Yeast PI31 inhibits the proteasome by a direct multisite mechanism.

Proteasome inhibitors are widely used as therapeutics and research tools, and typically target one of the three active sites, each present twice in the proteasome complex. An endogeneous proteasome inhibitor, PI31, was identified 30 years ago, but its inhibitory mechanism has remained unclear. Here, we identify the mechanism of Saccharomyces cerevisiae PI31, also known as Fub1. Using cryo-electron microscopy (cryo-EM), we show that the conserved carboxy-terminal domain of Fub1 is present inside the proteasome's barrel-shaped core particle (CP), where it simultaneously interacts with all six active sites. Targeted mutations of Fub1 disrupt proteasome inhibition at one active site, while leaving the other sites unaffected. Fub1 itself evades degradation through distinct mechanisms at each active site. The gate that allows substrates to access the CP is constitutively closed, and Fub1 is enriched in mutant CPs with an abnormally open gate, suggesting that Fub1 may function to neutralize aberrant proteasomes, thereby ensuring the fidelity of proteasome-mediated protein degradation.

Structures of chaperone-associated assembly intermediates reveal coordinated mechanisms of proteasome biogenesis.

The proteasome mediates most selective protein degradation. Proteolysis occurs within the 20S core particle (CP), a barrel-shaped chamber with an α7ß7ß7α7 configuration. CP biogenesis proceeds through an ordered multistep pathway requiring five chaperones, Pba1-4 and Ump1. Using Saccharomyces cerevisiae, we report high-resolution structures of CP assembly intermediates by cryogenic-electron microscopy. The first structure corresponds to the 13S particle, which consists of a complete α-ring, partial ß-ring (ß2-4), Ump1 and Pba1/2. The second structure contains two additional subunits (ß5-6) and represents a later pre-15S intermediate. These structures reveal the architecture and positions of Ump1 and ß2/ß5 propeptides, with important implications for their functions. Unexpectedly, Pba1's N terminus extends through an open CP pore, accessing the CP interior to contact Ump1 and the ß5 propeptide. These results reveal how the coordinated activity of Ump1, Pba1 and the active site propeptides orchestrate key aspects of CP assembly.

Solution Structure of the Cuz1 AN1 Zinc Finger Domain: An Exposed LDFLP Motif Defines a Subfamily of AN1 Proteins.

Zinc binding domains are common and versatile protein structural motifs that mediate diverse cellular functions. Among the many structurally distinct families of zinc finger (ZnF) proteins, the AN1 domain remains poorly characterized. Cuz1 is one of two AN1 ZnF proteins in the yeast S. cerevisiae, and is a stress-inducible protein that functions in protein degradation through direct interaction with the proteasome and Cdc48. Here we report the solution structure of the Cuz1 AN1 ZnF which reveals a compact C6H2 zinc-coordinating domain that resembles a two-finger hand holding a tri-helical clamp. A central phenylalanine residue sits between the two zinc-coordinating centers. The position of this phenylalanine, just before the penultimate zinc-chelating cysteine, is strongly conserved from yeast to man. This phenylalanine shows an exceptionally slow ring-flipping rate which likely contributes to the high rigidity and stability of the AN1 domain. In addition to the zinc-chelating residues, sequence analysis of Cuz1 indicates a second highly evolutionarily conserved motif. This LDFLP motif is shared with three human proteins-Zfand1, AIRAP, and AIRAP-L-the latter two of which share similar cellular functions with Cuz1. The LDFLP motif, while embedded within the zinc finger domain, is surface exposed, largely uninvolved in zinc chelation, and not required for the overall fold of the domain. The LDFLP motif was dispensable for Cuz1's major known functions, proteasome- and Cdc48-binding. These results provide the first structural characterization of the AN1 zinc finger domain, and suggest that the LDFLP motif may define a sub-family of evolutionarily conserved AN1 zinc finger proteins.

Proteomic Analysis Identifies Ribosome Reduction as an Effective Proteotoxic Stress Response.

Stress responses are adaptive cellular programs that identify and mitigate potentially dangerous threats. Misfolded proteins are a ubiquitous and clinically relevant stress. Trivalent metalloids, such as arsenic, have been proposed to cause protein misfolding. Using tandem mass tag-based mass spectrometry, we show that trivalent arsenic results in widespread reorganization of the cell from an anabolic to a catabolic state. Both major pathways of protein degradation, the proteasome and autophagy, show increased abundance of pathway components and increased functional output, and are required for survival. Remarkably, cells also showed a down-regulation of ribosomes at the protein level. That this represented an adaptive response and not an adverse toxic effect was indicated by enhanced survival of ribosome mutants after arsenic exposure. These results suggest that a major source of toxicity of trivalent arsenic derives from misfolding of newly synthesized proteins and identifies ribosome reduction as a rapid, effective, and reversible proteotoxic stress response.

