ATP-binding sites in brain p97/VCP (valosin-containing protein), a multifunctional AAA ATPase.
VCP (valosin-containing protein) or p97 is a member of the AAA family (ATPases associated with a variety of cellular activities family), a diverse group of proteins sharing a key conserved AAA module containing duplicate putative ATP-binding sites. Although the functions of the AAA family are related to their putative ATP-binding sites, the binding of ATP to these sites has not yet been demonstrated. In the present study, the ATP-binding site(s) of brain VCP was characterized using the photoreactive ATP analogue, BzATP [3'- O -(4-benzoylbenzoyl)ATP]. Photo-activation of Bz-[alpha-(32)P]ATP resulted in its covalent binding to a 97-kDa purified soluble or membrane-associated protein, identified by amino acid sequencing as VCP. Bz-[alpha-(32)P]ATP covalently bound to the purified homo-hexameric VCP with an apparent high affinity (74-111 nM). A molar stoichiometry of 2.23+/-0.14 BzATP bound per homo-hexameric VCP (n =6) was determined using different methods for analysis of radiolabelling and protein determination. Nucleotides inhibited the binding of Bz-[alpha-(32)P]ATP to VCP with the following efficiency: BzATP>ATP>ADP>>adenosine 5'-[beta,gamma-imido]triphosphate>or=adenosine 5'-[beta,gamma-methylene]triphosphate, whereas AMP, GTP and CTP were ineffective. VCP was observed to possess very low ATPase activity, with nucleotide specificity similar to that for BzATP binding. Conformational changes induced by an alternating site mechanism for ATP binding are suggested as a molecular mechanism for coupling ATP binding to the diverse activities of the AAA family.
Department of Life Sciences and Zlotowski Center for Neuroscience, Ben Gurion University of the Negev, Beer Sheva, Israel.
Ran
R
Zalk
Varda
V
Shoshan-Barmatz
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism*; D000345:Affinity Labels / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000378:metabolism; D002458:Cell Fractionation; D002462:Cell Membrane / Q000737:chemistry / Q000201:enzymology; D002540:Cerebral Cortex / Q000737:chemistry / Q000166:cytology / Q000201:enzymology; D006801:Humans; D006868:Hydrolysis; D051379:Mice; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000378:metabolism; D010449:Peptide Mapping; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D051381:Rats; D012756:Sheep; D013194:Staining and Labeling; D000074405:Valosin Containing Protein
D000345:Affinity Labels; D018797:Cell Cycle Proteins; D009097:Multienzyme Complexes; D009419:Nerve Tissue Proteins; C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C000614178:VCP protein, human; D000074405:Valosin Containing Protein; C000614179:Vcp protein, mouse; C000614180:Vcp protein, rat