The eukaryotic proteasome mediates degradation of polyubiquitinated proteins. Here we report the single-particle cryoelectron microscopy (cryo-EM) structures of the endogenous 26S proteasome from Saccharomyces cerevisiae at 4.6-to 6.3-Å resolution. The fine features of the cryo-EM maps allow modeling of 18 subunits in the regulatory particle and 28 in the core particle. The proteasome exhibits two distinct conformational states, designated M1 and M2, which correspond to those reported previously for the proteasome purified in the presence of ATP-γS and ATP, respectively. These conformations also correspond to those of the proteasome in the presence and absence of exogenous substrate. Structure-guided biochemical analysis reveals enhanced deubiquitylating enzyme activity of Rpn11 upon assembly of the lid. Our structures serve as a molecular basis for mechanistic understanding of proteasome function.protein degradation | proteasome | cryo-EM | structure T he eukaryotic ubiquitin-proteasome system is responsible for the degradation of polyubiquitinated proteins (1). The 26S proteasome consists of one 20S core particle (CP) and two 19S regulatory particles (RPs). The RP is divided into the lid and base assembly intermediates (1). The lid comprises nine Rpn subunits in yeast (Rpn3/5/6/7/8/9/11/12/15) and the base comprises three Rpn subunits (Rpn1/2/13) and six ATPases (Rpt1-6). Rpn10, which consists of an N-terminal von Willebrand factor A (VWA) domain and multiple C-terminal ubiquitin-interacting motifs (UIM), connects the lid and the base. Polyubiquitin (poly-Ub) chains from substrate are recognized by the RP, leading to unfolding of the substrate and its translocation into the CP, where it is degraded.The main function of the lid is to remove poly-Ub chains from the substrate (1). The released Ub chains are recycled via further cleavage into Ub monomers. Six of the nine Rpn subunits in the lid (Rpn3/5/6/7/9/12) contain a solenoid fold followed by a proteasome-CSN-eIF3 (PCI) domain of varying lengths; for Rpn8 and Rpn11, each has an Mpr1-Pad1-N-terminal (MPN) domain. Among all Rpn subunits, Rpn11 is the only deubiquitylating enzyme (DUB); it cleaves the isopeptide bond between the carboxyl terminus of Ub and the e-amino group of Lys in the substrate (2, 3). Except for Rpn15/ Sem1/Dss1, the C-terminal sequences of the other eight Rpn subunits in the lid form a helix bundle, which dictates lid assembly (4, 5).In the base, the six Rpt subunits form a hexameric ring. Powered by ATP hydrolysis, the Rpt ring is responsible for substrate unfolding and translocation of the unfolded substrate through the narrow RP central channel into the CP for degradation (6-8). The barrel-shaped CP consists of two outer α-rings and two inner β-rings, each containing seven subunits (α1-7 or β1-7). X-ray structures of the CP at atomic resolution have been reported for archaeabacteria (9), yeast (10), and mammals (11).Crystallization of the RP or the 26S proteasome is hampered by its dynamic nature. Improvement of cryo-EM technologies has ...
Acid-sensing ion channels (ASICs) are neuronal voltage-independent Na+ channels that are activated by extracellular acidification. ASICs play essential roles in a wide range of physiological processes, including sodium homeostasis, synaptic plasticity, neurodegeneration, and sensory transduction. Mambalgins, a family of three-finger toxins isolated from black mamba venom, specifically inhibit ASICs to exert strong analgesic effects in vivo, thus are thought to have potential therapeutic values against pain. However, the interaction and inhibition mechanism of mambalgin on ASICs remains elusive. Here, we report a cryo-electron microscopy (cryo-EM) structure of chicken ASIC1a (cASIC1a) in complex with mambalgin-1 toxin at 5.4 Å resolution. Our structure provides the first experimental evidence that mambalgin-1 interacts directly with the extracellular thumb domain of cASIC1a, rather than inserting into the acid-sensing pocket, as previously reported. Binding of mambalgin-1 leads to relocation of the thumb domain that could disrupt the acidic pocket of cASIC1a, illustrating an unusual inhibition mechanism of toxins on ASIC channels through an allosteric effect. These findings establish a structural basis for the toxicity of the mambalgins, and provide crucial insights for the development of new optimized inhibitors of ASICs.
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