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

2015 ◽  
Vol 112 (20) ◽  
pp. 6371-6376 ◽  
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.

scholarly journals The AAA+ ATPase p97, a cellular multitool

2017 ◽  
Vol 474 (17) ◽  
pp. 2953-2976 ◽  
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.

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

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.

scholarly journals Structural basis of protein translocation by the Vps4-Vta1 AAA ATPase

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Nicole Monroe ◽  
Han Han ◽  
Peter S Shen ◽  
Wesley I Sundquist ◽  
Christopher P Hill
Keyword(s):  
Protein Translocation ◽  
Cellular Membrane ◽  
The Other ◽  
Structural Basis ◽  
Atp Binding ◽  
Fission Reactions ◽  
Aaa Atpase ◽  
Membrane Fission ◽  
Substrate Peptide ◽  
Aaa Atpases

Many important cellular membrane fission reactions are driven by ESCRT pathways, which culminate in disassembly of ESCRT-III polymers by the AAA ATPase Vps4. We report a 4.3 Å resolution cryo-EM structure of the active Vps4 hexamer with its cofactor Vta1, ADP·BeFx, and an ESCRT-III substrate peptide. Four Vps4 subunits form a helix whose interfaces are consistent with ATP binding, is stabilized by Vta1, and binds the substrate peptide. The fifth subunit approximately continues this helix but appears to be dissociating. The final Vps4 subunit completes a notched-washer configuration as if transitioning between the ends of the helix. We propose that ATP binding propagates growth at one end of the helix while hydrolysis promotes disassembly at the other end, so that Vps4 ‘walks’ along ESCRT-III until it encounters the ordered N-terminal domain to destabilize the ESCRT-III lattice. This model may be generally applicable to other protein-translocating AAA ATPases.

scholarly journals Structural basis for dynamic regulation of the human 26S proteasome

2016 ◽  
Vol 113 (46) ◽  
pp. 12991-12996 ◽  
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.

scholarly journals Biosynthesis of the dystonia-associated AAA+ ATPase torsinA at the endoplasmic reticulum

2006 ◽  
Vol 401 (2) ◽  
pp. 607-612 ◽  
Author(s):  
Anna C. Callan ◽  
Sandra Bunning ◽  
Owen T. Jones ◽  
Stephen High ◽  
Eileithyia Swanton
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Protein ◽  
Transmembrane Domain ◽  
Signal Sequence ◽  
Integral Membrane Protein ◽  
Signal Peptidase ◽  
Aaa Atpase ◽  
C Terminus ◽  
Membrane Spanning ◽  
Aaa Atpases

TorsinA is a widely expressed AAA+ (ATPases associated with various cellular activities) ATPase of unknown function. Previous studies have described torsinA as a type II protein with a cleavable signal sequence, a single membrane spanning domain, and its C-terminus located in the ER (endoplasmic reticulum) lumen. However, in the present study we show that torsinA is not in fact an integral membrane protein. Instead we find that the mature protein associates peripherally with the ER membrane, most likely through an interaction with an integral membrane protein. Consistent with this model, we provide evidence that the signal peptidase complex cleaves the signal sequence of torsinA, and we show that the region previously suggested to form a transmembrane domain is translocated into the lumen of the ER. The finding that torsinA is a peripheral, and not an integral membrane protein as previously thought, has important implications for understanding the function of this novel ATPase.

scholarly journals The regulatory function of the AAA4 ATPase domain of cytoplasmic dynein

2020 ◽  
Author(s):  
Xinglei Liu ◽  
Lu Rao ◽  
Arne Gennerich
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Essential Role ◽  
Cytoplasmic Dynein ◽  
Regulatory Function ◽  
Atpase Domain ◽  
Single Molecule Fluorescence ◽  
Mechanochemical Cycle ◽  
Aaa Atpases

