Nonequilibrium Isotope Exchange Reveals a Catalytically Significant Enzyme–Phosphate Complex in the ATP Hydrolysis Pathway of the AAA+ ATPase Magnesium Chelatase

bEBPs (bacterial enhancer-binding proteins) are AAA+ (ATPase associated with various cellular activities) transcription activators that activate gene transcription through a specific bacterial σ factor, σ54. σ54–RNAP (RNA polymerase) binds to promoter DNA sites and forms a stable closed complex, unable to proceed to transcription. The closed complex must be remodelled using energy from ATP hydrolysis provided by bEBPs to melt DNA and initiate transcription. Recently, large amounts of structural and biochemical data have produced insights into how ATP hydrolysis within the active site of bEBPs is coupled to the re-modelling of the closed complex. In the present article, we review some of the key nucleotides, mutations and techniques used and how they have contributed towards our understanding of the function of bEBPs.

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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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Long-range intramolecular allostery and regulation in the dynein-like AAA protein Mdn1

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2002792117 ◽

2020 ◽

Vol 117 (31) ◽

pp. 18459-18469

Author(s):

Keith J. Mickolajczyk ◽

Paul Dominic B. Olinares ◽

Yiming Niu ◽

Nan Chen ◽

Sara E. Warrington ◽

...

Keyword(s):

Electron Microscopy ◽

Mass Spectrometry ◽

Protein Function ◽

Atp Hydrolysis ◽

Native Mass Spectrometry ◽

Chemical Probes ◽

Rich Region ◽

Aaa Atpase ◽

Aaa Protein ◽

Bimolecular Interaction

Mdn1 is an essential mechanoenzyme that uses the energy from ATP hydrolysis to physically reshape and remodel, and thus mature, the 60S subunit of the ribosome. This massive (>500 kDa) protein has an N-terminal AAA (ATPase associated with diverse cellular activities) ring, which, like dynein, has six ATPase sites. The AAA ring is followed by large (>2,000 aa) linking domains that include an ∼500-aa disordered (D/E-rich) region, and a C-terminal substrate-binding MIDAS domain. Recent models suggest that intramolecular docking of the MIDAS domain onto the AAA ring is required for Mdn1 to transmit force to its ribosomal substrates, but it is not currently understood what role the linking domains play, or why tethering the MIDAS domain to the AAA ring is required for protein function. Here, we use chemical probes, single-particle electron microscopy, and native mass spectrometry to study the AAA and MIDAS domains separately or in combination. We find that Mdn1 lacking the D/E-rich and MIDAS domains retains ATP and chemical probe binding activities. Free MIDAS domain can bind to the AAA ring of this construct in a stereo-specific bimolecular interaction, and, interestingly, this binding reduces ATPase activity. Whereas intramolecular MIDAS docking appears to require a treatment with a chemical inhibitor or preribosome binding, bimolecular MIDAS docking does not. Hence, tethering the MIDAS domain to the AAA ring serves to prevent, rather than promote, MIDAS docking in the absence of inducing signals.

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

Structure ◽

10.1016/j.str.2015.10.026 ◽

2016 ◽

Vol 24 (1) ◽

pp. 127-139 ◽

Cited By ~ 35

Author(s):

Petra Hänzelmann ◽

Hermann Schindelin

Keyword(s):

Atp Hydrolysis ◽

Structural Basis ◽

Aaa Atpase

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Fundamental Characteristics of AAA+ Protein Family Structure and Function

Archaea ◽

10.1155/2016/9294307 ◽

2016 ◽

Vol 2016 ◽

pp. 1-12 ◽

Cited By ~ 34

Author(s):

Justin M. Miller ◽

Eric J. Enemark

Keyword(s):

Atp Hydrolysis ◽

Molecular Machines ◽

Ring Structure ◽

Structure And Function ◽

Central Channel ◽

Aaa Atpase ◽

Cellular Events ◽

Critical Components ◽

And Function ◽

Aaa Protein

Many complex cellular events depend on multiprotein complexes known as molecular machines to efficiently couple the energy derived from adenosine triphosphate hydrolysis to the generation of mechanical force. Members of the AAA+ ATPase superfamily (ATPases Associated with various cellular Activities) are critical components of many molecular machines. AAA+ proteins are defined by conserved modules that precisely position the active site elements of two adjacent subunits to catalyze ATP hydrolysis. In many cases, AAA+ proteins form a ring structure that translocates a polymeric substrate through the central channel using specialized loops that project into the central channel. We discuss the major features of AAA+ protein structure and function with an emphasis on pivotal aspects elucidated with archaeal proteins.

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Coordination of Substrate Binding and ATP Hydrolysis in Vps4-Mediated ESCRT-III Disassembly

Molecular Biology of the Cell ◽

10.1091/mbc.e10-06-0512 ◽

2010 ◽

Vol 21 (19) ◽

pp. 3396-3408 ◽

Cited By ~ 35

Author(s):

Brian A. Davies ◽

Ishara F. Azmi ◽

Johanna Payne ◽

Anna Shestakova ◽

Bruce F. Horazdovsky ◽

...

