Figure 3
H3KC 36me3 MLA nucleosomes and peptides bind the PSIP1-PWWP domain with comparable affinities to their native counterparts (a) Recombinant MLA nucleosomes bind to endogenous PSIP1. Nucleosomes were immobilized on beads using biotinylated 601 DNA and incubated with HeLa nuclear extract. Bound proteins were analyzed by immunoblot with the indicated antibodies and by Coomassie blue staining to control for the amount of histones. (b) PSIP1-PWWP binds preferentially to H3K36me3 and H3KC36me3 peptides. Indicated peptides were biotinylated and immobilized, incubated with GST-PWWP lysates and analyzed by immunoblot with GST antibodies. (c) H3K36me3 (lower panels) and H3KC36me3 peptides (upper panels) bind with similar affinity to same pocket in PSIP1-PWWP. An overlay of sections of the 2D 1H-15N HSQC spectrum is shown for each NMR titration point. Assignments of resonances of interest are indicated on top; color-coding is indicated in the figure. (d) Chemical shift perturbation (CSP)-derived binding curves and best fits for H3K36me3 and H3KC36me3 peptide. Only points up to 4 mM of peptide were included in the fit. Curves for a selection of residues are shown; color-coding is indicated in the figure.