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

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.

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

Figure 2

Sir3 N-terminal acetylation stabilizes the interaction of Sir3 BAH with the NCP (a) Superposition of the structures of the N-terminally acetylated (pink) and unacetylated (grey) BAH in complex with the NCP. (b) Detailed view of Ala2 (A2), N-ter-acetyl group (Nt-Ac), Loop3 and Helix8. (c) Detailed view of the interactions between the acetylated N-terminus of Sir3 and Loop3. (e) Close-up view of Sir3 Helix8–histone core interactions. (d) Detailed view of the acetylated Sir3 N-terminus.