Fig. 5: Four distinct substrate-processing states of the midnolin-26S proteasome complex.
From: Structural dynamics of the midnolin-proteasome during ubiquitin-independent substrate turnover
a Cryo-EM density maps of the AAA+ motor in all processing-state (PS) conformations, pseudo-colored by RPT subunit identity. Substrate-disengaged seam subunits exhibit reduced local resolution, likely attributable to heightened conformational flexibility and heterogeneous vertical positioning along the spiral staircase architecture (spanning the base to apex). Notably, PSRPT6 and PSRPT2 display pronounced inter-subunit density discontinuities at the seam, a feature consistent with the presence of two substrate-disengaged seam subunits undergoing continuous dynamic motions. b Conformational landscape of the proteasomal AAA+ motor with four distinct spiral-staircase registers during asymmetric ATP hydrolysis and active substrate processing. Atomic models show the AAA+ ATPase domains in different colors with the engaged substrate in the central channel, bound ATP depicted in yellow, ADP in cyan, magnesium ions in green. The states are organized into a logical sequence according to event-driven transitions and their associated processes. The schematics depict the spiral-staircase arrangements and RPT contacts with the substrate. c Spiral staircase organization of pore-1 loop aromatic residues (tyrosine for RPT1-4, phenylalanine for RPT5 and RPT6) across processing states. Measured distances between the substrate backbone and the Cα atom of the pore-1 loop aromatic residue are annotated for disengaged seam subunits. These seam subunits occupy variable vertical positions, with some localized near the staircase base and others positioned toward the apex. The spatial assignment of pore-loop residues in these subunits remains approximate due to their elevated mobility and resultant lower resolution. d, e Cryo-EM densities and atomic models around ADP binding site in RPT3. The density of the magnesium ion at the ADP binding site in PSRPT1 (d) is visible when compared to PSRPT5 (e). f Structural superimposition shows the movement of the pore-1 loop between PSRPT1 and PSRPT5. Specifically, Lys222, Phe223 in RPT6 and their coordinated substrate residue shift downwards by 2.4 Å and 2.3 Å, respectively.
