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Molecular-motor coordinationThe proteasome is a cytosolic molecular machine that recognizes and degrades unneeded or damaged proteins that have been tagged with ubiquitin. A heterohexameric adenosine triphosphatase motor pulls the substrate into the proteolytic chamber, while at the same time, a protein located at the entrance of this motor removes the ubiquitin. De la Peña et al. trapped the substrate inside the motor by inhibiting removal of ubiquitin. This allowed them to determine cryo–electron microscopy structures in the presence of substrate and adenosine triphosphate (ATP). The findings distinguish three sequential conformational states that show how ATP binding, hydrolysis, and phosphate release are coordinated between the six subunits of the motor to cause the conformational changes that translocate the substrate through the proteasome.Science, this issue p. eaav0725INTRODUCTIONAs the major protease in eukaryotic cells and the final component of the ubiquitin-proteasome system, the 26S proteasome is responsible for protein homeostasis and the regulation of numerous vital processes. Misfolded, damaged, or obsolete regulatory proteins are marked for degradation by the attachment of polyubiquitin chains, which bind to ubiquitin receptors of the proteasome. A heterohexameric ring of AAA+ (ATPases associated with diverse cellular activities) subunits then uses conserved pore loops to engage, mechanically unfold, and translocate protein substrates into a proteolytic core for cleavage while the deubiquitinase Rpn11 removes substrate-attached ubiquitin chains.RATIONALEDespite numerous structural and functional studies, the mechanisms by which adenosine triphosphate (ATP) hydrolysis drives the conformational changes responsible for protein degradation remained elusive. Structures of related homohexameric AAA+ motors, in which bound substrates were stabilized with ATP analogs or hydrolysis-eliminating mutations, revealed snapshots of ATPase subunits in different nucleotide states and spiral-staircase arrangements of pore loops around the substrate. These structures gave rise to “hand-over-hand” translocation models by inferring how individual subunits may progress through various substrate-binding conformations. However, the coordination of ATP-hydrolysis steps and their mechanochemical coupling to propelling substrate were unknown.RESULTSWe present the cryo–electron microscopy (cryo-EM) structures of the actively ATP-hydrolyzing, substrate-engaged 26S proteasome with four distinct motor conformations. Stalling substrate translocation at a defined position by inhibiting deubiquitination led to trapped states in which the substrate-attached ubiquitin remains functionally bound to the Rpn11 deubiquitinase, and the scissile isopeptide bond of ubiquitin is aligned with the substrate-translocation trajectory through the AAA+ motor. Our structures suggest a ubiquitin capture mechanism, in which mechanical pulling on the substrate by the AAA+ motor delivers ubiquitin modifications directly into the Rpn11 catalytic groove and accelerates isopeptide cleavage for efficient, cotranslocational deubiquitination.These structures also show how the substrate polypeptide traverses from the Rpn11 deubiquitinase, through the AAA+ motor, and into the core peptidase. The proteasomal motor thereby adopts staircase arrangements with five substrate-engaged subunits and one disengaged subunit. Four of the substrate-engaged subunits are ATP bound, whereas the subunit at the bottom of the staircase and the disengaged subunit are bound to adenosine diphosphate (ADP).CONCLUSIONOf the four distinct motor states we observed, three apparently represent sequential stages of ATP binding, hydrolysis, and substrate translocation and hence reveal the coordination of individual steps in the ATPase cycle and their mechanochemical coupling with translocation. ATP hydrolysis occurs in the fourth substrate-engaged subunit from the top, concomitantly with exchange of ADP for ATP in the disengaged subunit. The subsequent transition, which is likely triggered by phosphate release from the fourth, posthydrolysis subunit of the staircase, then involves major conformational changes of the entire ATPase hexamer. The bottom ADP-bound subunit is displaced and the previously disengaged subunit binds the substrate at the top of the staircase, while the four engaged subunits move downward as a rigid body and translocate substrate toward the peptidase. Our likely consecutive proteasome conformations, together with previously determined substrate-free structures, suggest a sequential progression of ATPase subunits through the ATP-hydrolysis cycle. We hypothesize that, in general, hexameric AAA+ translocases function by this sequential mechanism.The 26S proteasome is the primary eukaryotic degradation machine and thus is critically involved in numerous cellular processes. The heterohexameric adenosine triphosphatase (ATPase) motor of the proteasome unfolds and translocates targeted protein substrates into the open gate of a proteolytic core while a proteasomal deubiquitinase concomitantly removes substrate-attached ubiquitin chains. However, the mechanisms by which ATP hydrolysis drives the conformational changes responsible for these processes have remained elusive. Here we present the cryo–electron microscopy structures of four distinct conformational states of the actively ATP-hydrolyzing, substrate-engaged 26S proteasome. These structures reveal how mechanical substrate translocation accelerates deubiquitination and how ATP-binding, -hydrolysis, and phosphate-release events are coordinated within the AAA+ (ATPases associated with diverse cellular activities) motor to induce conformational changes and propel the substrate through the central pore.