AbstractCytoplasmic dynein is the primary motor for microtubule minus-end-directed transport and is indispensable to eukaryotic cells. Although each motor domain of dynein contains three active AAA+ ATPases (AAA1, 3, and 4), only the functions of AAA1 and 3 are known. Here, we use single-molecule fluorescence and optical tweezers studies to elucidate the role of AAA4 in dynein’s mechanochemical cycle. We demonstrate that AAA4 controls the priming stroke of the motion-generating linker, which connects the dimerizing tail of the motor to the AAA+ ring. Before ATP binds to AAA4, dynein remains incapable of generating motion. However, when AAA4 is bound to ATP, the gating of AAA1 by AAA3 prevails and dynein motion can occur. Thus, AAA1, 3, and 4 work together to regulate dynein function. Our work elucidates an essential role for AAA4 in dynein’s stepping cycle and underscores the complexity and crosstalk among the motor’s multiple AAA+ domains.

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

2020 ◽  
Vol 3 (1) ◽  
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.

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

2011 ◽  
Vol 286 (18) ◽  
pp. 15815-15820 ◽  
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.

scholarly journals Dynein activation in vivo is regulated by the nucleotide states of its AAA3 domain

2021 ◽  
Author(s):  
Rongde Qiu ◽  
Jun Zhang ◽  
Jeremy D. Rotty ◽  
Xin Xiang
Keyword(s):  
Atp Hydrolysis ◽  
Cytoplasmic Dynein ◽  
The Other ◽  
Atp Binding ◽  
Motor Activation ◽  
Early Endosomes ◽  
Arginine Finger ◽  
Atp Binding And Hydrolysis

SummaryCytoplasmic dynein is activated by dynactin and cargo adapters in vitro, and the activation also needs LIS1 (Lissencephaly 1) in vivo. How this process is regulated remains unclear. Here we found in Aspergillus nidulans that a dynein AAA4 arginine-finger mutation bypasses the requirement of LIS1 for dynein activation driven by the early endosomal adapter HookA. As the AAA4 arginine-finger is implicated in AAA3 ATP hydrolysis, we examined AAA3 mutants defective in ATP binding and hydrolysis respectively. Astonishingly, blocking AAA3 ATP hydrolysis allows dynein activation by dynactin in the absence of LIS1 or HookA. As a consequence, dynein accumulates at microtubule minus ends while early endosomes stay near the plus ends. On the other hand, blocking AAA3 ATP binding abnormally prevents LIS1 from being dissociated from dynein upon motor activation. Thus, the AAA3 ATPase cycle regulates the coordination between dynein activation and cargo binding as well as the dynamic dynein-LIS1 interaction.

scholarly journals The regulatory function of the AAA4 ATPase domain of cytoplasmic dynein

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Xinglei Liu ◽  
Lu Rao ◽  
Arne Gennerich
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Essential Role ◽  
Cytoplasmic Dynein ◽  
Regulatory Function ◽  
Atpase Domain ◽  
Single Molecule Fluorescence ◽  
Mechanochemical Cycle ◽  
Aaa Atpases

AbstractCytoplasmic dynein is the primary motor for microtubule minus-end-directed transport and is indispensable to eukaryotic cells. Although each motor domain of dynein contains three active AAA+ ATPases (AAA1, 3, and 4), only the functions of AAA1 and 3 are known. Here, we use single-molecule fluorescence and optical tweezers studies to elucidate the role of AAA4 in dynein’s mechanochemical cycle. We demonstrate that AAA4 controls the priming stroke of the motion-generating linker, which connects the dimerizing tail of the motor to the AAA+ ring. Before ATP binds to AAA4, dynein remains incapable of generating motion. However, when AAA4 is bound to ATP, the gating of AAA1 by AAA3 prevails and dynein motion can occur. Thus, AAA1, 3, and 4 work together to regulate dynein function. Our work elucidates an essential role for AAA4 in dynein’s stepping cycle and underscores the complexity and crosstalk among the motor’s multiple AAA+ domains.

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