Structural Basis of ATP Hydrolysis and Intersubunit Signaling in the AAA+ ATPase p97

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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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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Structural basis of nucleosome assembly by the Abo1 AAA+ ATPase histone chaperone

Nature Communications ◽

10.1038/s41467-019-13743-9 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 7

Author(s):

Carol Cho ◽

Juwon Jang ◽

Yujin Kang ◽

Hiroki Watanabe ◽

Takayuki Uchihashi ◽

...

Keyword(s):

High Speed ◽

Atp Hydrolysis ◽

Structural Features ◽

Structural Basis ◽

Dependent Manner ◽

Histone Chaperones ◽

Nucleosome Assembly ◽

Chromatin Dynamics ◽

Aaa Atpase

AbstractThe fundamental unit of chromatin, the nucleosome, is an intricate structure that requires histone chaperones for assembly. ATAD2 AAA+ ATPases are a family of histone chaperones that regulate nucleosome density and chromatin dynamics. Here, we demonstrate that the fission yeast ATAD2 homolog, Abo1, deposits histone H3–H4 onto DNA in an ATP-hydrolysis-dependent manner by in vitro reconstitution and single-tethered DNA curtain assays. We present cryo-EM structures of an ATAD2 family ATPase to atomic resolution in three different nucleotide states, revealing unique structural features required for histone loading on DNA, and directly visualize the transitions of Abo1 from an asymmetric spiral (ATP-state) to a symmetric ring (ADP- and apo-states) using high-speed atomic force microscopy (HS-AFM). Furthermore, we find that the acidic pore of ATP-Abo1 binds a peptide substrate which is suggestive of a histone tail. Based on these results, we propose a model whereby Abo1 facilitates H3–H4 loading by utilizing ATP.

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

10.7554/elife.24487.059 ◽

2017 ◽

Keyword(s):

Protein Translocation ◽

Structural Basis ◽

Aaa Atpase

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Kinetic and structural mechanism for DNA unwinding by a non-hexameric helicase

Nature Communications ◽

10.1038/s41467-021-27304-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Sean P. Carney ◽

Wen Ma ◽

Kevin D. Whitley ◽

Haifeng Jia ◽

Timothy M. Lohman ◽

...

Keyword(s):

Optical Tweezers ◽

Single Molecule ◽

Atp Hydrolysis ◽

Variable Number ◽

Structural Basis ◽

Variable Step Size ◽

Dna Unwinding ◽

Step Size ◽

Structural Mechanism ◽

Dynamics Simulations

AbstractUvrD, a model for non-hexameric Superfamily 1 helicases, utilizes ATP hydrolysis to translocate stepwise along single-stranded DNA and unwind the duplex. Previous estimates of its step size have been indirect, and a consensus on its stepping mechanism is lacking. To dissect the mechanism underlying DNA unwinding, we use optical tweezers to measure directly the stepping behavior of UvrD as it processes a DNA hairpin and show that UvrD exhibits a variable step size averaging ~3 base pairs. Analyzing stepping kinetics across ATP reveals the type and number of catalytic events that occur with different step sizes. These single-molecule data reveal a mechanism in which UvrD moves one base pair at a time but sequesters the nascent single strands, releasing them non-uniformly after a variable number of catalytic cycles. Molecular dynamics simulations point to a structural basis for this behavior, identifying the protein-DNA interactions responsible for strand sequestration. Based on structural and sequence alignment data, we propose that this stepping mechanism may be conserved among other non-hexameric helicases.

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Structural basis for the destabilization of F-actin by phosphate release following ATP hydrolysis

Journal of Molecular Biology ◽

10.1016/0022-2836(92)90520-t ◽

1992 ◽

Vol 227 (4) ◽

pp. 1043-1053 ◽

Cited By ~ 91

Author(s):

Albina Orlova ◽

Edward H. Egelman

Keyword(s):

Atp Hydrolysis ◽

Structural Basis ◽

Phosphate Release

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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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