Structural basis for dynamic regulation of the human 26S proteasome

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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Nucleotide-Driven Triple-State Remodeling of the AAA-ATPase Channel in the Activated Human 26S Proteasome

10.1101/132613 ◽

2017 ◽

Cited By ~ 1

Author(s):

Yanan Zhu ◽

Wei Li Wang ◽

Daqi Yu ◽

Qi Ouyang ◽

Ying Lu ◽

...

Keyword(s):

Conformational Changes ◽

26S Proteasome ◽

Eukaryotic Cells ◽

Alternative States ◽

Nucleotide Exchange ◽

Core Particle ◽

Aaa Atpase ◽

The Core ◽

Gate Opening ◽

Proteolytic Chamber

SUMMARYThe proteasome is a sophisticated ATP-dependent molecular machine responsible for protein degradation in all eukaryotic cells. It remains elusive how conformational changes of the AAA-ATPase unfoldase in the regulatory particle (RP) control the gating of substrate-translocation channel to the proteolytic chamber of the core particle (CP). Here we report three alternative states of the ATP-γS-bound human proteasome, in which the CP gate is asymmetrically open, visualized by cryo-EM at near-atomic resolutions. Only four nucleotides are stably bound to the AAA-ATPase ring in the open-gate states. Concerted nucleotide exchange gives rise to a back-and-forth wobbling motion of the AAA-ATPase channel, coincident with remarkable transitions of their pore loops between the spiral staircase and saddle-shaped circle topologies. Gate opening in the CP is thus controlled with nucleotide-driven remodeling of the AAA-ATPase unfoldase. These findings demonstrate an elegant mechanism of allosteric coordination among sub-machines within the holoenzyme that is crucial for substrate translocation.

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Structural insights into the functional cycle of the ATPase module of the 26S proteasome

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1621129114 ◽

2017 ◽

Vol 114 (6) ◽

pp. 1305-1310 ◽

Cited By ~ 88

Author(s):

Marc Wehmer ◽

Till Rudack ◽

Florian Beck ◽

Antje Aufderheide ◽

Günter Pfeifer ◽

...

Keyword(s):

Atp Hydrolysis ◽

Ubiquitin Proteasome System ◽

26S Proteasome ◽

Core Particle ◽

Nucleotide Analogs ◽

The Core ◽

Cryo Electron Microscopy ◽

Intracellular Proteins ◽

Ubiquitin Proteasome ◽

Structural Insights

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.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1817752116 ◽

2018 ◽

Vol 116 (2) ◽

pp. 534-539 ◽

Cited By ~ 35

Author(s):

Parijat Majumder ◽

Till Rudack ◽

Florian Beck ◽

Radostin Danev ◽

Günter Pfeifer ◽

...

Keyword(s):

26S Proteasome ◽

Structural Basis ◽

Particle Analysis ◽

Aaa Atpase ◽

Cellular Processes ◽

Domains Of Life ◽

Atpase Subunits ◽

Domain Motions ◽

And Control ◽

Aaa Atpases

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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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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Substrate-engaged 26S proteasome structures reveal mechanisms for ATP-hydrolysis–driven translocation

Science ◽

10.1126/science.aav0725 ◽

2018 ◽

Vol 362 (6418) ◽

pp. eaav0725 ◽

Cited By ~ 109

Author(s):

Andres H. de la Peña ◽

Ellen A. Goodall ◽

Stephanie N. Gates ◽

Gabriel C. Lander ◽

Andreas Martin

Keyword(s):

Electron Microscopy ◽

Conformational Changes ◽

Adenosine Triphosphatase ◽

Atp Hydrolysis ◽

26S Proteasome ◽

Phosphate Release ◽

Protein Substrates ◽

Cellular Processes ◽

Cryo Electron Microscopy ◽

Aaa Atpases

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.

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Structural Basis for Katanin Self-Assembly

10.1101/197558 ◽

2017 ◽

Author(s):

Stanley Nithiananatham ◽

Francis J. McNally ◽

Jawdat Al-Bassam

Keyword(s):

Conformational Changes ◽

Self Assembly ◽

Bound States ◽

Atp Hydrolysis ◽

Regulatory Subunit ◽

Structural Basis ◽

Inhibition Mechanism ◽

Atpase Subunit ◽

Aaa Atpase ◽

Microtubule Severing

SUMMARYThe reorganization of microtubules in mitosis, meiosis and development requires the microtubule-severing activity of katanin. Katanin is composed of a AAA ATPase subunit and a regulatory subunit. Microtubule severing requires ATP hydrolysis by katanin’s conserved AAA ATPase domains. Whereas other AAA ATPases form stable hexamers, we show that wild-type katanin only forms heterodimers and heterotetramers. Heterododecamers were only observed for an ATP hydrolysis deficient mutant in the presence of ATP, suggesting an auto-inhibition mechanism that prevents oligomerization. X-ray structures of katanin’s AAA ATPase in monomeric nucleotide-free and pseudo-oligomeric ADP-bound states reveal conformational changes in AAA subdomains and N and C-terminal expansion segments that explain this auto-inhibition of assembly. These data lead to a model in which self-inhibited heterodimers bind to a microtubule, then transition into an assembly-competent conformation upon ATP binding. Microtubule-bound heterododecamers then promote tubulin extraction from the microtubule prior to oligomer dissociation.

