Structural Mapping of Missense Mutations in the Pex1/Pex6 Complex

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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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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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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Cytoplasmic dynein regulates its attachment to microtubules via nucleotide state-switched mechanosensing at multiple AAA domains

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1417422112 ◽

2015 ◽

Vol 112 (20) ◽

pp. 6371-6376 ◽

Cited By ~ 70

Author(s):

Matthew P. Nicholas ◽

Florian Berger ◽

Lu Rao ◽

Sibylle Brenner ◽

Carol Cho ◽

...

Keyword(s):

Optical Tweezers ◽

Atp Hydrolysis ◽

Cytoplasmic Dynein ◽

Atp Binding ◽

Mechanical Tension ◽

Main Site ◽

Aaa Atpase ◽

C Terminus ◽

Directed Motility ◽

Aaa Atpases

Cytoplasmic dynein is a homodimeric microtubule (MT) motor protein responsible for most MT minus-end–directed motility. Dynein contains four AAA+ ATPases (AAA: ATPase associated with various cellular activities) per motor domain (AAA1–4). The main site of ATP hydrolysis, AAA1, is the only site considered by most dynein motility models. However, it remains unclear how ATPase activity and MT binding are coordinated within and between dynein’s motor domains. Using optical tweezers, we characterize the MT-binding strength of recombinant dynein monomers as a function of mechanical tension and nucleotide state. Dynein responds anisotropically to tension, binding tighter to MTs when pulled toward the MT plus end. We provide evidence that this behavior results from an asymmetrical bond that acts as a slip bond under forward tension and a slip-ideal bond under backward tension. ATP weakens MT binding and reduces bond strength anisotropy, and unexpectedly, so does ADP. Using nucleotide binding and hydrolysis mutants, we show that, although ATP exerts its effects via binding AAA1, ADP effects are mediated by AAA3. Finally, we demonstrate “gating” of AAA1 function by AAA3. When tension is absent or applied via dynein’s C terminus, ATP binding to AAA1 induces MT release only if AAA3 is in the posthydrolysis state. However, when tension is applied to the linker, ATP binding to AAA3 is sufficient to “open” the gate. These results elucidate the mechanisms of dynein–MT interactions, identify regulatory roles for AAA3, and help define the interplay between mechanical tension and nucleotide state in regulating dynein motility.

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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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Atomic structure of the mitochondrial inner membrane AAA+ protease YME1 reveals the mechanism of substrate processing

10.1101/189316 ◽

2017 ◽

Cited By ~ 4

Author(s):

Cristina Puchades ◽

Anthony J. Rampello ◽

Mia Shin ◽

Christopher J. Giuliano ◽

R. Luke Wiseman ◽

...

Keyword(s):

Large Scale ◽

Atp Hydrolysis ◽

Inner Mitochondrial Membrane ◽

Inner Membrane ◽

Conserved Residues ◽

Mitochondrial Inner Membrane ◽

Translocation Mechanism ◽

Substrate Processing ◽

Aaa Atpases ◽

Proteolytic Chamber

AbstractWe present the first atomic model of a substrate-bound inner mitochondrial membrane AAA+ quality control protease, YME1. Our ~3.4 Å cryo-EM structure reveals how the ATPases form a closed spiral staircase encircling an unfolded substrate, directing it toward the flat, symmetric protease ring. Importantly, the structure reveals how three coexisting nucleotide states allosterically induce distinct positioning of tyrosines in the central channel, resulting in substrate engagement and translocation to the negatively charged proteolytic chamber. This tight coordination by a network of conserved residues defines a sequential, around-the-ring ATP hydrolysis cycle that results in step-wise substrate translocation. Furthermore, we identify a hinge-like linker that accommodates the large-scale nucleotide-driven motions of the ATPase spiral independently of the contiguous planar proteolytic base. These results define the first molecular mechanism for a mitochondrial inner membrane AAA+ protease and reveal a translocation mechanism likely conserved for other AAA+ ATPases.

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Structural insights into ATP hydrolysis by the MoxR ATPase RavA and the LdcI-RavA cage-like complex

Communications Biology ◽

10.1038/s42003-020-0772-0 ◽

2020 ◽

Vol 3 (1) ◽

Cited By ~ 5

Author(s):

Matthew Jessop ◽

Benoit Arragain ◽

Roger Miras ◽

Angélique Fraudeau ◽

Karine Huard ◽

...

Keyword(s):

Crystal Structure ◽

Atp Hydrolysis ◽

Current Knowledge ◽

Structural Similarity ◽

Lysine Decarboxylase ◽

E Coli ◽

Aaa Atpase ◽

Closed Ring ◽

Aaa Atpases ◽

Structural Insights

AbstractThe hexameric MoxR AAA+ ATPase RavA and the decameric lysine decarboxylase LdcI form a 3.3 MDa cage, proposed to assist assembly of specific respiratory complexes in E. coli. Here, we show that inside the LdcI-RavA cage, RavA hexamers adopt an asymmetric spiral conformation in which the nucleotide-free seam is constrained to two opposite orientations. Cryo-EM reconstructions of free RavA reveal two co-existing structural states: an asymmetric spiral, and a flat C2-symmetric closed ring characterised by two nucleotide-free seams. The closed ring RavA state bears close structural similarity to the pseudo two-fold symmetric crystal structure of the AAA+ unfoldase ClpX, suggesting a common ATPase mechanism. Based on these structures, and in light of the current knowledge regarding AAA+ ATPases, we propose different scenarios for the ATP hydrolysis cycle of free RavA and the LdcI-RavA cage-like complex, and extend the comparison to other AAA+ ATPases of clade 7.

