Figure 3. Degradation activities for base variants with a Walker-B EQ mutation in individual Rpt subunits correlate with the subunit's position in the spiral staircase arrangement of the base.

(a) In-vitro degradation rates for reconstituted proteasomes containing base variants with single Rpt subunits fixed in a permanent “ATP-bound” state by the EQ mutation. Degradation under multiple-turnover (gray) and single-turnover (black) conditions was monitored by the loss of fluorescence resulting from degradation of a polyubiquitinated GFP fusion substrate. Degradation activities were measured relative to reconstituted proteasome containing wild-type (WT) recombinant base. Errors for multiple-turnover degradation rates were estimated to be ±10% (s.d.) of the mean WT value based on repeat measurements (n = 3 technical replicates). The circular diagram is an alternative representation of the multiple-turnover data, with the line thickness corresponding to the observed degradation activities for a mutation in the respective subunit. (b) The large AAA+ subdomains of Rpt1–6 adopt distinct spiral staircase arrangements in the absence and presence of substrate. Shown are cartoon representations of the pre-engaged (top) and substrate-engaged (bottom) staircases based on cryoEM reconstructions^9,20, with the individual Rpt subunits splayed out and their pore-facing side pointing to the right. In the pre-engaged spiral, the small AAA+ subdomains of Rpt1–6 are arranged in a relatively planar fashion, while the large AAA+ subdomains are differentially lifted out of the ring plane, resulting in a pronounced spiral staircase with Rpt3 in the highest and Rpt2 in the lowest position. In the substrate-engaged spiral, the small and large AAA+ subdomains are mostly level, and the staircase orientation of pore loops primarily originates from differential rotations of subdomains in the plane of the ring.