Fluorescence spectroscopy of soluble E. coli SPase I Δ2-75 reveals conformational changes in response to ligand binding.

The bacterial Sec pathway is responsible for the translocation of secretory preproteins. During the later stages of transport, the membrane-embedded signal peptidase I (SPase I) cleaves the signal peptide from a preprotein. We used tryptophan fluorescence spectroscopy of a soluble, catalytically active E. coli SPase I Δ2-75 enzyme to study its dynamic conformational changes while in solution and when interacting with lipids and signal peptides. We generated four single Trp SPase I Δ2-75 mutants, W261, W284, W300, and W310. Based on fluorescence quenching experiments, W300 and W310 were found to be more solvent accessible than W261 and W284 in the absence of ligands. W300 and W310 inserted into lipids, consistent with their location at the enzyme's proposed membrane-interface region, while the solvent accessibilities of W261, W284, and W300 were modified in the presence of signal peptide, suggesting propagation of structural changes beyond the active site in response to peptide binding. The signal peptide binding affinity for the enzyme was measured via FRET experiments and the Kd determined to be 4.4 µM. The location of the peptide with respect to the enzyme was also established; this positioning is crucial for the peptide to gain access to the enzyme active site as it emerges from the translocon into the membrane bilayer. These studies reveal enzymatic structural changes required for preprotein proteolysis as it interacts with its two key partners, the signal peptide and membrane phospholipids.

Mapping of the SecA signal peptide binding site and dimeric interface by using the substituted cysteine accessibility method.

SecA is an ATPase nanomotor critical for bacterial secretory protein translocation. Secretory proteins carry an amino-terminal signal peptide that is recognized and bound by SecA followed by its transfer across the SecYEG translocon. While this process is crucial for the onset of translocation, exactly where the signal peptide interacts with SecA is unclear. SecA protomers also interact among themselves to form dimers in solution, yet the oligomeric interface and the residues involved in dimerization are unknown. To address these issues, we utilized the substituted cysteine accessibility method (SCAM); we generated a library of 23 monocysteine SecA mutants and probed for the accessibility of each mutant cysteine to maleimide-(polyethylene glycol)2-biotin (MPB), a sulfhydryl-labeling reagent, both in the presence and absence of a signal peptide. Dramatic differences in MPB labeling were observed, with a select few mutants located at the preprotein cross-linking domain (PPXD), the helical wing domain (HWD), and the helical scaffold domain (HSD), indicating that the signal peptide binds at the groove formed between these three domains. The exposure of this binding site is varied under different conditions and could therefore provide an ideal mechanism for preprotein transfer into the translocon. We also identified residues G793, A795, K797, and D798 located at the two-helix finger of the HSD to be involved in dimerization. Adenosine-5'-(γ-thio)-triphosphate (ATPγS) alone and, more extensively, in conjunction with lipids and signal peptides strongly favored dimer dissociation, while ADP supports dimerization. This study provides key insight into the structure-function relationships of SecA preprotein binding and dimer dissociation.

Signal peptidase I: cleaving the way to mature proteins.

Signal peptidase I (SPase I) is critical for the release of translocated preproteins from the membrane as they are transported from a cytoplasmic site of synthesis to extracytoplasmic locations. These proteins are synthesized with an amino-terminal extension, the signal sequence, which directs the preprotein to the Sec- or Tat-translocation pathway. Recent evidence indicates that the SPase I cleaves preproteins as they emerge from either pathway, though the steps involved are unclear. Now that the structure of many translocation pathway components has been elucidated, it is critical to determine how these components work in concert to support protein translocation and cleavage. Molecular modeling and NMR studies have provided insight on how the preprotein docks on SPase I in preparation for cleavage. This is a key area for future work since SPase I enzymes in a variety of species have now been identified and the inhibition of these enzymes by antibiotics is being pursued. The eubacterial SPase I is essential for cell viability and belongs to a unique group of serine endoproteases which utilize a Ser-Lys catalytic dyad instead of the prototypical Ser-His-Asp triad used by eukaryotes. As such, SPase I is a desirable antimicrobial target. Advances in our understanding of how the preprotein interfaces with SPase I during the final stages of translocation will facilitate future development of inhibitors that display a high efficacy against SPase I function.