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

Open Biology ◽  
2014 ◽  
Vol 4 (3) ◽  
pp. 130142 ◽  
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.

scholarly journals Conformational heterogeneity of the AAA ATPase p97 characterized by single particle cryo-EM

2014 ◽  
Vol 70 (a1) ◽  
pp. C853-C853
Author(s):  
Driss Mountassif ◽  
Lucien Fabre ◽  
Kaustuv Basu ◽  
Mihnea Bostina ◽  
Slavica Jonic ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Conformational Changes ◽  
Single Particle ◽  
Molecular Mechanisms ◽  
Protein Complexes ◽  
Normal Mode Analysis ◽  
Single Amino Acid ◽  
Mode Analysis ◽  
Conformational Heterogeneity ◽  
Aaa Atpase

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.

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

2008 ◽  
Vol 36 (1) ◽  
pp. 68-71 ◽  
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.

scholarly journals Role of conformational heterogeneity in ligand recognition by viral RNA molecules

2021 ◽  
Author(s):  
Lev Levintov ◽  
Harish Vashisth
Keyword(s):  
Conformational Changes ◽  
Ribonucleic Acid ◽  
Viral Rna ◽  
Ligand Recognition ◽  
Conformational Heterogeneity ◽  
Rna Molecules ◽  
Environmental Stimuli

Ribonucleic acid (RNA) molecules are known to undergo conformational changes in response to various environmental stimuli including temperature, pH, and ligands. In particular, viral RNA molecules are a key example...

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

2007 ◽  
Vol 27 (19) ◽  
pp. 6581-6592 ◽  
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.

scholarly journals Periprotein lipidomes of Saccharomyces cerevisiae provide a flexible environment for conformational changes of membrane proteins

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Joury S van 't Klooster ◽  
Tan-Yun Cheng ◽  
Hendrik R Sikkema ◽  
Aike Jeucken ◽  
Branch Moody ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Small Molecules ◽  
Conformational Changes ◽  
Direct Detection ◽  
Protein Complexes ◽  
Lateral Diffusion ◽  
Low Ph ◽  
Lipid Particles ◽  
High Degree

Yeast tolerates a low pH and high solvent concentrations. The permeability of the plasma membrane (PM) for small molecules is low and lateral diffusion of proteins is slow. These findings suggest a high degree of lipid order, which raises the question of how membrane proteins function in such an environment. The yeast PM is segregated into the Micro-Compartment-of-Can1 (MCC) and Pma1 (MCP), which have different lipid compositions. We extracted proteins from these microdomains via stoichiometric capture of lipids and proteins in styrene-maleic-acid-lipid-particles (SMALPs). We purified SMALP-lipid-protein complexes by chromatography and quantitatively analyzed periprotein lipids located within the diameter defined by one SMALP. Phospholipid and sterol concentrations are similar for MCC and MCP, but sphingolipids are enriched in MCP. Ergosterol is depleted from this periprotein lipidome, whereas phosphatidylserine is enriched relative to the bulk of the plasma membrane. Direct detection of PM lipids in the 'periprotein space' supports the conclusion that proteins function in the presence of a locally disordered lipid state.

scholarly journals Simplified geometric representations of protein structures identify complementary interaction interfaces

2019 ◽  
Author(s):  
Caitlyn L. McCafferty ◽  
Edward M. Marcotte ◽  
David W. Taylor
Keyword(s):  
Conformational Changes ◽  
Protein Interaction ◽  
Protein Function ◽  
Large Scale ◽  
Predictive Accuracy ◽  
Protein Complexes ◽  
Protein Structures ◽  
Molecular Properties ◽  
3D Structures ◽  
Geometric Representations