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Positive Cooperativity of the p97 AAA ATPase Is Critical for Essential Functions

Journal of Biological Chemistry ◽

10.1074/jbc.m110.201400 ◽

2011 ◽

Vol 286 (18) ◽

pp. 15815-15820 ◽

Cited By ~ 37

Author(s):

Shingo Nishikori ◽

Masatoshi Esaki ◽

Kunitoshi Yamanaka ◽

Shinya Sugimoto ◽

Teru Ogura

Keyword(s):

Caenorhabditis Elegans ◽

Atpase Activity ◽

Atp Hydrolysis ◽

Yeast Cells ◽

Growth Speed ◽

Hydrolysis Mechanism ◽

Aaa Atpase ◽

Physiological Temperature ◽

Positive Cooperativity ◽

Aaa Atpases

p97 is composed of two conserved AAA (ATPases associated with diverse cellular activities) domains, which form a tandem hexameric ring. We characterized the ATP hydrolysis mechanism of CDC-48.1, a p97 homolog of Caenorhabditis elegans. The ATPase activity of the N-terminal AAA domain was very low at physiological temperature, whereas the C-terminal AAA domain showed high ATPase activity in a coordinated fashion with positive cooperativity. The cooperativity and coordination are generated by different mechanisms because a noncooperative mutant still showed the coordination. Interestingly, the growth speed of yeast cells strongly related to the positive cooperativity rather than the ATPase activity itself, suggesting that the positive cooperativity is critical for the essential functions of p97.

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

10.21203/rs.3.rs-1181322/v1 ◽

2022 ◽

Author(s):

Ning Gao ◽

Chengying Ma ◽

Damu Wu ◽

Qian Chen

Keyword(s):

Atp Hydrolysis ◽

General Structure ◽

Ribosome Assembly ◽

Active Centers ◽

Independent Manner ◽

Aaa Atpase ◽

Assembly Factor ◽

Covalent Adducts ◽

Substrate Processing ◽

Aaa Protein

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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Analysis of the Structural Mechanism of ATP Inhibition at the AAA1 Subunit of Cytoplasmic Dynein-1 Using a Chemical “Toolkit”

International Journal of Molecular Sciences ◽

10.3390/ijms22147704 ◽

2021 ◽

Vol 22 (14) ◽

pp. 7704

Author(s):

Sayi’Mone Tati ◽

Laleh Alisaraie

Keyword(s):

Binding Affinity ◽

Atp Hydrolysis ◽

Critical Role ◽

Motor Protein ◽

Structural Data ◽

Retrograde Transport ◽

Cytoplasmic Dynein ◽

Motor Domain ◽

Conformational Search ◽

Structural Mechanism

Dynein is a ~1.2 MDa cytoskeletal motor protein that carries organelles via retrograde transport in eukaryotic cells. The motor protein belongs to the ATPase family of proteins associated with diverse cellular activities and plays a critical role in transporting cargoes to the minus end of the microtubules. The motor domain of dynein possesses a hexameric head, where ATP hydrolysis occurs. The presented work analyzes the structure–activity relationship (SAR) of dynapyrazole A and B, as well as ciliobrevin A and D, in their various protonated states and their 46 analogues for their binding in the AAA1 subunit, the leading ATP hydrolytic site of the motor domain. This study exploits in silico methods to look at the analogues’ effects on the functionally essential subsites of the motor domain of dynein 1, since no similar experimental structural data are available. Ciliobrevin and its analogues bind to the ATP motifs of the AAA1, namely, the walker-A (W-A) or P-loop, the walker-B (W-B), and the sensor I and II. Ciliobrevin A shows a better binding affinity than its D analogue. Although the double bond in ciliobrevin A and D was expected to decrease the ligand potency, they show a better affinity to the AAA1 binding site than dynapyrazole A and B, lacking the bond. In addition, protonation of the nitrogen atom in ciliobrevin A and D, as well as dynapyrazole A and B, at the N9 site of ciliobrevin and the N7 of the latter increased their binding affinity. Exploring ciliobrevin A geometrical configuration suggests the E isomer has a superior binding profile over the Z due to binding at the critical ATP motifs. Utilizing the refined structure of the motor domain obtained through protein conformational search in this study exhibits that Arg1852 of the yeast cytoplasmic dynein could involve in the “glutamate switch” mechanism in cytoplasmic dynein 1 in lieu of the conserved Asn in AAA+ protein family.

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Mitochondrial Lon protease is a gatekeeper for proteins newly imported into the matrix

Communications Biology ◽

10.1038/s42003-021-02498-z ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Yuichi Matsushima ◽

Kazuya Takahashi ◽

Song Yue ◽

Yuki Fujiyoshi ◽

Hideaki Yoshioka ◽

...

Keyword(s):

Protease Activity ◽

Matrix Protein ◽

Protein Import ◽

Atp Hydrolysis ◽

Lon Protease ◽

Mitochondrial Matrix ◽

The Matrix ◽

Specific Proteins ◽

Aggregation Activity ◽

Hydrolysis Activity

AbstractHuman ATP-dependent Lon protease (LONP1) forms homohexameric, ring-shaped complexes. Depletion of LONP1 causes aggregation of a broad range of proteins in the mitochondrial matrix and decreases the levels of their soluble forms. The ATP hydrolysis activity, but not protease activity, of LONP1 is critical for its chaperone-like anti-aggregation activity. LONP1 forms a complex with the import machinery and an incoming protein, and protein aggregation is linked with matrix protein import. LONP1 also contributes to the degradation of imported, aberrant, unprocessed proteins using its protease activity. Taken together, our results show that LONP1 functions as a gatekeeper for specific proteins imported into the mitochondrial matrix.

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