Fig. 3: The Ubl and Catch domains guide substrate to the pore entrance.
From: Structural dynamics of the midnolin-proteasome during ubiquitin-independent substrate turnover
a Cryo-EM map of MIDN-PSUbl (Ubl bound state), showing the midnolin Ubl domain (magenta) interacting with RPN11 (PSMD14) of the 19S regulatory particle. b Cryo-EM map of the midnolin/substrate density in the central channel, reaching the ATPase ring. c Cryo-EM map of MIDN-PSUbl_Catch (Ubl and Catch domains bound state). The midnolin Ubl and Catch domains (magenta) interacts with RPN11 (PSMD14) of the 19S regulatory particle and the Catch domain is positioned above the proteasome entry port. d A cut-through of the cryo-EM map of the midnolin/substrate density in the central channel, reaching the ATPase ring. e Atomic model showing the interaction between the 19S proteasome (surface) and the Ubl and Catch domains of midnolin. f The atomic model illustrates the binding interface between Ubl and the deubiquitinating enzyme RPN11. For comparison, the experimentally determined complex of canonical ubiquitin with RPN11 (PDB: 6MSG) is superimposed. g The molecular basis of Ubl (magenta) interaction with the RPN11 (light pink). h Time course of Suc-LLVY-AMC hydrolysis for the 26S proteasome alone (black), or with MBP-MIDNP100E + EGR1(101–200) (dark green), with MBP-MIDNP100E/V102E/104E + EGR1(101–200) (light green), with wild-type MBP-MIDN + EGR1(101–200) (red). The y-axis indicates relative fluorescence units (RFU). i Quantification of in-vitro peptidase activity results in (h), shown as RFU change over 90 min. Data are presented as mean ± SD (n = 3). **** p < 0.0001, *** p < 0.001, obtained by two-tailed one-way ANOVA followed by Tukey’s multiple comparisons test.
