Structural insights into the functional cycle of the ATPase module of the 26S proteasome

Abstract

In eukaryotic cells, the ubiquitin-proteasome system (UPS) is responsible for the regulated degradation of intracellular proteins. The 26S holocomplex comprises the core particle (CP), where proteolysis takes place, and one or two regulatory particles (RPs). The base of the RP is formed by a heterohexameric AAA + ATPase module, which unfolds and translocates substrates into the CP. Applying single-particle cryo-electron microscopy (cryo-EM) and image classification to samples in the presence of different nucleotides and nucleotide analogs, we were able to observe four distinct conformational states (s1 to s4). The resolution of the four conformers allowed for the construction of atomic models of the AAA + ATPase module as it progresses through the functional cycle. In a hitherto unobserved state (s4), the gate controlling access to the CP is open. The structures described in this study allow us to put forward a model for the 26S functional cycle driven by ATP hydrolysis.26S proteasome | cryo-electron microscopy | AAA + ATPase | integrative modeling | single-particle analysis I n eukaryotic cells, the ubiquitin-proteasome system (UPS) degrades proteins that are misfolded, damaged, or no longer needed (1). The 26S proteasome is a 2.5-MDa multisubunit complex comprising the barrel-shaped 20S core particle (CP), where degradation takes place, and one or two 19S regulatory particles (RPs), which bind to the ends of the CP (2-4). The CP is built of four coaxially stacked heteroheptameric rings of α-and β-subunits in the order of αββα (5). Three of the seven β-subunits are catalytically active; substrates are sequestered from the cellular environment in a chamber formed by the two β-rings (6, 7). This self-compartmentalization is a hallmark of many intracellular proteases (8). Substrate access to the proteolytic chamber is controlled by the α-subunit N-terminal extensions, forming a gate (3). Most of proteasome activators, including the RP, contain C-terminal hydrophobic-tyrosine-X (HbYX) motifs, which have been reported to insert into α-ring pockets, triggering gate opening (9-11).The RP is composed of at least 19 canonical subunits and interacts substoichiometrically with an array of proteasomeinteracting proteins that modulate RP function (3). The RP is divided into the "base" and the "lid" subcomplexes. The core of the base is formed by a heterohexameric ATPase associated with various cellular activities (AAA + ATPase), which is the driver of large-scale conformational dynamics of the RP. The AAA + ATPase prepares substrates for degradation in coordination with at least three ubiquitin receptors [26S proteasome non-ATPase regulatory subunit 1 (Rpn1), Rpn10, and Rpn13] (12-14) and a deubiquitylating subunit (Rpn11) (15, 16). Other subunits have structural roles, such as holding the CP and RP together, or in coordinating the movements needed to position the substrates above the pore of the AAA + ATPase for unfolding and translocation (17, 18). The AAA + ATPase is lined by aromatic-hydrophobic loops (pore-1 loops), wh...

In Situ Structure of Neuronal C9orf72 Poly-GA Aggregates Reveals Proteasome Recruitment

“…In contrast, the SPS2 class (23.1% of particles) was best fitted by the s4 model of actively translocating proteasomes (Table S1). This is remarkable because, in vitro , the s4 state was only recently detected in proteasomes incubated with non-hydrolysable nucleotide analogs (Wehmer et al, 2017), suggesting that it is normally a highly transient conformation. Thus, the interaction of poly-GA aggregates with the proteasome appears to stall its s4 conformation.…”

“…These additional particles were then visually inspected, aligned, classified and averaged again to produce a higher resolution average. The iterative application of this procedure yielded an average structure unequivocally corresponding to the 26S proteasome (Chen et al, 2016a; Huang et al, 2016; Schweitzer et al, 2016; Wehmer et al, 2017) for the smaller ring-like structures, and to the TRiC/CCT chaperonin (Leitner et al, 2012; Zang et al, 2016) for the larger ones (Figure 2, Figure 3, Figure S3A–C, Movie S1). Other large UPS components such as p97/VCP did not appear abundant at poly-GA aggregates.…”

“…Regulatory particles are responsible for substrate recognition, unfolding and translocation into the CP for proteolysis. Recent single particle cryo-EM studies have revealed how RP conformational dynamics are coupled to the functional cycle of the 26S complex (Chen et al, 2016a; Unverdorben et al, 2014; Wehmer et al, 2017). Initial binding of substrates to the 26S proteasome presumably occurs in a low energy ground state (s1) (see also Lu et al, 2015).…”

“…To this end we employed molecular dynamics flexible fitting (MDFF; Trabuco et al, 2009) initiated through the atomic models of the s1–4states of the yeast 26S proteasome (Figure 3C) (Wehmer et al, 2017). The s1 state was clearly the best fit for the GS1 and GS2 classes (Table S1).…”

“…The GS2 class (13.7% of particles) was overall similar to GS1, but the Rpn1 subunit pivoted 25° on its N-terminal region to shift its C-terminus towards the CP with respect to GS1 (Figure 4A–C). This is a novel conformation of Rpn1, a particularly dynamic subunit (Asano et al, 2015; Huang et al, 2016; Schweitzer et al, 2016; Wehmer et al, 2017) that serves as binding hub for 26S regulatory cofactors containing ubiquitin-like (UBL) domains (Elsasser et al, 2002; Leggett et al, 2002) and was recently identified as an ubiquitin receptor (Shi et al, 2016). …”