ABSTRACTProtein-protein interactions are critical to protein function, but three-dimensional (3D) arrangements of interacting proteins have proven hard to predict, even given the identities and 3D structures of the interacting partners. Specifically, identifying the relevant pairwise interaction surfaces remains difficult, often relying on shape complementarity with molecular docking while accounting for molecular motions to optimize rigid 3D translations and rotations. However, such approaches can be computationally expensive, and faster, less accurate approximations may prove useful for large-scale prediction and assembly of 3D structures of multi-protein complexes. We asked if a reduced representation of protein geometry retains enough information about molecular properties to predict pairwise protein interaction interfaces that are tolerant of limited structural rearrangements. Here, we describe a cuboid transformation of 3D protein accessible surfaces on which molecular properties such as charge, hydrophobicity, and mutation rate can be easily mapped, implemented in the MorphProt package. Pairs of surfaces are compared to rapidly assess partner-specific potential surface complementarity. On two available benchmarks of 85 overall known protein complexes, we observed F1 scores (a weighted combination of precision and recall) of 19-34% at correctly identifying protein interaction surfaces, comparable to more computationally intensive 3D docking methods in the annual Critical Assessment of PRedicted Interactions. Furthermore, we examined the effect of molecular motion through normal mode simulation on a benchmark receptor-ligand pair and observed no marked loss of predictive accuracy for distortions of up to 6 Å RMSD. Thus, a cuboid transformation of protein surfaces retains considerable information about surface complementarity, offers enhanced speed of comparison relative to more complex geometric representations, and exhibits tolerance to conformational changes.

scholarly journals Regulation of Clathrin-Mediated Endocytosis

2018 ◽  
Vol 87 (1) ◽  
pp. 871-896 ◽  
Author(s):  
Marcel Mettlen ◽  
Ping-Hung Chen ◽  
Saipraveen Srinivasan ◽  
Gaudenz Danuser ◽  
Sandra L. Schmid
Keyword(s):  
Conformational Changes ◽  
Posttranslational Modifications ◽  
Mammalian Cells ◽  
Environmental Changes ◽  
Protein Complexes ◽  
Adaptor Protein ◽  
Endocytic Pathway ◽  
Coat Proteins ◽  
Membrane Composition ◽  
Coated Pits

Clathrin-mediated endocytosis (CME) is the major endocytic pathway in mammalian cells. It is responsible for the uptake of transmembrane receptors and transporters, for remodeling plasma membrane composition in response to environmental changes, and for regulating cell surface signaling. CME occurs via the assembly and maturation of clathrin-coated pits that concentrate cargo as they invaginate and pinch off to form clathrin-coated vesicles. In addition to the major coat proteins, clathrin triskelia and adaptor protein complexes, CME requires a myriad of endocytic accessory proteins and phosphatidylinositol lipids. CME is regulated at multiple steps—initiation, cargo selection, maturation, and fission—and is monitored by an endocytic checkpoint that induces disassembly of defective pits. Regulation occurs via posttranslational modifications, allosteric conformational changes, and isoform and splice-variant differences among components of the CME machinery, including the GTPase dynamin. This review summarizes recent findings on the regulation of CME and the evolution of this complex process.

Abstract 85: The AAA-ATPase p97 is a Critical Regulator of Cardiac Homeostasis

2017 ◽  
Vol 121 (suppl_1) ◽  
Author(s):  
Matthew J Brody ◽  
Michelle A Sargent ◽  
Jeffery D Molkentin
Keyword(s):  
Quality Control ◽  
Protein Quality ◽  
Protein Complexes ◽  
Protein Quality Control ◽  
Ubiquitin Proteasome System ◽  
Cellular Protein ◽  
Dominant Negative ◽  
Aaa Atpase ◽  
And Function ◽  
Thrombospondin 4

