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The N-terminal acetylation of Sir3 stabilizes its binding to the nucleosome core particle.

Arnaudo N, Fernández IS, McLaughlin SH, Peak-Chew SY, Rhodes D, Martino F - Nat. Struct. Mol. Biol. (2013)

Bottom Line: The N-terminal acetylation of Sir3 is essential for heterochromatin establishment and maintenance in yeast, but its mechanism of action is unknown.The crystal structure of the N-terminally acetylated BAH domain of Saccharomyces cerevisiae Sir3 bound to the nucleosome core particle reveals that the N-terminal acetylation stabilizes the interaction of Sir3 with the nucleosome.Additionally, we present a new method for the production of protein-nucleosome complexes for structural analysis.

View Article: PubMed Central - PubMed

Affiliation: Structural Studies Division, Medical Research Council-Laboratory of Molecular Biology, Cambridge, UK.

ABSTRACT
The N-terminal acetylation of Sir3 is essential for heterochromatin establishment and maintenance in yeast, but its mechanism of action is unknown. The crystal structure of the N-terminally acetylated BAH domain of Saccharomyces cerevisiae Sir3 bound to the nucleosome core particle reveals that the N-terminal acetylation stabilizes the interaction of Sir3 with the nucleosome. Additionally, we present a new method for the production of protein-nucleosome complexes for structural analysis.

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Sir3 N-terminal acetylation increases Sir3 BAH affinity for NCPs(a) Native-PAGE of fluorescently-labeled NCPs incubated with increasing concentrations of unacetylated (black, BAH) or acetylated (red, Nt-Ac BAH) BAH. Asterisks indicate the titration point with the largest difference in substrate saturation between the acetylated and unacetylated BAH. (b) Quantification of Sir3 BAH binding to NCPs from panel a. The mean value (± standard deviation) of the percentage of unbound NCPs from three independent experiments is plotted against the BAH concentration. The concentration of BAH required for 50% binding to the NCP is indicated by a dashed line
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Figure 1: Sir3 N-terminal acetylation increases Sir3 BAH affinity for NCPs(a) Native-PAGE of fluorescently-labeled NCPs incubated with increasing concentrations of unacetylated (black, BAH) or acetylated (red, Nt-Ac BAH) BAH. Asterisks indicate the titration point with the largest difference in substrate saturation between the acetylated and unacetylated BAH. (b) Quantification of Sir3 BAH binding to NCPs from panel a. The mean value (± standard deviation) of the percentage of unbound NCPs from three independent experiments is plotted against the BAH concentration. The concentration of BAH required for 50% binding to the NCP is indicated by a dashed line

Mentions: To investigate whether the N-terminal acetylation of Sir3 regulates its binding to the NCP we expressed a long version of the BAH spanning Sir3 first 380 amino acids. This is the N-terminal fragment of Sir3 that best complements the silencing defect of a sir3Δ strain 13. We expressed the BAH both in insect cells where Met1 is removed and the Ala2 N-terminus acetylated and in bacteria where Met1 is cleaved but Ala2 is not acetylated. MALDI analysis and SDS-PAGE confirmed that the two proteins are indistinguishable except for the N-terminal acetylation present only in the protein expressed in insect cells (Supplementary Fig. 1a-c). Quantitative EMSAs (Electro Mobility Shift Assay) revealed that the N-terminal acetylation increases the affinity of the BAH for the NCP by at least a factor of two (Fig. 1a,b). To understand how the N-terminal acetylation affects the chromatin binding properties of Sir3, we determined the crystal structure of the N-terminally acetylated BAH bound to the NCP. Since conventional matrix-based purification methods often cause sample heterogeneity and losses, we developed a new matrix-free method. In this method, the BAH-NCP complexes were precipitated with PEG and re-suspended in a buffer at a concentration suitable for crystallization trials with a final recovery of 90% of the initial complex. The purified complexes were highly homogeneous as revealed by the presence of only one well-defined band in native gels (Supplementary Fig. 2a, lane 3). Importantly, we isolated only BAH-NCP complexes and not free components, since no free NCPs or free BAH precipitate at the PEG concentration used (Supplementary Fig. 2a, lane 4 and Supplementary Fig. 2b, lane 2). We analyzed the subunit composition of the purified BAH-NCP complex against known amounts of the NCP and the BAH. From this analysis we estimated that the complex has a stoichiometry of two molecules of BAH per NCP (Supplementary Fig. 2c,d).


