title: Analysis of nucleotide binding to P97 reveals the properties of a tandem AAA hexameric ATPase.
abstract: p97, an essential chaperone in endoplasmic reticulum-associated degradation and organelle biogenesis, contains two AAA domains (D1 and D2) and assembles as a stable hexamer. We present a quantitative analysis of nucleotide binding to both D1 and D2 domains of p97, the first detailed study of nucleotide binding to both AAA domains for this type of AAA+ ATPase. We report that adenosine 5'-O-(thiotriphosphate) (ATPgammaS) binds with similar affinity to D1 and D2, but ADP binds with higher affinity to D1 than D2, offering an explanation for the higher ATPase activity in D2. Stoichiometric measurements suggest that although both ADP and ATPgammaS can saturate all 6 nucleotide binding sites in D1, only 3-4 of the 6 D2 sites can bind ATPgammaS simultaneously. ATPgammaS binding triggers a downstream cooperative conformational change of at least three monomers, which involves conserved arginine fingers and is necessary for ATP hydrolysis.
authors.affiliation: Division of Molecular Biosciences, Imperial College London, South Kensington, London SW7 2AZ, United Kingdom.
authors.forename: Louise C
authors.initials: LC
authors.lastname: Briggs
authors.forename: Geoff S
authors.initials: GS
authors.lastname: Baldwin
authors.forename: Non
authors.initials: N
authors.lastname: Miyata
authors.forename: Hisao
authors.initials: H
authors.lastname: Kondo
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Zhang
authors.forename: Paul S
authors.initials: PS
authors.lastname: Freemont
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000502:physiology*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D004305:Dose-Response Relationship, Drug; D006868:Hydrolysis; D007700:Kinetics; D051379:Mice; D008968:Molecular Conformation; D009687:Nuclear Proteins / Q000737:chemistry / Q000502:physiology*; D009711:Nucleotides / Q000737:chemistry; D011485:Protein Binding; D051381:Rats
chemical_list: D009687:Nuclear Proteins; D009711:Nucleotides; C022571:adenosine 5'-O-(3-thiotriphosphate); D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C404942:p97 ATPase