p97 is a AAA-ATPase that plays critical roles in a myriad of cellular protein quality control processes, including the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway that targets misfolded proteins in the ER for degradation in the cytosol by the ubiquitin proteasome system. Mutations in p97 cause a multisystem degenerative proteinopathy disorder called inclusion body myopathy with Paget disease of bone and frontotemporal dementia (IBMPFD) that includes pathologies of the nervous system, skeletal muscle, bone, and heart. Previous studies in the laboratory into the mechanisms whereby thrombospondin 4 has its cardioprotective effects and enhanced ERAD activity identified p97 as a direct interacting partner. This observation suggested that p97 itself could be an important cardioprotective effector by benefiting protein quality control in the heart. To address this hypothesis here we generated cardiac-specific transgenic mice overexpressing wildtype p97 or a p97 K524A mutant with deficient ATPase activity, the latter of which functioned as a dominant negative. Mice overexpressing wildtype p97 exhibit normal cardiac structure and function while mutant p97 overexpressing mice develop cardiomyopathy, upregulate several ERAD complex components, and have elevated levels of ubiquitinated proteins. Proteomics and immunoprecipitation assays identified overwhelming interactions between endogenous p97 and a number of interesting protein complexes that suggest unique functions for this protein in regulating protein quality control in the heart. The results and novel regulatory relationships will be presented, which suggests entirely unique pathways whereby p97 functions in the heart.

scholarly journals Structure and mechanism of the ESCRT pathway AAA+ ATPase Vps4

2019 ◽  
Vol 47 (1) ◽  
pp. 37-45 ◽  
Author(s):  
Han Han ◽  
Christopher P. Hill
Keyword(s):  
Cell Biology ◽  
Cellular Membrane ◽  
Structural Features ◽  
Transport Pathways ◽  
Endosomal Sorting ◽  
Aaa Atpase ◽  
Protein Substrates ◽  
Membrane Fission ◽  
Aaa Atpases ◽  
Equivalent Mechanism

Abstract The progression of ESCRT (Endosomal Sorting Complexes Required for Transport) pathways, which mediate numerous cellular membrane fission events, is driven by the enzyme Vps4. Understanding of Vps4 mechanism is, therefore, of fundamental importance in its own right and, moreover, it is highly relevant to the understanding of many related AAA+ ATPases that function in multiple facets of cell biology. Vps4 unfolds its ESCRT-III protein substrates by translocating them through its central hexameric pore, thereby driving membrane fission and recycling of ESCRT-III subunits. This mini-review focuses on recent advances in Vps4 structure and mechanism, including ideas about how Vps4 translocates and unfolds ESCRT-III subunits. Related AAA+ ATPases that share structural features with Vps4 and likely utilize an equivalent mechanism are also discussed.

scholarly journals Multiple conformations facilitate PilT function in the type IV pilus

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Matthew McCallum ◽  
Samir Benlekbir ◽  
Sheryl Nguyen ◽  
Stephanie Tammam ◽  
John L. Rubinstein ◽  
...  
Keyword(s):  
Single Particle ◽  
Protein Complexes ◽  
Comprehensive Model ◽  
Type Iv Pilus ◽  
Electron Cryomicroscopy ◽  
X Ray ◽  
Functional Studies ◽  
Type Iv ◽  
Coevolution Analysis ◽  
Multiple Conformations

AbstractType IV pilus-like systems are protein complexes that polymerize pilin fibres. They are critical for virulence in many bacterial pathogens. Pilin polymerization and depolymerization are powered by motor ATPases of the PilT/VirB11-like family. This family is thought to operate with C2 symmetry; however, most of these ATPases crystallize with either C3 or C6 symmetric conformations. The relevance of these conformations is unclear. Here, we determine the X-ray structures of PilT in four unique conformations and use these structures to classify the conformation of available PilT/VirB11-like family member structures. Single particle electron cryomicroscopy (cryoEM) structures of PilT reveal condition-dependent preferences for C2,C3, and C6 conformations. The physiologic importance of these conformations is validated by coevolution analysis and functional studies of point mutants, identifying a rare gain-of-function mutation that favours the C2 conformation. With these data, we propose a comprehensive model of PilT function with broad implications for PilT/VirB11-like family members.

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