Cryo-EM structures of the archaeal PAN-proteasome reveal an around-the-ring ATPase cycle

Proteasomes occur in all three domains of life, and are the principal molecular machines for the regulated degradation of intracellular proteins. They play key roles in the maintenance of protein homeostasis, and control vital cellular processes. While the eukaryotic 26S proteasome is extensively characterized, its putative evolutionary precursor, the archaeal proteasome, remains poorly understood. The primordial archaeal proteasome consists of a 20S proteolytic core particle (CP), and an AAA-ATPase module. This minimal complex degrades protein unassisted by non-ATPase subunits that are present in a 26S proteasome regulatory particle (RP). Using cryo-EM single-particle analysis, we determined structures of the archaeal CP in complex with the AAA-ATPase PAN (proteasome-activating nucleotidase). Five conformational states were identified, elucidating the functional cycle of PAN, and its interaction with the CP. Coexisting nucleotide states, and correlated intersubunit signaling features, coordinate rotation of the PAN-ATPase staircase, and allosterically regulate N-domain motions and CP gate opening. These findings reveal the structural basis for a sequential around-the-ring ATPase cycle, which is likely conserved in AAA-ATPases.

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Structural basis for dynamic regulation of the human 26S proteasome

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1614614113 ◽

2016 ◽

Vol 113 (46) ◽

pp. 12991-12996 ◽

Cited By ~ 96

Author(s):

Shuobing Chen ◽

Jiayi Wu ◽

Ying Lu ◽

Yong-Bei Ma ◽

Byung-Hoon Lee ◽

...

Keyword(s):

Conformational Changes ◽

Ground State ◽

Atp Hydrolysis ◽

26S Proteasome ◽

Structural Basis ◽

Core Particle ◽

Dynamic Regulation ◽

Aaa Atpase ◽

The Core ◽

Aaa Atpases

The proteasome is the major engine of protein degradation in all eukaryotic cells. At the heart of this machine is a heterohexameric ring of AAA (ATPases associated with diverse cellular activities) proteins that unfolds ubiquitylated target proteins that are concurrently translocated into a proteolytic chamber and degraded into peptides. Using cryoelectron microscopy, we determined a near–atomic-resolution structure of the 2.5-MDa human proteasome in its ground state, as well as subnanometer-resolution structures of the holoenzyme in three alternative conformational states. The substrate-unfolding AAA-ATPase channel is narrowed by 10 inward-facing pore loops arranged into two helices that run in parallel with each other, one hydrophobic in character and the other highly charged. The gate of the core particle was unexpectedly found closed in the ground state and open in only one of the alternative states. Coordinated, stepwise conformational changes of the regulatory particle couple ATP hydrolysis to substrate translocation and regulate gating of the core particle, leading to processive degradation.

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Specific lid-base contacts in the 26S proteasome control the conformational switching required for substrate engagement and degradation

10.1101/687921 ◽

2019 ◽

Cited By ~ 1

Author(s):

Eric R. Greene ◽

Ellen A. Goodall ◽

Andres H. de la Peña ◽

Mary E. Matyskiela ◽

Gabriel C. Lander ◽

...

Keyword(s):

Conformational Changes ◽

26S Proteasome ◽

Conformational Switching ◽

Atpase Subunit ◽

Aaa Atpase ◽

Cellular Processes ◽

Substrate Processing ◽

And Control ◽

Early Degradation ◽

Insight Into

AbstractThe 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 proteasome’s regulatory particle, 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.

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The AAA+ ATPase p97, a cellular multitool

Biochemical Journal ◽

10.1042/bcj20160783 ◽

2017 ◽

Vol 474 (17) ◽

pp. 2953-2976 ◽

Cited By ~ 42

Author(s):

Lasse Stach ◽

Paul S. Freemont

Keyword(s):

