Figure 2. ATP binds to the CTD of Sirt1.

(a) Photoaffinity-labelling of recombinant Sirt1, BSA and recombinant Sirt2. Photoaffinity-labelling of 8-azido-[α-³²P]ATP was visualized by autoradiography (left panel) and total protein levels were visualized by Coomassie staining (right panel). (b) His-tagged Sirt1, Sirt3 or Sirt6 were incubated with empty agarose beads or ATP-conjugated agarose beads in the absence or presence of 10 mM ATP for 4 h. Bound proteins were eluted by SDS sample buffer and visualized by immunoblotting with a His-tag specific antibody. (c) Photoaffinity-labelling of recombinant Sirt1 and BSA with 8-azido-[α-³²P]ATP in the presence of 150–500 mM NaCl. Photoaffinity-labelling of 8-azido-[α-³²P]ATP was visualized by autoradiography (top panel) and total protein levels were visualized by Coomassie staining (bottom panel). (d) Visualization of ATP-binding by mobility-shift. Sirt1 and BSA were photoaffinity-labelled with 0–8 mM of 8-azido-ATP, electrophoresed in SDS–PAGE and visualized by Coomassie staining. (e) The concentration dependence of ATP binding to Sirt1. ATP binding was measured by using the gel filtration technique and K_d was calculated to be ∼4.6 mM (n=3). (f) A schematic diagram of mouse Sirt1 fragments. The deacetylase domain is shown as a black rectangle. The CTD of Sirt1 binds to ATP. Photoaffinity-labelling of fragments F1, F2 and BSA with 8-azido-[α-³²P]ATP is shown. (g) Tryptic digest of Sirt1 photoaffinity-labelled with 8-azido-[α-³²P]ATP was electrophoresed in 20% SDS–PAGE. The peptide labelled with 8-azido-[α-³²P]ATP was visualized with autoradiography (right) and all of the peptides were visualized by Coomassie staining (left). (h) The mass of the photoaffinity-labelled peptide (*, g) was determined by MS/MS. The absolute intensity of the peak is indicated on the right Y axis. The sequence analysis of the peptide is shown in Supplementary Table 1. (i) Endogenous Sirt1 also binds ATP. Permeabilized HeLa cells were incubated with [α-³²P]ATP and endogenous Sirt1 was immunoprecipitated after photoaffinity-labelling with ultraviolet radiation. Endogenous Sirt1 labelled with [α-³²P]ATP was detected by autoradiography (right) and immunoblotting with anti-Sirt1 antibody (left).