Survival of M. tuberculosis in host macrophages requires the eukaryotic-type protein kinase G, PknG, but the underlying mechanism has remained unknown. Here, we show that PknG is an integral component of a novel redox homeostatic system, RHOCS, which includes the ribosomal protein L13 and RenU, a Nudix hydrolase encoded by a gene adjacent to pknG. Studies in M. smegmatis showed that PknG expression is uniquely induced by NADH, which plays a key role in metabolism and redox homeostasis. In vitro, RenU hydrolyses FAD, ADP-ribose and NADH, but not NAD+. Absence of RHOCS activities in vivo causes NADH and FAD accumulation, and increased susceptibility to oxidative stress. We show that PknG phosphorylates L13 and promotes its cytoplasmic association with RenU, and the phosphorylated L13 accelerates the RenU-catalyzed NADH hydrolysis. Importantly, interruption of RHOCS leads to impaired mycobacterial biofilms and reduced survival of M. tuberculosis in macrophages. Thus, RHOCS represents a checkpoint in the developmental program required for mycobacterial growth in these environments.

The Anfinsen hypothesis, the demonstration of which led to the Nobel prize in Chemistry, posits that all information required to determine a proteins’ three dimensional structure is contained within its amino acid sequence. This suggests that it should be possible, in theory, to fold any protein in vitro . In practice, however, protein production by refolding is challenging because suitable refolding conditions must be empirically determined for each protein and can be painstaking. Here we demonstrate, using a variety of proteins, that differential scanning fluorimetry (DSF) can be used to determine and optimize conditions that favor proper protein folding in a rapid and high-throughput fashion. The resulting method, which we deem DSF guided refolding (DGR), thus enables the production of aggregation-prone and disulfide-containing proteins by refolding from E. coli inclusion bodies, which would not normally be amenable to production in bacteria.

The 20S proteasome (20S) facilitates turnover of most eukaryotic proteins. Substrate entry into the 20S first requires opening of gating loops through binding of HbYX motifs that are present at the C-termini of certain proteasome activators (PAs). The HbYX motif has been predominantly characterized in the archaeal 20S, whereas little is known about the sequence preferences of the human 20S ( h 20S). Here, we synthesize and screen ~120 HbYX-like peptides, revealing unexpected differences from the archaeal system and defining the h 20S recognition sequence as the Y-F/Y (YФ) motif. To gain further insight, we create a functional chimera of the optimized sequence, NLSYYT, fused to the model activator, PA26 E102A . A cryo-EM structure of PA26 E102A - h 20S is used to identify key interactions, including non-canonical contacts and gate-opening mechanisms. Finally, we demonstrate that the YФ sequence preferences are tuned by valency, allowing multivalent PAs to sample greater sequence space. These results expand the model for termini-mediated gating and provide a template for the design of h 20S activators. The proteasome complexes, composed of 20S core particles and one or two regulatory particles (proteasome activators), degrade most eukaryotic proteins. Here, the authors identify a sequence motif and resolve its interactions mediating the activation of the human 20S proteasome.

The 26S proteasome is essential for proteostasis and the regulation of vital processes through ATP-dependent degradation of ubiquitinated substrates. To accomplish the multi-step degradation process, the proteasome’s regulatory particle, consisting of lid and base subcomplexes, undergoes major conformational changes whose origin is unknown. Investigating the Saccharomyces cerevisiae proteasome, we found that peripheral interactions between the lid subunit Rpn5 and the base AAA+ ATPase ring are important for stabilizing the substrate-engagement-competent state and coordinating the conformational switch to processing states upon substrate engagement. Disrupting these interactions perturbs the conformational equilibrium and interferes with degradation initiation, while later processing steps remain unaffected. Similar defects in early degradation steps are observed when eliminating hydrolysis in the ATPase subunit Rpt6, whose nucleotide state seems to control proteasome conformational transitions. These results provide important insight into interaction networks that coordinate conformational changes with various stages of degradation, and how modulators of conformational equilibria may influence substrate turnover.

Given the broad range of substrates hydrolyzed by Nudix (nucleoside diphosphate linked to X) enzymes, identification of sequence and structural elements that correctly predict a Nudix substrate or characterize a family is key to correctly annotate the myriad of Nudix enzymes. Here, we present the structure determination and characterization of Bd3179 -- a Nudix hydrolase from Bdellovibrio bacteriovorus-that we show localized in the periplasmic space of this obligate Gram-negative predator. We demonstrate that the enzyme is a nucleoside diphosphate sugar hydrolase (NDPSase) and has a high degree of sequence and structural similarity to a canonical ADP-ribose hydrolase and to a nucleoside diphosphate sugar hydrolase (1.4 and 1.3 Å Cα RMSD respectively). Examination of the structural elements conserved in both types of enzymes confirms that an aspartate-X-lysine motif on the C-terminal helix of the α-β-α NDPSase fold differentiates NDPSases from ADPRases.

