Zalk Ran, Shoshan-Barmatz Varda
Department of Life Sciences and Zlotowski Center for Neuroscience, Ben Gurion University of the Negev, Beer Sheva, Israel.
Biochem J. 2003 Sep 1;374(Pt 2):473-80. doi: 10.1042/BJ20030219.
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.
含缬酪肽蛋白（VCP）或p97是AAA家族（与多种细胞活动相关的ATP酶家族）的成员，这是一组多样的蛋白质，共享一个关键的保守AAA模块，其中包含重复的假定ATP结合位点。尽管AAA家族的功能与其假定的ATP结合位点相关，但尚未证实ATP与这些位点的结合。在本研究中，使用光反应性ATP类似物BzATP [3'-O-（4-苯甲酰苯甲酰基）ATP]对脑VCP的ATP结合位点进行了表征。Bz-[α-（32）P]ATP的光激活导致其与一种97 kDa的纯化可溶性或膜相关蛋白共价结合，经氨基酸测序鉴定为VCP。Bz-[α-（32）P]ATP以明显的高亲和力（74-111 nM）与纯化的同型六聚体VCP共价结合。使用不同的放射性标记分析和蛋白质测定方法，确定每个同型六聚体VCP（n = 6）结合的BzATP的摩尔化学计量比为2.23±0.14。核苷酸以以下效率抑制Bz-[α-（32）P]ATP与VCP的结合：BzATP>ATP>ADP>>腺苷5'-[β,γ-亚氨基]三磷酸>或=腺苷5'-[β,γ-亚甲基]三磷酸，而AMP、GTP和CTP无效。观察到VCP具有非常低的ATP酶活性，其核苷酸特异性与BzATP结合的特异性相似。ATP结合的交替位点机制诱导的构象变化被认为是将ATP结合与AAA家族的多种活动偶联的分子机制。
