Interaction between the AAA+ATPase p97 and its cofactor ataxin3 in health and disease: Nucleotide-induced conformational changes regulate cofactor binding

p97, a member of the AAA (ATPase Associated with various Activities) ATPase family, is essential and centrally involved in a wide variety of cellular processes. Single amino-acid substitutions in p97 have been associated with the severe degenerative disorder of Inclusion Body Myopathy associated with Paget disease of bone and Frontotemporal Dementia (IBMPFD) as well as amytropic leteral sclerosis (ALS). Current models propose that p97 acts as a motor transmitting the energy from the ATPase cycle to conformational changes of substrate protein complexes causing segregation, remodeling or translocation. Mutations at the interface between the N and the D1 domains impact the ATPase activity and the conformation of D2 on the opposite side of the protein complex, suggesting intermolecular communication. Because of limited structural information, the molecular mechanisms on how p97 drives its activities and the molecular basis for transmission of information within the molecule remain elusive. Structural heterogeneity is observed in vitro and is likely relevant for the in vivo biological function of p97. Single particle cryo-EM is the method of choice to study a flexible complex. The technique allows study in solution and also deals with sample heterogeneity by image classification. We have set-up the characterization of the conformational heterogeneity in WT and disease relevant p97 mutant using multi-likelihood classification and Hybrid Electron Microscopy Normal Mode Analysis HEMNMA. The multi-likelihood analysis shows a link between the conformations of the N and D2 domains. HEMNMA allows the analysis of the asymmetry of the conformational changes. Together these studies describe the structural flexibility of p97 and the coupling of the ATPase activity with conformational changes in health and in disease. Study of this model system also allows the development of new methods to understand the conformational heterogeneity of other protein complexes.

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Inter-ring rotations of AAA ATPase p97 revealed by electron cryomicroscopy

Open Biology ◽

10.1098/rsob.130142 ◽

2014 ◽

Vol 4 (3) ◽

pp. 130142 ◽

Cited By ~ 20

Author(s):

Heidi O. Yeung ◽

Andreas Förster ◽

Cecilia Bebeacua ◽

Hajime Niwa ◽

Caroline Ewens ◽

...

Keyword(s):

Conformational Changes ◽

Protein Complexes ◽

Cell Cycle Control ◽

Conformational Heterogeneity ◽

Electron Cryomicroscopy ◽

Aaa Atpase ◽

Protein Substrates ◽

Ubiquitin System ◽

Aaa Protein ◽

Ubiquitylated Protein

The type II AAA+ protein p97 is involved in numerous cellular activities, including endoplasmic reticulum-associated degradation, transcription activation, membrane fusion and cell-cycle control. These activities are at least in part regulated by the ubiquitin system, in which p97 is thought to target ubiquitylated protein substrates within macromolecular complexes and assist in their extraction or disassembly. Although ATPase activity is essential for p97 function, little is known about how ATP binding or hydrolysis is coupled with p97 conformational changes and substrate remodelling. Here, we have used single-particle electron cryomicroscopy (cryo-EM) to study the effect of nucleotides on p97 conformation. We have identified conformational heterogeneity within the cryo-EM datasets from which we have resolved two major p97 conformations. A comparison of conformations reveals inter-ring rotations upon nucleotide binding and hydrolysis that may be linked to the remodelling of target protein complexes.

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Voltage clamp fluorimetry studies of mammalian voltage-gated K+ channel gating

Biochemical Society Transactions ◽

10.1042/bst0351080 ◽

2007 ◽

Vol 35 (5) ◽

pp. 1080-1082 ◽

Cited By ~ 14

Author(s):

T.W. Claydon ◽

D. Fedida

Keyword(s):

Ion Channel ◽

Potassium Channel ◽

Real Time ◽

Voltage Clamp ◽

Conformational Changes ◽

Channel Gating ◽

K Channel ◽

Voltage Gated ◽

Kv Channels ◽

Health And Disease

VCF (voltage clamp fluorimetry) provides a powerful technique to observe real-time conformational changes that are associated with ion channel gating. The present review highlights the insights such experiments have provided in understanding Kv (voltage-gated potassium) channel gating, with particular emphasis on the study of mammalian Kv1 channels. Further applications of VCF that would contribute to our understanding of the modulation of Kv channels in health and disease are also discussed.