Atp Hydrolysis ◽

Current Status ◽

Cellular Functions ◽

Aaa Atpase ◽

Cellular Processes ◽

Proteotoxic Stress ◽

Binding Domains ◽

Wide Range ◽

Aaa Atpases ◽

Substrate Proteins

The AAA+ (ATPases associated with diverse cellular activities) ATPase p97 is essential to a wide range of cellular functions, including endoplasmic reticulum-associated degradation, membrane fusion, NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation and chromatin-associated processes, which are regulated by ubiquitination. p97 acts downstream from ubiquitin signaling events and utilizes the energy from ATP hydrolysis to extract its substrate proteins from cellular structures or multiprotein complexes. A multitude of p97 cofactors have evolved which are essential to p97 function. Ubiquitin-interacting domains and p97-binding domains combine to form bi-functional cofactors, whose complexes with p97 enable the enzyme to interact with a wide range of ubiquitinated substrates. A set of mutations in p97 have been shown to cause the multisystem proteinopathy inclusion body myopathy associated with Paget's disease of bone and frontotemporal dementia. In addition, p97 inhibition has been identified as a promising approach to provoke proteotoxic stress in tumors. In this review, we will describe the cellular processes governed by p97, how the cofactors interact with both p97 and its ubiquitinated substrates, p97 enzymology and the current status in developing p97 inhibitors for cancer therapy.

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New insights into the human 26S proteasome function and regulation

10.1101/2021.10.03.462214 ◽

2021 ◽

Author(s):

Miglė Kišonaitė ◽

Pavel Afanasyev ◽

Jonida Tafilaku ◽

Ana Toste Rêgo ◽

Paula C. A. da Fonseca

Keyword(s):

High Resolution ◽

Structural Data ◽

26S Proteasome ◽

Aaa Atpase ◽

Physiological Processes ◽

Allosteric Communication ◽

Atpase Subunits ◽

Strict Regulation ◽

Major Conformation

SummaryThe 26S proteasome is a protease complex essential for proteostasis and strict regulation of diverse critical physiological processes, the mechanisms of which are still not fully described. The human 26S proteasome purification was optimized without exogenous nucleotides, to preserve the endogenous nucleotide occupancy and conformation of its AAA-ATPase subunits. This unveiled important effects on the proteasome function and structure resulting from exposure to Ca2+ or Mg2+, with important physiological implications. This sample, with an added model degron designed to mimic the minimum canonical ubiquitin signal for proteasomal recognition, was analysed by high-resolution cryo-EM. Two proteasome conformations were resolved, with only one capable of degron binding. The structural data show that this occurs without major conformation rearrangements and allows to infer into the allosteric communication between ubiquitin degron binding and the peptidase activities. These results revise existing concepts on the 26S proteasome function and regulation, opening important opportunities for further research.

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Structural basis of protein translocation by the Vps4-Vta1 AAA ATPase

eLife ◽

10.7554/elife.24487 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 78

Author(s):

Nicole Monroe ◽

Han Han ◽

Peter S Shen ◽

Wesley I Sundquist ◽

Christopher P Hill

Keyword(s):

Protein Translocation ◽

Cellular Membrane ◽

The Other ◽

Structural Basis ◽

Atp Binding ◽

Fission Reactions ◽

Aaa Atpase ◽

Membrane Fission ◽

Substrate Peptide ◽

Aaa Atpases

Many important cellular membrane fission reactions are driven by ESCRT pathways, which culminate in disassembly of ESCRT-III polymers by the AAA ATPase Vps4. We report a 4.3 Å resolution cryo-EM structure of the active Vps4 hexamer with its cofactor Vta1, ADP·BeFx, and an ESCRT-III substrate peptide. Four Vps4 subunits form a helix whose interfaces are consistent with ATP binding, is stabilized by Vta1, and binds the substrate peptide. The fifth subunit approximately continues this helix but appears to be dissociating. The final Vps4 subunit completes a notched-washer configuration as if transitioning between the ends of the helix. We propose that ATP binding propagates growth at one end of the helix while hydrolysis promotes disassembly at the other end, so that Vps4 ‘walks’ along ESCRT-III until it encounters the ordered N-terminal domain to destabilize the ESCRT-III lattice. This model may be generally applicable to other protein-translocating AAA ATPases.

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Conserved prolines in the coiled coil-OB domain linkers of proteasomal ATPases facilitate eukaryotic proteasome base assembly

10.1101/2020.11.13.381962 ◽

2020 ◽

Author(s):

Chin Leng Cheng ◽

Michael K Wong ◽

Yanjie Li ◽

Mark Hochstrasser

Keyword(s):

