Structural dynamics of AAA+ ATPase Drg1 and mechanism of benzo-diazaborine inhibition

Abstract The AAA+ ATPase Drg1 is a ribosome assembly factor in yeast, and functions to release Rlp24, another assembly factor, from the pre-60S particle just exported from nucleus to initiate its further cytoplasmic maturation. Being a type II AAA+ protein with two ATPase domains (D1 and D2), its activity in ribosome assembly can be inhibited by a drug molecule diazaborine. In human, mutations of Drg1 homologue has been linked to a disease condition called epilepsy, hearing loss, and mental retardation syndrome. Although the general structure of Drg1 hexamer was recently reported, its complete structure and dynamic conformational rearrangements driven by ATP-hydrolysis are poorly understood. Here, we report a comprehensive structural characterization of Drg1 hexamers in different nucleotide-binding and benzo-diazaborine treated states. Our data show that Drg1 hexamers transits between two extreme conformations, characterized by a planar or helical arrangement of its six protomers. By forming covalent adducts with the ATP molecules in the active centers of both D1 and D2, benzo-diazaborine locks Drg1 hexamers in a more symmetric and non-productive conformation. In addition, we obtained the structure of a mutant Drg1 hexamer (Walker B mutations) with a polypeptide trapped in the central channel, representing a 3D snapshot of its functional, substrate-processing form. Conserved pore loops on the ATPase domains of Drg1 form a spiral staircase to interact with the substrate through a sequence-independent manner. These results suggest that Drg1, similar as Cdc48/p97, acts as a molecular unfoldase to remodel pre-60S particles, and benzo-diazaborine inhibits both the inter-protomer and inter-ring communication to disable the conformational cycling of Drg1 protomers required for the unfolding activity.

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From the common molecular basis of the AAA protein to various energy-dependent and -independent activities of AAA proteins

Biochemical Society Transactions ◽

10.1042/bst0360068 ◽

2008 ◽

Vol 36 (1) ◽

pp. 68-71 ◽

Cited By ~ 11

Author(s):

Teru Ogura ◽

Yuka Matsushita-Ishiodori ◽

Ai Johjima ◽

Masayo Nishizono ◽

Shingo Nishikori ◽

...

Keyword(s):

Atp Hydrolysis ◽

Protein Complexes ◽

Facilitated Diffusion ◽

Independent Manner ◽

Aromatic Residue ◽

Aaa Atpase ◽

Central Pore ◽

Aaa Protein

AAA (ATPase associated with various cellular activities) proteins remodel substrate proteins and protein complexes upon ATP hydrolysis. Substrate remodelling is diverse, e.g. proteolysis, unfolding, disaggregation and disassembly. In the oligomeric ring of the AAA protein, there is a conserved aromatic residue which lines the central pore. Functional analysis indicates that this conserved residue in AAA proteases is involved in threading unfolded polypeptides. Katanin and spastin have microtubule-severing activity. These AAA proteins also possess a conserved aromatic residue at the central pore, suggesting its importance in their biological activity. We have constructed pore mutants of these AAA proteins and have obtained in vivo and in vitro results indicating the functional importance of the pore motif. Degradation of casein by the Escherichia coli AAA protease, FtsH, strictly requires ATP hydrolysis. We have constructed several chimaeric proteases by exchanging domains of FtsH and its homologues from Caenorhabditis elegans mitochondria, and examined their ATPase and protease activities in vitro. Interestingly, it has been found that some chimaeras are able to degrade casein in an ATP-independent manner. The proteolysis is supported by either ATP[S] (adenosine 5′-[γ-thio]triphosphate) or ADP, as well as ATP. It is most likely that substrate translocation in these chimaeras occurs by facilitated diffusion. We have also investigated the roles of C. elegans p97 homologues in aggregation/disaggregation of polyglutamine repeats, and have found that p97 prevents filament formation of polyglutamine proteins in an ATP-independent fashion.

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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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The Crystal Structure of the Ubiquitin-like Domain of Ribosome Assembly Factor Ytm1 and Characterization of Its Interaction with the AAA-ATPase Midasin

Journal of Biological Chemistry ◽

10.1074/jbc.m115.693259 ◽

2015 ◽

Vol 291 (2) ◽

pp. 882-893 ◽

Cited By ~ 14

Author(s):

Erin M. Romes ◽

Mack Sobhany ◽

Robin E. Stanley

Keyword(s):

Crystal Structure ◽

Ribosome Assembly ◽

Aaa Atpase ◽

Assembly Factor

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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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Dynamic functional assembly of the Torsin AAA+ ATPase and its modulation by LAP1

Molecular Biology of the Cell ◽

10.1091/mbc.e17-05-0281 ◽

2017 ◽

Vol 28 (21) ◽

pp. 2765-2772 ◽

Cited By ~ 16

Author(s):

Anna R. Chase ◽

Ethan Laudermilch ◽

Jimin Wang ◽

Hideki Shigematsu ◽

Takeshi Yokoyama ◽

...

