10. The low-affinity GR DBD–DNA interaction involves only one sequence-specific contact: Arg447 contacts guanine 856, outside the nGRE consensus sequence (Supplementary Fig. 1a). Mutation of guanine 856 does not affect GR binding to the high-affinity site (Table 1), and Lys442 and Val443 do not sufficiently penetrate the major groove to facilitate sequence-specific DNA contacts (Supplementary Fig. 1b). As a result, the DNA major groove at the low-affinity site contains more waters than the high-affinity site. Recognition of the nGRE high-affinity site requires a more specific contacts and a greater hydrophobic interaction surface than either the low-affinity nGRE site or (+)GRE sequences, as confirmed by PISA26.


The structural basis of direct glucocorticoid-mediated transrepression.

Hudson WH, Youn C, Ortlund EA - Nat. Struct. Mol. Biol. (2012)

GR employs unique interactions to recognize the high-affinity site within nGREs(a) Close up view of the high-affinity GR–TSLP nGRE interaction with side chains and nucleotide depicted as sticks (O, red; N, blue). Hydrogen bonds and van der Waals interactions are represented by red and black dashed lines, respectively. Three base-specific contacts are present between GR and the high-affinity nGRE binding site. Val443 makes two hydrophobic contacts, and Lys442 donates a hydrogen bond to guanine 849. (b) Arg447 makes unique non-specific interactions with DNA at the high-affinity nGRE binding site. In contrast, Arg447 makes base-specific contacts with a guanine base when bound to a (+)GRE (orange; PDB FYL)12. c) 2Fo-Fc electron density (blue mesh) contoured at 1σ showing the conformation of the lever arm residues in TSLP nGRE-bound GR alone and (d) superimposed on (+)GRE-bound GR (orange).
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Related In: Results  -  Collection

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Figure 2: GR employs unique interactions to recognize the high-affinity site within nGREs(a) Close up view of the high-affinity GR–TSLP nGRE interaction with side chains and nucleotide depicted as sticks (O, red; N, blue). Hydrogen bonds and van der Waals interactions are represented by red and black dashed lines, respectively. Three base-specific contacts are present between GR and the high-affinity nGRE binding site. Val443 makes two hydrophobic contacts, and Lys442 donates a hydrogen bond to guanine 849. (b) Arg447 makes unique non-specific interactions with DNA at the high-affinity nGRE binding site. In contrast, Arg447 makes base-specific contacts with a guanine base when bound to a (+)GRE (orange; PDB FYL)12. c) 2Fo-Fc electron density (blue mesh) contoured at 1σ showing the conformation of the lever arm residues in TSLP nGRE-bound GR alone and (d) superimposed on (+)GRE-bound GR (orange).
Mentions: This suspected high-affinity GR DBD–nGRE DNA interaction involves three base specific contacts within the major groove (Fig. 2a): Val443 makes hydrophobic contacts with cytosine 846 and thymine 847 and Lys442 donates a hydrogen bond to N7 of guanine 849. Mutation of this guanine to adenine increases the Kd of GR for the high affinity site, confirming the identity of the high-affinity GR binding site (Table 1). In a previous study, the identical mutation ablates the repressive ability of the mouse TSLP nGRE10. Likewise, mutation of Lys442 significantly diminishes nGRE binding (Table 1). Unlike its DNA-reading function in (+)GRE structures, the Arg447 side chain is prevented from making base-specific contacts due to a steric clash with thymine 845 (Fig. 2b). The repositioned Arg447 instead make hydrophobic interactions with this base and ionic interactions with the cytosine 844 backbone phosphate. Mutation of thymine 845 to guanine, which would permit the “active” conformation of Arg447, abrogates transrepression10. The low-affinity GR DBD–DNA interaction involves only one sequence-specific contact: Arg447 contacts guanine 856, outside the nGRE consensus sequence (Supplementary Fig. 1a). Mutation of guanine 856 does not affect GR binding to the high-affinity site (Table 1), and Lys442 and Val443 do not sufficiently penetrate the major groove to facilitate sequence-specific DNA contacts (Supplementary Fig. 1b). As a result, the DNA major groove at the low-affinity site contains more waters than the high-affinity site. Recognition of the nGRE high-affinity site requires a more specific contacts and a greater hydrophobic interaction surface than either the low-affinity nGRE site or (+)GRE sequences, as confirmed by PISA26.

Bottom Line: The nGRE differs dramatically from activating response elements, and the mechanism driving GR binding and transrepression is unknown.To unravel the mechanism of nGRE-mediated transrepression by the GR, we characterized the interaction between GR and an nGRE in the thymic stromal lymphopoietin (TSLP) promoter.We show using structural and mechanistic approaches that nGRE binding is a new mode of sequence recognition by human GR and that nGREs prevent receptor dimerization through a unique GR-binding orientation and strong negative cooperativity, ensuring the presence of monomeric GR at repressive elements.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia, USA.

ABSTRACT
A newly discovered negative glucocorticoid response element (nGRE) mediates DNA-dependent transrepression by the glucocorticoid receptor (GR) across the genome and has a major role in immunosuppressive therapy. The nGRE differs dramatically from activating response elements, and the mechanism driving GR binding and transrepression is unknown. To unravel the mechanism of nGRE-mediated transrepression by the GR, we characterized the interaction between GR and an nGRE in the thymic stromal lymphopoietin (TSLP) promoter. We show using structural and mechanistic approaches that nGRE binding is a new mode of sequence recognition by human GR and that nGREs prevent receptor dimerization through a unique GR-binding orientation and strong negative cooperativity, ensuring the presence of monomeric GR at repressive elements.

Show MeSH