Protein Quality ◽

Coiled Coil ◽

26S Proteasome ◽

Single Type ◽

Mutant Form ◽

Peptide Bonds ◽

Atpase Subunit ◽

Aaa Atpase ◽

Growth Defects ◽

Atpase Subunits

AbstractThe proteasome is a large protease complex that degrades both misfolded and regulatory proteins. In eukaryotes, the 26S proteasome contains six different AAA+ ATPase subunits, Rpt1-Rpt6, which form a hexameric ring as part of the base subcomplex that drives unfolding and translocation of substrates into the proteasome core. Archaeal proteasomes contain only a single type of ATPase subunit, the proteasome-activating nucleotidase (PAN), which forms a trimer-of-dimers and is homologous to the eukaryotic Rpt subunits. A key PAN proline residue (P91) forms cis and trans peptide bonds in successive subunits around the ring, allowing efficient dimerization through upstream coiled coils. The importance of the equivalent Rpt prolines in eukaryotic proteasome assembly was unknown. We show an equivalent proline is strictly conserved in Rpt3 (in S. cerevisiae, P93) and Rpt5 (P76), well conserved in Rpt2 (P103), and loosely conserved in Rpt1 (P96) in deeply divergent eukaryotes, but in no case is its mutation strongly deleterious to yeast growth. However, the rpt2-P103A, rpt3-P93A, and rpt5-P76A mutations all cause synthetic defects with specific base assembly chaperone deletions. The Rpt5-P76A mutation decreases the levels of the protein and induces a mild proteasome assembly defect. The yeast rpt2-P103A rpt5-P76A double mutant has strong growth defects attributable to defects in proteasome base formation. Several Rpt subunits in this mutant form aggregates that are cleared, at least in part, by the Hsp42-mediated protein quality control (PQC) machinery. We propose that the conserved Rpt linker prolines promote efficient 26S proteasome base assembly by facilitating specific ATPase heterodimerization.

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Structure of the human 26S proteasome at a resolution of 3.9 Å

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1608050113 ◽

2016 ◽

Vol 113 (28) ◽

pp. 7816-7821 ◽

Cited By ~ 123

Author(s):

Andreas Schweitzer ◽

Antje Aufderheide ◽

Till Rudack ◽

Florian Beck ◽

Günter Pfeifer ◽

...

Keyword(s):

Ubiquitin Proteasome System ◽

Structural Features ◽

26S Proteasome ◽

The Other ◽

Aaa Atpase ◽

Loop Region ◽

C Terminus ◽

Proteasome Subunits ◽

Atpase Subunits ◽

Substrate Processing

Protein degradation in eukaryotic cells is performed by the Ubiquitin-Proteasome System (UPS). The 26S proteasome holocomplex consists of a core particle (CP) that proteolytically degrades polyubiquitylated proteins, and a regulatory particle (RP) containing the AAA-ATPase module. This module controls access to the proteolytic chamber inside the CP and is surrounded by non-ATPase subunits (Rpns) that recognize substrates and deubiquitylate them before unfolding and degradation. The architecture of the 26S holocomplex is highly conserved between yeast and humans. The structure of the human 26S holocomplex described here reveals previously unidentified features of the AAA-ATPase heterohexamer. One subunit, Rpt6, has ADP bound, whereas the other five have ATP in their binding pockets. Rpt6 is structurally distinct from the other five Rpt subunits, most notably in its pore loop region. For Rpns, the map reveals two main, previously undetected, features: the C terminus of Rpn3 protrudes into the mouth of the ATPase ring; and Rpn1 and Rpn2, the largest proteasome subunits, are linked by an extended connection. The structural features of the 26S proteasome observed in this study are likely to be important for coordinating the proteasomal subunits during substrate processing.

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AAA+ ATPases in Protein Degradation: Structures, Functions and Mechanisms

Biomolecules ◽

10.3390/biom10040629 ◽

2020 ◽

Vol 10 (4) ◽

pp. 629 ◽

Cited By ~ 5

Author(s):

Shuwen Zhang ◽

Youdong Mao

Keyword(s):

Protein Degradation ◽

Conformational Changes ◽

Dynamic Models ◽

Three Dimensional ◽

26S Proteasome ◽

Aaa Atpase ◽

Substrate Protein ◽

Function Relationship ◽

Atp Binding And Hydrolysis ◽

Aaa Atpases

Adenosine triphosphatases (ATPases) associated with a variety of cellular activities (AAA+), the hexameric ring-shaped motor complexes located in all ATP-driven proteolytic machines, are involved in many cellular processes. Powered by cycles of ATP binding and hydrolysis, conformational changes in AAA+ ATPases can generate mechanical work that unfolds a substrate protein inside the central axial channel of ATPase ring for degradation. Three-dimensional visualizations of several AAA+ ATPase complexes in the act of substrate processing for protein degradation have been resolved at the atomic level thanks to recent technical advances in cryogenic electron microscopy (cryo-EM). Here, we summarize the resulting advances in structural and biochemical studies of AAA+ proteases in the process of proteolysis reactions, with an emphasis on cryo-EM structural analyses of the 26S proteasome, Cdc48/p97 and FtsH-like mitochondrial proteases. These studies reveal three highly conserved patterns in the structure–function relationship of AAA+ ATPase hexamers that were observed in the human 26S proteasome, thus suggesting common dynamic models of mechanochemical coupling during force generation and substrate translocation.

