Observing Protein Degradation by the PAN-20S Proteasome by Time-Resolved Neutron Scattering

Abstract

The proteasome is a key player of regulated protein degradation in all kingdoms of life. Although recent atomic structures have provided snapshots on a number of conformations, data on substrate states and populations during the active degradation process in solution remain scarce. Here, we use time-resolved small-angle neutron scattering of a deuterium-labeled GFPssrA substrate and an unlabeled archaeal PAN-20S system to obtain direct structural information on substrate states during ATP-driven unfolding and subsequent proteolysis in solution. We find that native GFPssrA structures are degraded in a biexponential process, which correlates strongly with ATP hydrolysis, the loss of fluorescence, and the buildup of small oligopeptide products. Our solution structural data support a model in which the substrate is directly translocated from PAN into the 20S proteolytic chamber, after a first, to our knowledge, successful unfolding process that represents a point of no return and thus prevents dissociation of the complex and the release of harmful, aggregation-prone products.

Figure 1

Experimental SANS curves as a function of time. Shown are d-GFP alone (A), d-GFP in the presence of h-PAN (B), and d-GFP in the presence of h-PAN and h-20S (C) on a double logarithmic plot. Continuous lines display fits to the atomic Protein Data Bank model with CRYSON (38). Insets in (A) and (C) show the respective Guinier fits used to extract radii of gyration (R_G). GFP was perdeuterated in all cases and had a strong contrast in the 42% D₂O buffers. PAN and 20S were in their natural, hydrogenated state and effectively invisible in the 42% D₂O (and are therefore depicted as a transparent ribbon). All samples were measured at 55°C and in the presence of ATP. The arrows indicate the direction of the change of I(q) over time. In (C), the experimental points of the last curve (45 min) are connected by a line to facilitate their visualization at high angles. A total of 90 SANS frames of 30 s were recorded for all samples. For reasons of clarity, only a selection of frames is shown. Likewise, error bars are shown only for the first and last data set in case (B). Typical error bars of the data sets in cases (A) and (C) are shown in Fig. S12. The error bars shown represent statistical errors of the solvent-subtracted SANS intensities as obtained by the program PRIMUS. To see this figure in color, go online.

Figure 2

Time dependence of R_G, I(0), and fluorescence. Shown are GFP alone (A), GFP in the presence of PAN (B), and GFP in the presence of PAN and 20S (C). All samples were measured at 55°C and in the presence of ATP. Because of the contrast matching of h-PAN and h-20S, the SANS curves report exclusively on (d-)GFP and derived species and states (native, unfolded, hydrolyzed products and aggregates) in solution. R_G and I(0) were extracted from the data in Fig. 1 with the Guinier approximation (see Materials and Methods); the online fluorescence intensities are provided in arbitrary units. Continuous lines represent linear or biexponential (Eq. 1) fits to the data. A total of 90 SANS and fluorescence frames were recorded for all samples. For reasons of clarity, all frames were used for t < 8 min (to fit the fast processes in B and C), but only one out of 10 was used for t > 8 min. The error bars of the I(0) and R_G values were obtained by Guinier fits using the program PRIMUS. The fluorescence error bars are statistical errors obtained by Gaussian fits. To see this figure in color, go online.

Figure 3

Time-dependent populations of native GFP, a representative decapeptide product, and the putative intermediate state. The respective populations (volume fractions) were fitted with the program OLIGOMER (36) based on form factors (theoretical curves) of native GFP, the representative decapeptide product, and the model of the putative intermediate state. The structural model of the intermediate was generated based on cryo-EM and single-molecule data (see Materials and Methods; (18,42)). Likewise, the PAN and 20S structures shown in this figure were exceptionally taken from the cryo-EM study of their eukaryotic homologs and are depicted as a transparent ribbon to indicate that their SANS signal was contrast matched by the solvent at 42% D₂O. The continuous lines represent the fits with a single exponential decay function (Eq. 1 with t₂ = 0). To see this figure in color, go online.

Figure 4

Comparison of characteristic time constants from different experimental data. Shown are the characteristic time constants t₁ and t₂ (Eq. 1) from fits of I(0) SANS intensities (indicative of molecular weight), fluorescence at 509 nm (indicative of the percentage of folded state), ATP consumption, and OLIGOMER populations (Table 1). All data refer to GFP in the presence of both PAN and 20S. In the case of OLIGOMER, a single exponential decay was sufficient to fit the data (Fig. 3); the corresponding characteristic time can be considered as an average of the t₁ and t₂ measured in the other experiments. The t₁ and t₂ error bars were obtained from fits of the experimental data by using Eq. 1 with the Igor Pro Software. To see this figure in color, go online.

Figure 5

Schematic overview of GFP degradation process by PAN and 20S. Shown is the proposed mechanism of GFP degradation and interaction with the PAN-20S complex, based on TR-SANS data, coupled to online fluorescence. Our data are compatible with a trial-and-error model of unfruitful unfolding attempts and substrate release in a form (denominated “destabilized GFP” and distinct from the bound, intermediate form) that has no propensity to aggregate. At this stage, dissociation of the PAN-20S complex is still possible. Once unfolding is successfully engaged, the process has reached a “point of no return,” and substrate is no longer released in solution, neither on the N-terminal nor on the C-terminal side of PAN. This implies that the substrate is efficiently transferred through the central PAN channel into the 20S protease catalytic chamber and hydrolyzed subsequently. The elongated intermediate substrate, in the process of translocation, might indeed act as a “tether” and keep PAN and 20S tightly associated and thus prevent the release of harmful, aggregation-prone substrate states. To see this figure in color, go online.