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The Role of Electrostatic Interactions in Binding of Histone H3K4me2/3 to the Sgf29 Tandem Tudor Domain.

Pieters BJ, Meulenbroeks E, Belle R, Mecinović J - PLoS ONE (2015)

Bottom Line: Several reader domain proteins that specifically recognize methyllysine-containing histones contain the negatively-charged aspartate or glutamate residues as part of the aromatic cage.Small uncharged and large aromatic substitutions on the Asp266 site resulted in a significant decrease in binding affinities for both H3K4me3 and H3K4me2, demonstrating the role of the negative charge of Asp266 in the readout process by Sgf29.This study emphasizes the essential contribution of electrostatic interactions to the overall binding affinity, and reveals that the underlying mechanisms for the recognition of Kme2/3 depend on the composition and arrangement of the aromatic cage.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecules and Materials, Radboud University Nijmegen, Nijmegen, The Netherlands.

ABSTRACT
Several reader domain proteins that specifically recognize methyllysine-containing histones contain the negatively-charged aspartate or glutamate residues as part of the aromatic cage. Herein, we report thermodynamic analyses for the recognition of histone H3K4me3 and H3K4me2 by the tandem tudor domain of Sgf29 and its recognition site variants. Small uncharged and large aromatic substitutions on the Asp266 site resulted in a significant decrease in binding affinities for both H3K4me3 and H3K4me2, demonstrating the role of the negative charge of Asp266 in the readout process by Sgf29. This study emphasizes the essential contribution of electrostatic interactions to the overall binding affinity, and reveals that the underlying mechanisms for the recognition of Kme2/3 depend on the composition and arrangement of the aromatic cage.

No MeSH data available.


Tm curves of A) wild-type Sgf29 and its D266 variants and B) Sgf29 and its Y238 and Y245 variants.[a]
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pone.0139205.g003: Tm curves of A) wild-type Sgf29 and its D266 variants and B) Sgf29 and its Y238 and Y245 variants.[a]

Mentions: Additionally, differential scanning fluorimetry (DSF) was employed as a secondary method to determine the structural stability of the Sgf29 variants (Fig 3, Table 1). Using DSF, WT Sgf29 was shown to have a Tm of 57.8°C. Variant proteins have similar or decreased Tm values when compared to WT Sgf29, ranging from 53.9–57.7°C. The introduction of the negatively-charged Glu at the D266 site gave indistinguishable Tm values relative to WT Sgf29. In addition, the introduction of aromatic residues only had a small influence on Tm, as substitution of D266 with Trp, Phe or Tyr only decreased Tm by 2.3, 3.3 or 3.9°C, respectively. Collectively, all Tm values can be considered to be within close proximity to the Tm for WT Sgf29. These data, combined with CD spectra indicate that D266A, F, Y and W variants have altered tertiary structures, which do not substantially affect the stability of the variant proteins. Because the D266 residue is located in an unstructured region on the surface of the reader domain, alterations made to this residue do not appear to affect the more structurally and thermodynamically stable β-sheet rich regions. These structured regions make up the major part of the Sgf29 reader domain, explaining the subtle changes in Tm values for the D266X variants. Similarly, we observed similar Tm values for all Y238F variants that display altered and unchanged CD spectra relative to WT Sgf29, suggesting that different conformations of the D266 containing loop region do not substantially affect the stability of this variant. Y245F variant displays similar Tm value as the related Y238F.


The Role of Electrostatic Interactions in Binding of Histone H3K4me2/3 to the Sgf29 Tandem Tudor Domain.

Pieters BJ, Meulenbroeks E, Belle R, Mecinović J - PLoS ONE (2015)

Tm curves of A) wild-type Sgf29 and its D266 variants and B) Sgf29 and its Y238 and Y245 variants.[a]
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4589357&req=5

pone.0139205.g003: Tm curves of A) wild-type Sgf29 and its D266 variants and B) Sgf29 and its Y238 and Y245 variants.[a]
Mentions: Additionally, differential scanning fluorimetry (DSF) was employed as a secondary method to determine the structural stability of the Sgf29 variants (Fig 3, Table 1). Using DSF, WT Sgf29 was shown to have a Tm of 57.8°C. Variant proteins have similar or decreased Tm values when compared to WT Sgf29, ranging from 53.9–57.7°C. The introduction of the negatively-charged Glu at the D266 site gave indistinguishable Tm values relative to WT Sgf29. In addition, the introduction of aromatic residues only had a small influence on Tm, as substitution of D266 with Trp, Phe or Tyr only decreased Tm by 2.3, 3.3 or 3.9°C, respectively. Collectively, all Tm values can be considered to be within close proximity to the Tm for WT Sgf29. These data, combined with CD spectra indicate that D266A, F, Y and W variants have altered tertiary structures, which do not substantially affect the stability of the variant proteins. Because the D266 residue is located in an unstructured region on the surface of the reader domain, alterations made to this residue do not appear to affect the more structurally and thermodynamically stable β-sheet rich regions. These structured regions make up the major part of the Sgf29 reader domain, explaining the subtle changes in Tm values for the D266X variants. Similarly, we observed similar Tm values for all Y238F variants that display altered and unchanged CD spectra relative to WT Sgf29, suggesting that different conformations of the D266 containing loop region do not substantially affect the stability of this variant. Y245F variant displays similar Tm value as the related Y238F.

Bottom Line: Several reader domain proteins that specifically recognize methyllysine-containing histones contain the negatively-charged aspartate or glutamate residues as part of the aromatic cage.Small uncharged and large aromatic substitutions on the Asp266 site resulted in a significant decrease in binding affinities for both H3K4me3 and H3K4me2, demonstrating the role of the negative charge of Asp266 in the readout process by Sgf29.This study emphasizes the essential contribution of electrostatic interactions to the overall binding affinity, and reveals that the underlying mechanisms for the recognition of Kme2/3 depend on the composition and arrangement of the aromatic cage.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecules and Materials, Radboud University Nijmegen, Nijmegen, The Netherlands.

ABSTRACT
Several reader domain proteins that specifically recognize methyllysine-containing histones contain the negatively-charged aspartate or glutamate residues as part of the aromatic cage. Herein, we report thermodynamic analyses for the recognition of histone H3K4me3 and H3K4me2 by the tandem tudor domain of Sgf29 and its recognition site variants. Small uncharged and large aromatic substitutions on the Asp266 site resulted in a significant decrease in binding affinities for both H3K4me3 and H3K4me2, demonstrating the role of the negative charge of Asp266 in the readout process by Sgf29. This study emphasizes the essential contribution of electrostatic interactions to the overall binding affinity, and reveals that the underlying mechanisms for the recognition of Kme2/3 depend on the composition and arrangement of the aromatic cage.

No MeSH data available.