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Positive cooperativity of the p97 AAA ATPase is critical for essential functions.

ABSTRACT: 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.

SUBMITTER: Nishikori S

PROVIDER: S-EPMC3091191 | biostudies-other | 2011 May

REPOSITORIES: biostudies-other

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Positive cooperativity of the p97 AAA ATPase is critical for essential functions.

Nishikori Shingo S Esaki Masatoshi M Yamanaka Kunitoshi K Sugimoto Shinya S Ogura Teru T

The Journal of biological chemistry 20110318 18

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 diff ...[more]

PMID: 21454554

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AAA ATPase p97/VCP: cellular functions, disease and therapeutic potential.

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

Project description: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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Subunit Interactions and cooperativity in the microtubule-severing AAA ATPase spastin.

Project description:Spastin is a hexameric ring AAA ATPase that severs microtubules. To see whether the ring complex funnels the energy of multiple ATP hydrolysis events to the site of mechanical action, we investigate here the cooperativity of spastin. Several lines of evidence indicate that interactions among two subunits dominate the cooperative behavior: (i) the ATPase activity shows a sigmoidal dependence on the ATP concentration; (ii) ATPγS displays a mixed-inhibition behavior for normal ATP turnover; and (iii) inactive mutant subunits inhibit the activity of spastin in a hyperbolic dependence, characteristic for two interacting species. A quantitative model based on neighbor interactions fits mutant titration experiments well, suggesting that each subunit is mainly influenced by one of its neighbors. These observations are relevant for patients suffering from SPG4-type hereditary spastic paraplegia and explain why single amino acid exchanges lead to a dominant negative phenotype. In severing assays, wild type spastin is even more sensitive toward the presence of inactive mutants than in enzymatic assays, suggesting a weak coupling of ATPase and severing activity.

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The AAA + ATPase valosin-containing protein (VCP)/p97/Cdc48 interaction network in Leishmania.

Project description:Valosin-containing protein (VCP)/p97/Cdc48 is an AAA + ATPase associated with many ubiquitin-dependent cellular pathways that are central to protein quality control. VCP binds various cofactors, which determine pathway selectivity and substrate processing. Here, we used co-immunoprecipitation and mass spectrometry studies coupled to in silico analyses to identify the Leishmania infantum VCP (LiVCP) interactome and to predict molecular interactions between LiVCP and its major cofactors. Our data support a largely conserved VCP protein network in Leishmania including known but also novel interaction partners. Network proteomics analysis confirmed LiVCP-cofactor interactions and provided novel insights into cofactor-specific partners and the diversity of LiVCP complexes, including the well-characterized VCP-UFD1-NPL4 complex. Gene Ontology analysis coupled with digitonin fractionation and immunofluorescence studies support cofactor subcellular compartmentalization with either cytoplasmic or organellar or vacuolar localization. Furthermore, in silico models based on 3D homology modeling and protein-protein docking indicated that the conserved binding modules of LiVCP cofactors, except for NPL4, interact with specific binding sites in the hexameric LiVCP protein, similarly to their eukaryotic orthologs. Altogether, these results allowed us to build the first VCP protein interaction network in parasitic protozoa through the identification of known and novel interacting partners potentially associated with distinct VCP complexes.

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The role of the N-domain in the ATPase activity of the mammalian AAA ATPase p97/VCP.

Project description:p97/valosin-containing protein (VCP) is a type II ATPase associated with various cellular activities that forms a homohexamer with each protomer containing an N-terminal domain (N-domain); two ATPase domains, D1 and D2; and a disordered C-terminal region. Little is known about the role of the N-domain or the C-terminal region in the p97 ATPase cycle. In the p97-associated human disease inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia, the majority of missense mutations are located at the N-domain D1 interface. Structure-based predictions suggest that such mutations affect the interaction of the N-domain with D1. Here we have tested ten major inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia-linked mutants for ATPase activity and found that all have increased activity over the wild type, with one mutant, p97(A232E), having three times higher activity. Further mutagenesis of p97(A232E) shows that the increase in ATPase activity is mediated through D2 and requires both the N-domain and a flexible ND1 linker. A disulfide mutation that locks the N-domain to D1 in a coplanar position reversibly abrogates ATPase activity. A cryo-EM reconstruction of p97(A232E) suggests that the N-domains are flexible. Removal of the C-terminal region also reduces ATPase activity. Taken together, our data suggest that the conformation of the N-domain in relation to the D1-D2 hexamer is directly linked to ATP hydrolysis and that the C-terminal region is required for hexamer stability. This leads us to propose a model where the N-domain adopts either of two conformations: a flexible conformation compatible with ATP hydrolysis or a coplanar conformation that is inactive.

| S-EPMC3318706 | biostudies-literature

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