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-[α-32P]ATP resulted in its covalent binding to a 97-kDa purified soluble or membrane-associated protein, identified by amino acid sequencing as VCP. Bz-[α-32P]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-[α-32P]ATP to VCP with the following efficiency: BzATP>ATP>ADP≫adenosine 5′-[β,γ-imido]triphosphate≥adenosine 5′-[β,γ-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.
- 3′-O-(4-benzoylbenzoyl)ATP (BzATP)
- nucleotide-binding site
- valosin-containing protein (VCP)
Abbreviations used: AAA family, ATPases associated with a variety of cellular activities family; BzATP, 3′-O-(4-benzoylbenzoyl)ATP; LC-MS/MS, liquid chromatography-tandem MS; NSF, N-ethylmaleimide-sensitive fusion protein; p[CH2]ppA, adenosine 5′-[β,γ-methylene]triphosphate; p[NH]ppA, adenosine 5′-[β,γ-imido]triphosphate; SR, sarcoplasmic reticulum; TER-ATPase, transitional endoplasmic reticulum-ATPase; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; VCP, valosin-containing protein.
- The Biochemical Society, London ©2003