The N-terminal acetylation of Sir3 stabilizes its binding to the nucleosome core particle.

Arnaudo N, Fernández IS, McLaughlin SH, Peak-Chew SY, Rhodes D, Martino F - Nat. Struct. Mol. Biol. (2013)

Sir3 N-terminal acetylation increases Sir3 BAH affinity for NCPs(a) Native-PAGE of fluorescently-labeled NCPs incubated with increasing concentrations of unacetylated (black, BAH) or acetylated (red, Nt-Ac BAH) BAH. Asterisks indicate the titration point with the largest difference in substrate saturation between the acetylated and unacetylated BAH. (b) Quantification of Sir3 BAH binding to NCPs from panel a. The mean value (± standard deviation) of the percentage of unbound NCPs from three independent experiments is plotted against the BAH concentration. The concentration of BAH required for 50% binding to the NCP is indicated by a dashed line
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Related In: Results  -  Collection

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Figure 1: Sir3 N-terminal acetylation increases Sir3 BAH affinity for NCPs(a) Native-PAGE of fluorescently-labeled NCPs incubated with increasing concentrations of unacetylated (black, BAH) or acetylated (red, Nt-Ac BAH) BAH. Asterisks indicate the titration point with the largest difference in substrate saturation between the acetylated and unacetylated BAH. (b) Quantification of Sir3 BAH binding to NCPs from panel a. The mean value (± standard deviation) of the percentage of unbound NCPs from three independent experiments is plotted against the BAH concentration. The concentration of BAH required for 50% binding to the NCP is indicated by a dashed line
Mentions: To investigate whether the N-terminal acetylation of Sir3 regulates its binding to the NCP we expressed a long version of the BAH spanning Sir3 first 380 amino acids. This is the N-terminal fragment of Sir3 that best complements the silencing defect of a sir3Δ strain 13. We expressed the BAH both in insect cells where Met1 is removed and the Ala2 N-terminus acetylated and in bacteria where Met1 is cleaved but Ala2 is not acetylated. MALDI analysis and SDS-PAGE confirmed that the two proteins are indistinguishable except for the N-terminal acetylation present only in the protein expressed in insect cells (Supplementary Fig. 1a-c). Quantitative EMSAs (Electro Mobility Shift Assay) revealed that the N-terminal acetylation increases the affinity of the BAH for the NCP by at least a factor of two (Fig. 1a,b). To understand how the N-terminal acetylation affects the chromatin binding properties of Sir3, we determined the crystal structure of the N-terminally acetylated BAH bound to the NCP. Since conventional matrix-based purification methods often cause sample heterogeneity and losses, we developed a new matrix-free method. In this method, the BAH-NCP complexes were precipitated with PEG and re-suspended in a buffer at a concentration suitable for crystallization trials with a final recovery of 90% of the initial complex. The purified complexes were highly homogeneous as revealed by the presence of only one well-defined band in native gels (Supplementary Fig. 2a, lane 3). Importantly, we isolated only BAH-NCP complexes and not free components, since no free NCPs or free BAH precipitate at the PEG concentration used (Supplementary Fig. 2a, lane 4 and Supplementary Fig. 2b, lane 2). We analyzed the subunit composition of the purified BAH-NCP complex against known amounts of the NCP and the BAH. From this analysis we estimated that the complex has a stoichiometry of two molecules of BAH per NCP (Supplementary Fig. 2c,d).

Bottom Line: The N-terminal acetylation of Sir3 is essential for heterochromatin establishment and maintenance in yeast, but its mechanism of action is unknown.The crystal structure of the N-terminally acetylated BAH domain of Saccharomyces cerevisiae Sir3 bound to the nucleosome core particle reveals that the N-terminal acetylation stabilizes the interaction of Sir3 with the nucleosome.Additionally, we present a new method for the production of protein-nucleosome complexes for structural analysis.

View Article: PubMed Central - PubMed

Affiliation: Structural Studies Division, Medical Research Council-Laboratory of Molecular Biology, Cambridge, UK.

ABSTRACT
The N-terminal acetylation of Sir3 is essential for heterochromatin establishment and maintenance in yeast, but its mechanism of action is unknown. The crystal structure of the N-terminally acetylated BAH domain of Saccharomyces cerevisiae Sir3 bound to the nucleosome core particle reveals that the N-terminal acetylation stabilizes the interaction of Sir3 with the nucleosome. Additionally, we present a new method for the production of protein-nucleosome complexes for structural analysis.

Show MeSH