Figure 6: Recognition of the nucleosome by the RING1B–PCGF–UbcH5c∼Ub complexes.

(a) Ubiquitination of Histone H2A in mononucleosomes (left) or ‘octamers’ (right; see Methods) by the different RING1B–PCGF E3s, Top: western blot using α-H2A K119ub antibody, Bottom: western blot using α-H3 antibody (b) LANA peptide inhibition of RING1B–PCGF ubiquitination of Histone H2A. Each RING1B–PCGF was tested in the absence of LANA peptide, or in the presence of wild-type or non-binding (LRS) mutant peptide; see Methods for sequences). Product was detected by western blot using α-H2A K119ub antibody. (c) Structure of the nucleosome core particle at left showing location of acidic surface patches (H2A E92 and surrounding residues form the major patch; the smaller patch is formed by H3 E73, H3 D77 and H4 D24), location of the site of ubiquitination of H2A by PCGF ligases, and bound LANA peptide in cyan (PDB: 1ZLA⁵¹). The location of charged surface patches on RING1B–PCGF RING dimers is shown above. (d) Model of the RING1B–PCGF4–UbcH5c nucleosome complex with Ub in a transfer competent position. The model is based on the required proximity of the E2 active site cysteine and H2A K118/119, as well as the requirement of the H2A acidic patch for RING1B–PCGF ligase activity⁴⁹ and the LANA peptide inhibition data shown in b. RING1B is positioned over the H2A acidic patch. (Note that this is not a docked model, thus detailed interactions are not predicted, only the approximate positions of the various proteins. An experimental structure has been reported recently⁵² for this E2-E3-substrate complex without ubiquitin present.) Electrostatic potential molecular surface of the histone octamer shown in c and d was generated from the structure of the nucleosome core particle (PDB: 1AOI⁷¹) using the Adaptive Poisson-Boltzman Solver (APBS) plugin in PyMOL (Schrödinger LLC), contoured to ±4.0 kT/e.