Keyword(s):

Atp Hydrolysis ◽

Substrate Binding ◽

Multivesicular Body ◽

Virus Budding ◽

Aaa Atpase ◽

Enveloped Virus ◽

Dynamic Assembly

ESCRT-III undergoes dynamic assembly and disassembly to facilitate membrane exvagination processes including multivesicular body (MVB) formation, enveloped virus budding, and membrane abscission during cytokinesis. The AAA-ATPase Vps4 is required for ESCRT-III disassembly, however the coordination of Vps4 ATP hydrolysis with ESCRT-III binding and disassembly is not understood. Vps4 ATP hydrolysis has been proposed to execute ESCRT-III disassembly as either a stable oligomer or an unstable oligomer whose dissociation drives ESCRT-III disassembly. An in vitro ESCRT-III disassembly assay was developed to analyze Vps4 function during this process. The studies presented here support a model in which Vps4 acts as a stable oligomer during ATP hydrolysis and ESCRT-III disassembly. Moreover, Vps4 oligomer binding to ESCRT-III induces coordination of ATP hydrolysis at the level of individual Vps4 subunits. These results suggest that Vps4 functions as a stable oligomer that acts upon individual ESCRT-III subunits to facilitate ESCRT-III disassembly.

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Toward an understanding of the Cdc48/p97 ATPase

F1000Research ◽

10.12688/f1000research.11683.1 ◽

2017 ◽

Vol 6 ◽

pp. 1318 ◽

Cited By ~ 50

Author(s):

Nicholas Bodnar ◽

Tom Rapoport

Keyword(s):

Atp Hydrolysis ◽

Critical Role ◽

Ring Structure ◽

Cellular Systems ◽

Aaa Atpase ◽

Double Ring ◽

Terminal Domain ◽

D2 Domain ◽

Central Pore

A conserved AAA+ ATPase, called Cdc48 in yeast and p97 or VCP in metazoans, plays an essential role in many cellular processes by segregating polyubiquitinated proteins from complexes or membranes. For example, in endoplasmic reticulum (ER)-associated protein degradation (ERAD), Cdc48/p97 pulls polyubiquitinated, misfolded proteins out of the ER and transfers them to the proteasome. Cdc48/p97 consists of an N-terminal domain and two ATPase domains (D1 and D2). Six Cdc48 monomers form a double-ring structure surrounding a central pore. Cdc48/p97 cooperates with a number of different cofactors, which bind either to the N-terminal domain or to the C-terminal tail. The mechanism of Cdc48/p97 action is poorly understood, despite its critical role in many cellular systems. Recent in vitro experiments using yeast Cdc48 and its heterodimeric cofactor Ufd1/Npl4 (UN) have resulted in novel mechanistic insight. After interaction of the substrate-attached polyubiquitin chain with UN, Cdc48 uses ATP hydrolysis in the D2 domain to move the polypeptide through its central pore, thereby unfolding the substrate. ATP hydrolysis in the D1 domain is involved in substrate release from the Cdc48 complex, which requires the cooperation of the ATPase with a deubiquitinase (DUB). Surprisingly, the DUB does not completely remove all ubiquitin molecules; the remaining oligoubiquitin chain is also translocated through the pore. Cdc48 action bears similarities to the translocation mechanisms employed by bacterial AAA ATPases and the eukaryotic 19S subunit of the proteasome, but differs significantly from that of a related type II ATPase, the NEM-sensitive fusion protein (NSF). Many questions about Cdc48/p97 remain unanswered, including how it handles well-folded substrate proteins, how it passes substrates to the proteasome, and how various cofactors modify substrates and regulate its function.

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Utilization of positional isotope exchange experiments to evaluate reversibility of ATP hydrolysis catalyzed by Escherichia coli Lon proteaseThis paper is one of a selection of papers published in this special issue entitled 8th International Conference on AAA Proteins and has undergone the Journal's usual peer review process.

Biochemistry and Cell Biology ◽

10.1139/o09-117 ◽

2010 ◽

Vol 88 (1) ◽

pp. 119-128 ◽

Cited By ~ 4

Author(s):

Jennifer Thomas ◽

Jennifer Fishovitz ◽

Irene Lee

Keyword(s):

Escherichia Coli ◽

Atpase Activity ◽

Isotope Exchange ◽

Atp Hydrolysis ◽

Peer Review Process ◽

Lon Protease ◽

Peptidase Activity ◽

E Coli ◽

Atpase Reaction ◽

Half Site

Lon protease, also known as protease La, is an ATP-dependent serine protease. Despite the presence of a proteolytic Ser–Lys dyad, the enzyme only catalyzes protein degradation in the presence of ATP. Lon possesses an intrinsic ATPase activity that is stimulated by protein and certain peptide substrates. Through sequence alignment and analysis, it is concluded that Lon belongs to the AAA+ protein family. Previous kinetic characterization of the ATPase domain of Escherichia coli Lon protease implicates a half-site reactivity model in which only 50% of the ATP bound to Lon are hydrolyzed to yield ADP; the remaining ATPase sites remain bound with ATP and are considered non-catalytic. In this model, it is implied that ATP hydrolysis is irreversible. To further evaluate the proposed half-site reactivity model, the reversibility of the ATPase activity of E. coli Lon was evaluated by positional isotope exchange experiments. The ATPase reactions were conducted in the 18O-enriched buffer such that the extent of 18O incorporation into inorganic phosphate generated from ATP hydrolysis could be used to evaluate the extent of reversibility in ATP hydrolysis. Collectively, our experimental data reveal that the ATPase reaction catalyzed by E. coli Lon in the presence and absence of peptide substrate that stimulated the enzyme’s ATPase activity is irreversible. Therefore, the half-site ATPase reactivity of E. coli Lon is validated, and can be used to account for the kinetic mechanism of the ATP-dependent peptidase activity of the enzyme.

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