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...

There is growing appreciation for the fundamental role of structural dynamics in the function of macromolecules. In particular, the 26S proteasome, responsible for selective protein degradation in an ATP dependent manner, exhibits dynamic conformational changes that enable substrate processing. Recent cryo-electron microscopy (cryo-EM) work has revealed the conformational dynamics of the 26S proteasome and established the function of the different conformational states. Technological advances such as direct electron detectors and image processing algorithms allowed resolving the structure of the proteasome at atomic resolution. Here we will review those studies and discuss their contribution to our understanding of proteasome function.

Protective compounds must be present in chemically defined fermentation media in order to stabilize antibodies against the formation of HMW aggregates. An increase in chemical modifications is detectable in bioreactor stress models and over the course of cell culture fermentations; this increase is dependent on the expression rate, pH, temperature and fermentation time. Consequently, product heterogeneity increases during upstream processing, and this compromises the product quality.

Parvulins are small prolyl isomerases and serve as catalytic domains of folding enzymes. SurA (survival protein A) from the periplasm of Escherichia coli consists of an inactive (Par1) and an active (Par2) parvulin domain as well as a chaperone domain. In the absence of the chaperone domain, the folding activity of Par2 is virtually abolished. We created a chimeric protein by inserting the chaperone domain of SlyD, an unrelated folding enzyme from the FKBP family, into a loop of the isolated Par2 domain of SurA. This increased its folding activity 450-fold to a value higher than the activity of SurA, in which Par2 is linked with its natural chaperone domain. In the presence of both the natural and the foreign chaperone domain, the folding activity of Par2 was 1500-fold increased. Related and unrelated chaperone domains thus are similarly efficient in enhancing the folding activity of the prolyl isomerase Par2. A sequence analysis of various chaperone domains suggests that clusters of exposed methionine residues in mobile chain regions might be important for a generic interaction with unfolded protein chains. This binding is highly dynamic to allow frequent transfer of folding protein chains between chaperone and catalytic domains.

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