The Lys1010-Lys1325 fragment of the Wilson's disease protein binds nucleotides and interacts with the N-terminal domain of this protein in a copper-dependent manner
The Lys1010-Lys1325 fragment of the Wilson's disease protein binds nucleotides and interacts with the N-terminal domain of this protein in a copper-dependent manner
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DOI：
10.1074/jbc.m003238200
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发表时间：
2001-01-19
影响因子：
4.8
通讯作者：
Lutsenko, S
中科院分区：
文献类型：
--
作者：
Tsivkovskii, R;MacArthurs, BC;Lutsenko, S
肝豆状核变性是一种常染色体疾病，与组织中铜的大量积累有关，是由编码铜转运ATP酶（肝豆状核变性蛋白，WNDP）的基因突变引起的。在整个WNDP序列中，特别是在Lys（1010）-Lys（1325）片段中，已经鉴定出许多突变;然而，WNDP的生化特性和分子机制仍然很差。在此，WNDP的Lys（1010）-Lys（1325）片段被过表达、纯化，并显示形成独立折叠的ATP结合结构域（ATP-BD）。ATP-BD以高亲和力结合荧光ATP类似物三硝基苯基-ATP，ATP与三硝基苯基-ATP竞争结合位点; ADP和AMP似乎在与ATP分开的位点与ATP-BD结合。纯化的ATP-BD水解ATP并与WNDP的N-末端铜结合结构域（N-WNDP）特异性地相互作用。引人注目的是，铜结合到N-WNDP减少这些相互作用，这表明铜依赖的变化域域接触可能代表WNDP的调节机制。与此假设一致，N-WNDP诱导ATP-BD的构象变化，如在N-WNDP存在下ATP-BD的核苷酸结合性质的改变所证明的。重要的是，无铜和铜结合的N-WNDP对ATP-BD的影响是不相同的。这些结果的影响WNDP功能进行了讨论。
Wilson's disease, an autosomal disorder associated with vast accumulation of copper in tissues, is caused by mutations in a gene encoding a copper-transporting ATPase (Wilson's disease protein, WNDP), Numerous mutations have been identified throughout the WNDP sequence, particularly in the Lys(1010)-Lys(1325) segment; however, the biochemical properties and molecular mechanism of WNDP remain poorly characterized. Here, the Lys(1010)-Lys(1325) fragment of WNDP was overexpressed, purified, and shown to form an independently folded ATP-binding domain (ATP-BD). ATP-BD binds the fluorescent ATP analogue trinitrophenyl-ATP with high affinity, and ATP competes with trinitrophenyl-ATP for the binding site; ADP and AMP appear to bind to ATP-BD at the site separate from ATP. Purified ATP-BD hydrolyzes ATP and interacts specifically with the N-terminal copper-binding domain of WNDP (N-WNDP). Strikingly, copper binding to N-WNDP diminishes these interactions, suggesting that the copper-dependent change in domain-domain contact may represent the mechanism of WNDP regulation. In agreement with this hypothesis, N-WNDP induces conformational changes in ATP-BD as evidenced by the altered nucleotide binding properties of ATP-BD in the presence of N-WNDP, Significantly, the effects of copper-free and copper-bound N-WNDP on ATP-BD are not identical. The implications of these results for the WNDP function are discussed.