The proteasome is a cytosolic molecular machine that recognizes and degrades unneeded or damaged proteins that have been tagged with ubiquitin. A heterohexameric adenosine triphosphatase motor pulls the substrate into the proteolytic chamber, while at the same time, a protein located at the entrance of this motor removes the ubiquitin. De la Peña et al. trapped the substrate inside the motor by inhibiting removal of ubiquitin. This allowed them to determine cryo–electron microscopy structures in the presence of substrate and adenosine triphosphate (ATP). The findings distinguish three sequential conformational states that show how ATP binding, hydrolysis, and phosphate release are coordinated between the six subunits of the motor to cause the conformational changes that translocate the substrate through the proteasome. Science , this issue p. eaav0725 The structure of the yeast 26 S proteasome reveals sequential states of ATP binding, hydrolysis, and substrate translocation. The 26 S proteasome is the primary eukaryotic degradation machine and thus is critically involved in numerous cellular processes. The heterohexameric adenosine triphosphatase (ATPase) motor of the proteasome unfolds and translocates targeted protein substrates into the open gate of a proteolytic core while a proteasomal deubiquitinase concomitantly removes substrate-attached ubiquitin chains. However, the mechanisms by which ATP hydrolysis drives the conformational changes responsible for these processes have remained elusive. Here we present the cryo–electron microscopy structures of four distinct conformational states of the actively ATP-hydrolyzing, substrate-engaged 26 S proteasome. These structures reveal how mechanical substrate translocation accelerates deubiquitination and how ATP-binding, -hydrolysis, and phosphate-release events are coordinated within the AAA+ (ATPases associated with diverse cellular activities) motor to induce conformational changes and propel the substrate through the central pore.

The 26S proteasome is essential for protein homeostasis and the regulation of vital cellular processes through ATP-dependent degradation of ubiquitinated substrates. To accomplish the multi-step reaction of protein degradation, the regulatory particle of the proteasome, consisting of the lid and base subcomplexes, undergoes major conformational changes whose origin and control are largely unknown. Investigating the Saccharomyces cerevisiae 26S proteasome, we found that peripheral interactions between the lid subunit Rpn5 and the base AAA+ ATPase ring play critical roles in stabilizing the substrate-engagement-competent state and coordinating the conformational switch to processing states after a substrate has been engaged. Disrupting these interactions perturbs the conformational equilibrium and interferes with degradation initiation, while later steps of substrate processing remain unaffected. Similar defects in the early degradation steps are also observed when eliminating hydrolysis in the ATPase subunit Rpt6, whose nucleotide state seems to control conformational transitions of the proteasome. These results provide important insight into the network of interactions that coordinate conformational changes with various stages of proteasomal degradation, and how modulators of conformational equilibria may influence substrate turnover.

The 26S proteasome is the primary eukaryotic degradation machine and thus critically involved in numerous cellular processes. The hetero-hexameric ATPase motor of the proteasome unfolds and translocates targeted protein substrates into the open gate of a proteolytic core, while a proteasomal deubiquitinase concomitantly removes substrate-attached ubiquitin chains. However, the mechanisms by which ATP hydrolysis drives the conformational changes responsible for these processes have remained elusive. Here we present the cryo-EM structures of four distinct conformational states of the actively ATP-hydrolyzing, substrate-engaged 26S proteasome. These structures reveal how mechanical substrate translocation accelerates deubiquitination, and how ATP-binding, hydrolysis, and phosphate-release events are coordinated within the AAA+ motor to induce conformational changes and propel the substrate through the central pore.

The 20S proteasome (20S) facilitates turnover of most eukaryotic proteins. Substrate entry into the 20S first requires opening of gating loops through binding of HbYX motifs that are present at the C-termini of certain proteasome activators (PAs). The HbYX motif has been predominantly characterized in the archaeal 20S, whereas little is known about the sequence preferences of the human 20S (h20S). Here, we synthesized and screened ∼120 HbYX-like peptides, revealing unexpected differences from the archaeal system and defining the h20S recognition sequence as the Y-F/Y (YΦ) motif. To gain further insight, we created a functional chimera of the optimized sequence, NLSYYT, fused to the model activator, PA26E102A.A cryo-EM structure of PA26E102A-h20S identified key interactions, including non-canonical contacts and gate-opening mechanisms. Finally, we demonstrated that the YΦ sequence preferences are tuned by valency, allowing multivalent PAs to sample greater sequence space. These results expand the model for termini-mediated gating and provide a template for the design of h20S activators.