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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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Structural basis for DEAH-helicase activation by G-patch proteins

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1913880117 ◽

2020 ◽

Vol 117 (13) ◽

pp. 7159-7170 ◽

Cited By ~ 2

Author(s):

Michael K. Studer ◽

Lazar Ivanović ◽

Marco E. Weber ◽

Sabrina Marti ◽

Stefanie Jonas

Keyword(s):

Conformational Changes ◽

Ribosome Biogenesis ◽

Rna Binding ◽

Atp Hydrolysis ◽

Protein Complexes ◽

Adenosine Diphosphate ◽

Structural Basis ◽

Rna Helicases ◽

Catalytic Core ◽

Terminal Domains

RNA helicases of the DEAH/RHA family are involved in many essential cellular processes, such as splicing or ribosome biogenesis, where they remodel large RNA–protein complexes to facilitate transitions to the next intermediate. DEAH helicases couple adenosine triphosphate (ATP) hydrolysis to conformational changes of their catalytic core. This movement results in translocation along RNA, which is held in place by auxiliary C-terminal domains. The activity of DEAH proteins is strongly enhanced by the large and diverse class of G-patch activators. Despite their central roles in RNA metabolism, insight into the molecular basis of G-patch–mediated helicase activation is missing. Here, we have solved the structure of human helicase DHX15/Prp43, which has a dual role in splicing and ribosome assembly, in complex with the G-patch motif of the ribosome biogenesis factor NKRF. The G-patch motif binds in an extended conformation across the helicase surface. It tethers the catalytic core to the flexibly attached C-terminal domains, thereby fixing a conformation that is compatible with RNA binding. Structures in the presence or absence of adenosine diphosphate (ADP) suggest that motions of the catalytic core, which are required for ATP binding, are still permitted. Concomitantly, RNA affinity, helicase, and ATPase activity of DHX15 are increased when G-patch is bound. Mutations that detach one end of the tether but maintain overall binding severely impair this enhancement. Collectively, our data suggest that the G-patch motif acts like a flexible brace between dynamic portions of DHX15 that restricts excessive domain motions but maintains sufficient flexibility for catalysis.

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Stoichiometry of Nucleotide Binding to Proteasome AAA+ ATPase Hexamer Established by Native Mass Spectrometry

Molecular & Cellular Proteomics ◽

10.1074/mcp.ra120.002067 ◽

2020 ◽

Vol 19 (12) ◽

pp. 1997-2014

Author(s):

Yadong Yu ◽

Haichuan Liu ◽

Zanlin Yu ◽

H. Ewa Witkowska ◽

Yifan Cheng

Keyword(s):

Protein Unfolding ◽

Atp Hydrolysis ◽

Mass Measurement ◽

Site Occupancy ◽

Internal Standard ◽

Large Family ◽

Nucleotide Binding ◽

Native Mass Spectrometry ◽

Aaa Atpase ◽

Aaa Atpases

AAA+ ATPases constitute a large family of proteins that are involved in a plethora of cellular processes including DNA disassembly, protein degradation and protein complex disassembly. They typically form a hexametric ring-shaped structure with six subunits in a (pseudo) 6-fold symmetry. In a subset of AAA+ ATPases that facilitate protein unfolding and degradation, six subunits cooperate to translocate protein substrates through a central pore in the ring. The number and type of nucleotides in an AAA+ ATPase hexamer is inherently linked to the mechanism that underlies cooperation among subunits and couples ATP hydrolysis with substrate translocation. We conducted a native MS study of a monodispersed form of PAN, an archaeal proteasome AAA+ ATPase, to determine the number of nucleotides bound to each hexamer of the WT protein. We utilized ADP and its analogs (TNP-ADP and mant-ADP), and a nonhydrolyzable ATP analog (AMP-PNP) to study nucleotide site occupancy within the PAN hexamer in ADP- and ATP-binding states, respectively. Throughout all experiments we used a Walker A mutant (PANK217A) that is impaired in nucleotide binding as an internal standard to mitigate the effects of residual solvation on mass measurement accuracy and to serve as a reference protein to control for nonspecific nucleotide binding. This approach led to the unambiguous finding that a WT PAN hexamer carried – from expression host – six tightly bound ADP molecules that could be exchanged for ADP and ATP analogs. Although the Walker A mutant did not bind ADP analogs, it did bind AMP-PNP, albeit at multiple stoichiometries. We observed variable levels of hexamer dissociation and an appearance of multimeric species with the over-charged molecular ion distributions across repeated experiments. We posit that these phenomena originated during ESI process at the final stages of ESI droplet evolution.

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Structural basis for inhibition of the AAA-ATPase Drg1 by diazaborine

Nature Communications ◽

10.1038/s41467-021-23854-x ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Michael Prattes ◽

Irina Grishkovskaya ◽

Victor-Valentin Hodirnau ◽

Ingrid Rössler ◽

Isabella Klein ◽

...

Keyword(s):

Ribosome Biogenesis ◽

Atp Hydrolysis ◽

Ribosomal Subunit ◽

Covalent Bond ◽

Resistance Mechanisms ◽

Drug Binding ◽

Structural Basis ◽

Aaa Atpase ◽

Maturation Factor ◽

Key Factor

AbstractThe hexameric AAA-ATPase Drg1 is a key factor in eukaryotic ribosome biogenesis and initiates cytoplasmic maturation of the large ribosomal subunit by releasing the shuttling maturation factor Rlp24. Drg1 monomers contain two AAA-domains (D1 and D2) that act in a concerted manner. Rlp24 release is inhibited by the drug diazaborine which blocks ATP hydrolysis in D2. The mode of inhibition was unknown. Here we show the first cryo-EM structure of Drg1 revealing the inhibitory mechanism. Diazaborine forms a covalent bond to the 2′-OH of the nucleotide in D2, explaining its specificity for this site. As a consequence, the D2 domain is locked in a rigid, inactive state, stalling the whole Drg1 hexamer. Resistance mechanisms identified include abolished drug binding and altered positioning of the nucleotide. Our results suggest nucleotide-modifying compounds as potential novel inhibitors for AAA-ATPases.

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