Fig. 6. Acetylation inhibits UBA dimerization and directly increases the affinity of p62 for ubiquitin.

a The W12 H_ε1-N_ε1 region of the ¹H-¹⁵N HSQC spectra of ¹⁵N-labeled WT and acetylation-mimicking mutants of p62-UBA were measured at high (200 µM, blue) and low (20 µM, red) concentrations. b The dissociation constant of each of the indicated p62-UBA proteins was calculated based on the relative peak volumes for dimeric and monomeric species at different concentrations. The residues used for calculation are S399, L402, G405, S407, G411, W412, T419, and G425 H_ε1-N_ε1; the error bars indicate 1S.D. in the averaged relative peak volume at each protein concentration. c Representative trajectories of MD simulation for WT, K420 acetylated, K435 acetylated and K420/K435 acetylated p62-UBA dimers. Fluctuations in Cα RMSD of the dimer are plotted. d Structural model of the complex of p62-UBA and ubiquitin. The ubiquitin molecule is colored according to electrostatic potential, which is displayed on a scale from red (−3 kT/e) to blue (+3 kT/e). The side chain of K435 in p62-UBA is shown as blue spheres. e Paramagnetic relaxation enhancement for the transverse relaxation rate Γ₂ of backbone amide protons for ¹⁵N-labeled WT or K435Q mutant of p62-UBA in complex with isotopically unlabeled ubiquitin carrying a nitroxide spin radical tag at the E24C site. The error bars indicate 1S.D. in the fitting of Γ₂ rates. The yellow shaded area indicates the unstructured C-terminal tail of p62. Source data are provided as a Source Data file.