Fig. 1: Activation of h20S by hRpt5-derived peptides reveals unexpected SAR.

a Schematic of proteasome activation by the C-terminal HbYX motif of a model PA. HbYX motifs are known to dock into α-pockets located between subunits of the 20 S α-rings (gray). This interaction opens the gates of the 20 S, allowing substrate entry. The inset summarizes the interactions between PAN’s HbYX motif (orange) and the α-pocket (gray surface/cartoon) of the archaeal 20S (PDB ID: 3IPM). b Alanine scanning revealed residues of Rpt5 C-terminus that make key contributions to the stimulation of the h20S. Activation of the h20S (4 nM) was measured by an increase in the hydrolysis rate (RFU/s) of the fluorescent substrate, suc-LLVY-amc (10 µM) upon addition of hRpt5-derived peptides. Data are normalized to DMSO-treated h20S and plotted individually (n = 3). c Proteasome stimulation by peptides (250 µM) from an N-terminal truncation series. Results are normalized to ANLQYYA and the average of four independent experiments (open circles; n = 4) is plotted with error reported as s.e.m. P-values were calculated using two-tailed unpaired t-test: *p < 0.04. d Scatterplot of the relative activities of N-terminally acetylated hRpt5 hexapeptides (250 μM) sampling modifications along the P3 (green), P2 (blue), and P1 (orange) residues. Data are normalized to Ac-NLQYYA and plotted individually (n = 2). e Scatterplot of maximal activity of hexapeptides sampling all proteinogenic amino acids at the P4 position (purple). Data are normalized to Ac-NLQYYT and the average of three independent experiments (circles; n = 3) is plotted with error reported as s.e.m. f Combination of optimized residues (underlined) from the SAR campaign. Data are normalized to Opt5 (Ac-NLSYYT, star) and plotted individually (n = 3 or 4). EC₅₀ values were calculated from four independent experiments and reported as mean ± s.e.m.