Keyword(s):

Atpase Activity ◽

Nuclear Envelope ◽

Atp Hydrolysis ◽

Important Class ◽

Oligomeric State ◽

Aaa Atpase ◽

Dynamic Assembly ◽

Aaa Protein

TorsinA is an essential AAA+ ATPase requiring LAP1 or LULL1 as cofactors. The dynamics of the Torsin/cofactor system remain poorly understood, with previous models invoking Torsin/cofactor assemblies with fixed stoichiometries. Here we demonstrate that TorsinA assembles into homotypic oligomers in the presence of ATP. Torsin variants mutated at the “back” interface disrupt homo-oligomerization but still show robust ATPase activity in the presence of its cofactors. These Torsin mutants are severely compromised in their ability to rescue nuclear envelope defects in Torsin-deficient cells, suggesting that TorsinA homo-oligomers play a key role in vivo. Engagement of the oligomer by LAP1 triggers ATP hydrolysis and rapid complex disassembly. Thus the Torsin complex is a highly dynamic assembly whose oligomeric state is tightly controlled by distinctively localized cellular cofactors. Our discovery that LAP1 serves as a modulator of the oligomeric state of an AAA+ protein establishes a novel means of regulating this important class of oligomeric ATPases.

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Structural Mapping of Missense Mutations in the Pex1/Pex6 Complex

International Journal of Molecular Sciences ◽

10.3390/ijms20153756 ◽

2019 ◽

Vol 20 (15) ◽

pp. 3756 ◽

Cited By ~ 2

Author(s):

Anne Schieferdecker ◽

Petra Wendler

Keyword(s):

Protein Import ◽

Atp Hydrolysis ◽

Structural Data ◽

Homology Model ◽

Hereditary Diseases ◽

Missense Mutations ◽

Stabilizing Agents ◽

Aaa Atpase ◽

Substrate Processing ◽

Aaa Atpases

Peroxisome biogenesis disorders (PBDs) are nontreatable hereditary diseases with a broad range of severity. Approximately 65% of patients are affected by mutations in the peroxins Pex1 and Pex6. The proteins form the heteromeric Pex1/Pex6 complex, which is important for protein import into peroxisomes. To date, no structural data are available for this AAA+ ATPase complex. However, a wealth of information can be transferred from low-resolution structures of the yeast scPex1/scPex6 complex and homologous, well-characterized AAA+ ATPases. We review the abundant records of missense mutations described in PBD patients with the aim to classify and rationalize them by mapping them onto a homology model of the human Pex1/Pex6 complex. Several mutations concern functionally conserved residues that are implied in ATP hydrolysis and substrate processing. Contrary to fold destabilizing mutations, patients suffering from function-impairing mutations may not benefit from stabilizing agents, which have been reported as potential therapeutics for PBD patients.

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Essential ribosome assembly factor Fap7 regulates a hierarchy of RNA-protein interactions during small ribosomal subunit biogenesis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1306389110 ◽

2013 ◽

Vol 110 (38) ◽

pp. 15253-15258 ◽

Cited By ~ 29

Author(s):

U. A. Hellmich ◽

B. L. Weis ◽

A. Lioutikov ◽

J. P. Wurm ◽

M. Kaiser ◽

...

Keyword(s):

Protein Interactions ◽

Ribosomal Subunit ◽

Ribosome Assembly ◽

Small Ribosomal Subunit ◽

Assembly Factor

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Cytoplasmic Recycling of 60S Preribosomal Factors Depends on the AAA Protein Drg1

Molecular and Cellular Biology ◽

10.1128/mcb.00668-07 ◽

2007 ◽

Vol 27 (19) ◽

pp. 6581-6592 ◽

Cited By ~ 66

Author(s):

Brigitte Pertschy ◽

Cosmin Saveanu ◽

Gertrude Zisser ◽

Alice Lebreton ◽

Martin Tengg ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Nuclear Export ◽

Ribosome Biogenesis ◽

Dominant Negative ◽

Cytoplasmic Localization ◽

Atpase Domain ◽

Aaa Atpase ◽

Cytoplasmic Accumulation ◽

Functional Inactivation ◽

Aaa Protein

ABSTRACT Allelic forms of DRG1/AFG2 confer resistance to the drug diazaborine, an inhibitor of ribosome biogenesis in Saccharomyces cerevisiae. Our results show that the AAA-ATPase Drg1 is essential for 60S maturation and associates with 60S precursor particles in the cytoplasm. Functional inactivation of Drg1 leads to an increased cytoplasmic localization of shuttling pre-60S maturation factors like Rlp24, Arx1, and Tif6. Surprisingly, Nog1, a nuclear pre-60S factor, was also relocalized to the cytoplasm under these conditions, suggesting that it is a previously unsuspected shuttling preribosomal factor that is exported with the precursor particles and very rapidly reimported. Proteins that became cytoplasmic under drg1 mutant conditions were blocked on pre-60S particles at a step that precedes the association of Rei1, a later-acting preribosomal factor. A similar cytoplasmic accumulation of Nog1 and Rlp24 in pre-60S-bound form could be seen after overexpression of a dominant-negative Drg1 variant mutated in the D2 ATPase domain. We conclude that the ATPase activity of Drg1 is required for the release of shuttling proteins from the pre-60S particles shortly after their nuclear export. This early cytoplasmic release reaction defines a novel step in eukaryotic ribosome maturation.

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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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