Fig. 4: Midnolin without the Catch domain is less efficiently engaged by the proteasome.
From: Structural dynamics of the midnolin-proteasome during ubiquitin-independent substrate turnover
a Schematic representation of truncations introduced into the midnolin. bThe population of the processing state (PS) and the resting state (RS) proteasome pulled down by MIDN FL and MIDNΔ(112-336). c A low-pass filtered map (15 Å) shows the Ubl domain of midnolin density (magenta) at the N-ring entrance of 26S proteasome in the MIDNΔ(112-336)-26S proteasome complex. The model for midnolin is shown as cartoon. d Cryo-EM map of the midnolin IDR1 density in the central channel, translocating into the ATPase ring. The map was post-processed with EMReady for optimal visualization. e Atomic model of midnolin IDR1 and corresponding Cryo-EM map. f Cryo-EM density maps for pore-1 loop residues of RPTs interacting with IDR1. g Time course of Suc-LLVY-AMC hydrolysis for the 26S proteasome alone (black), or with Catch-deleted mutant [MBP-MIDN∆(112-336)] (purple), with Catch-replaced midnolin [MBP-MIDN(1-111)-EGR1(101–200)-MIDN(337-468)], with wild-type MBP-MIDN + EGR1(101–200) (red). h Quantification of in-vitro peptidase activity results in (g), shown as RFU change over 90 min. Data are presented as mean ± SD (n = 3). **** p < 0.0001, obtained by two-tailed one-way ANOVA followed by Tukey’s multiple comparisons test; ns, not significant.