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Structural Basis of Ist1 Function and Ist1–Did2 Interaction in the Multivesicular Body Pathway and Cytokinesis

Molecular Biology of the Cell ◽

10.1091/mbc.e09-05-0403 ◽

2009 ◽

Vol 20 (15) ◽

pp. 3514-3524 ◽

Cited By ~ 67

Author(s):

Junyu Xiao ◽

Xiao-Wei Chen ◽

Brian A. Davies ◽

Alan R. Saltiel ◽

David J. Katzmann ◽

...

Keyword(s):

Crystal Structure ◽

Mechanism Of Action ◽

Intramolecular Interaction ◽

Multivesicular Body ◽

Structural Motif ◽

Structural Basis ◽

Membrane Deformation ◽

Aaa Atpase ◽

Cellular Processes ◽

Escrt Machinery

The ESCRT machinery functions in several important eukaryotic cellular processes. The AAA-ATPase Vps4 catalyzes disassembly of the ESCRT-III complex and may regulate membrane deformation and vesicle scission as well. Ist1 was proposed to be a regulator of Vps4, but its mechanism of action was unclear. The crystal structure of the N-terminal domain of Ist1 (Ist1NTD) reveals an ESCRT-III subunit-like fold, implicating Ist1 as a divergent ESCRT-III family member. Ist1NTD specifically binds to the ESCRT-III subunit Did2, and cocrystallization of Ist1NTD with a Did2 fragment shows that Ist1 interacts with the Did2 C-terminal MIM1 (MIT-interacting motif 1) via a novel MIM-binding structural motif. This arrangement indicates a mechanism for intermolecular ESCRT-III subunit association and may also suggest one form of ESCRT-III subunit autoinhibition via intramolecular interaction.

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Deep manifold learning reveals hidden dynamics of proteasome autoregulation

10.1101/2020.12.22.423932 ◽

2020 ◽

Author(s):

Zhaolong Wu ◽

Shuwen Zhang ◽

Wei Li Wang ◽

Yinping Ma ◽

Yuanchen Dong ◽

...

Keyword(s):

Manifold Learning ◽

Conformational Changes ◽

26S Proteasome ◽

Nucleophilic Attack ◽

Nucleotide Exchange ◽

Polyubiquitin Chains ◽

Aaa Atpase ◽

Cellular Processes ◽

Systemic Analysis ◽

Manifold Embedding

AbstractThe 2.5-MDa 26S proteasome maintains proteostasis and regulates myriad cellular processes1. How polyubiquitylated substrate interactions regulate proteasome activity is not understood1,2. Here we introduce a deep manifold learning framework, named AlphaCryo4D, which enables atomic-level cryogenic electron microscopy (cryo-EM) reconstructions of nonequilibrium conformational continuum and reconstitutes ‘hidden’ dynamics of proteasome autoregulation in the act of substrate degradation. AlphaCryo4D integrates 3D deep residual learning3 with manifold embedding4 of free-energy landscapes5, which directs 3D clustering via an energy-based particle-voting algorithm. In blind assessments using simulated heterogeneous cryo-EM datasets, AlphaCryo4D achieved 3D classification accuracy three times that of conventional methods6–9 and reconstructed continuous conformational changes of a 130-kDa protein at sub-3-Å resolution. By using AlphaCryo4D to analyze a single experimental cryo-EM dataset2, we identified 64 conformers of the substrate-bound human 26S proteasome, revealing conformational entanglement of two regulatory particles in the doubly capped holoenzymes and their energetic differences with singly capped ones. Novel ubiquitin-binding sites are discovered on the RPN2, RPN10 and α5 subunits to remodel polyubiquitin chains for deubiquitylation and recycle. Importantly, AlphaCryo4D choreographs single-nucleotide-exchange dynamics of proteasomal AAA-ATPase motor during translocation initiation, which upregulates proteolytic activity by allosterically promoting nucleophilic attack. Our systemic analysis illuminates a grand hierarchical allostery for proteasome autoregulation.

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