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Structural characterization of the apo form and NADH binary complex of human lactate dehydrogenase

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004714005422 ◽

2014 ◽

Vol 70 (5) ◽

pp. 1484-1490 ◽

Cited By ~ 12

Author(s):

Sally Dempster ◽

Stephen Harper ◽

John E. Moses ◽

Ingrid Dreveny

Keyword(s):

Lactate Dehydrogenase ◽

Conformational Changes ◽

Active Site ◽

Anaerobic Respiration ◽

Crystal Form ◽

Binary Complex ◽

Cofactor Binding ◽

Key Enzyme ◽

Crystallization Conditions

Lactate dehydrogenase A (LDH-A) is a key enzyme in anaerobic respiration that is predominantly found in skeletal muscle and catalyses the reversible conversion of pyruvate to lactate in the presence of NADH. LDH-A is overexpressed in many tumours and has therefore emerged as an attractive target for anticancer drug discovery. Crystal structures of human LDH-A in the presence of inhibitors have been described, but currently no structures of the apo or binary NADH-bound forms are available for any mammalian LDH-A. Here, the apo structure of human LDH-A was solved at a resolution of 2.1 Å in space groupP4122. The active-site loop adopts an open conformation and the packing and crystallization conditions suggest that the crystal form is suitable for soaking experiments. The soaking potential was assessed with the cofactor NADH, which yielded a ligand-bound crystal structure in the absence of any inhibitors. The structures show that NADH binding induces small conformational changes in the active-site loop and an adjacent helix. A comparison with other eukaryotic apo LDH structures reveals the conservation of intra-loop interactions. The structures provide novel insight into cofactor binding and provide the foundation for soaking experiments with fragments and inhibitors.

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Conformational changes of the multifunction p97 AAA ATPase during its ATPase cycle

Natural Structural Biology ◽

10.1038/nsb872 ◽

2002 ◽

Vol 9 (12) ◽

pp. 950-957 ◽

Cited By ~ 156

Author(s):

Isabelle Rouiller ◽

Byron DeLaBarre ◽

Andrew P. May ◽

William I. Weis ◽

Axel T. Brunger ◽

...

Keyword(s):

Conformational Changes ◽

Aaa Atpase

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Specific lid-base contacts in the 26s proteasome control the conformational switching required for substrate degradation

eLife ◽

10.7554/elife.49806 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 5

Author(s):

Eric R Greene ◽

Ellen A Goodall ◽

Andres H de la Peña ◽

Mary E Matyskiela ◽

Gabriel C Lander ◽

...

Keyword(s):

Conformational Changes ◽

Degradation Process ◽

26S Proteasome ◽

Conformational Switching ◽

Conformational Switch ◽

Atpase Subunit ◽

Aaa Atpase ◽

Early Degradation ◽

Processing Steps ◽

Insight Into

The 26S proteasome is essential for proteostasis and the regulation of vital processes through ATP-dependent degradation of ubiquitinated substrates. To accomplish the multi-step degradation process, the proteasome’s regulatory particle, consisting of lid and base subcomplexes, undergoes major conformational changes whose origin is unknown. Investigating the Saccharomyces cerevisiae proteasome, we found that peripheral interactions between the lid subunit Rpn5 and the base AAA+ ATPase ring are important for stabilizing the substrate-engagement-competent state and coordinating the conformational switch to processing states upon substrate engagement. Disrupting these interactions perturbs the conformational equilibrium and interferes with degradation initiation, while later processing steps remain unaffected. Similar defects in early degradation steps are observed when eliminating hydrolysis in the ATPase subunit Rpt6, whose nucleotide state seems to control proteasome conformational transitions. These results provide important insight into interaction networks that coordinate conformational changes with various stages of degradation, and how modulators of conformational equilibria may influence substrate turnover.

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The CryoEM Structure of the Ribosome Maturation Factor Rea1

10.1101/455634 ◽

2018 ◽

Author(s):

Piotr Sosnowski ◽

Linas Urnavicius ◽

Andreas Boland ◽

Robert Fagiewicz ◽

Johan Busselez ◽

...

Keyword(s):

Amino Acid ◽

Atpase Activity ◽

Conformational Changes ◽

Ribosomal Subunit ◽

Force Generation ◽

Docking Site ◽

Aaa Atpase ◽

Helical Bundle ◽

Maturation Factor ◽

Assembly Factors

AbstractThe biogenesis of the 60S ribosomal subunit is initiated in the nucleus where rRNAs and proteins form pre-60S particles. These pre-60S particles mature by transiently interacting with various assembly factors. The ~5000 amino-acid AAA+ ATPase Rea1 (or Midasin) generates force to mechanically remove assembly factors from pre-60S particles, which promotes their export to the cytosol. Here we present three Rea1 cryoEM structures. We visualize the Rea1 engine, a hexameric ring of AAA+ domains, and identify an α-helical bundle of AAA2 as a major ATPase activity regulator. The α-helical bundle interferes with nucleotide induced conformational changes that create a docking site for the substrate binding MIDAS domain of Rea1 on the AAA+ ring. Furthermore, we reveal the architecture of the Rea1 linker, which is involved in force generation and extends from the AAA+ ring. The data presented here provide insights into the mechanism of one of the most complex ribosome maturation factors.

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