High yield heterologous expression of wild-type and mutant Cu+-ATPase (ATP7B, Wilson disease protein) for functional characterization of catalytic activity and serine residues undergoing copper-dependent phosphorylation.
High yield heterologous expression of wild-type and mutant Cu+-ATPase (ATP7B, Wilson disease protein) for functional characterization of catalytic activity and serine residues undergoing copper-dependent phosphorylation.
复制标题
DOI：
10.1074/jbc.m109.023341
复制
发表时间：
2009-08-07
期刊：
影响因子：
--
通讯作者：
Inesi G
中科院分区：
文献类型：
--
作者：
Pilankatta R;Lewis D;Adams CM;Inesi G
ATP 7 B是一种P型ATP酶，是维持铜稳态所必需的，与肝豆状核变性有关。除了对应于其他P型ATP酶的各种结构域之外，ATP 7 B还包括具有六个铜结合位点的N末端延伸（NMBD）。用腺病毒载体感染COS 1细胞，获得了野生型和突变型ATP 7 B的高效表达。与细胞匀浆的微粒体部分分离的ATP 7 B占总蛋白的10-20%。铜依赖性稳态ATP酶在37 °C，pH 6.0下产生30 nmol Pi/mg蛋白质/min。ATP对ATP 7 B的磷酸化作用以双相动力学发生，并且完全依赖于铜。碱不稳定性磷酸酶（P-ATP酶的催化中间体）占总磷蛋白的一小部分，并且被D1027 N（P结构域）或C983 A/C985 A（TM 6中的CXC铜结合基序）突变阻止。[32 P]磷酸酶在非放射性ATP的追逐下发生衰变，最初的爆发涉及碱不稳定的磷酸酶（在D1027 N和C983 A/C985 A突变体中不存在），并以缓慢的速度继续涉及耐碱性磷酸酶。如果铜螯合剂与ATP追逐一起加入，则初始爆发较小，并且完全抑制进一步的切割。通过蛋白水解和质谱分析表明，碱稳定的磷酸酶涉及Ser 478和Ser 481（NMBD），Ser 1121（“N”结构域）和Ser 1453（C末端），并且在离体（COS-1）和体外（微粒体）中以相同的模式发生。磷酸化和水解切割的总体铜依赖性表明了长期的构象效应，包括NMBD和头片段结构域的相互作用，对催化转换有很大影响。
ATP7B is a P-type ATPase required for copper homeostasis and related to Wilson disease of humans. In addition to various domains corresponding to other P-type ATPases, ATP7B includes an N terminus extension (NMBD) with six copper binding sites. We obtained high yield expression of WT and mutant ATP7B in COS1 cells infected with adenovirus vector. ATP7B, isolated with the microsomal fraction of cell homogenates, accounts for 10–20% of the total protein. Copper-dependent, steady-state ATPase yields 30 nmol of Pi/mg of protein/min at 37 °C, pH 6.0. ATP7B phosphorylation with ATP occurs with diphasic kinetics and is totally copper-dependent. Alkali labile phosphoenzyme (catalytic intermediate of P-ATPases) accounts for a small fraction of the total phosphoprotein and is prevented by D1027N (P domain) or C983A/C985A (CXC copper binding motif in TM6) mutations. Decay of [32P]phosphoenzyme following chase with non-radioactive ATP occurs with an initial burst involving alkali labile phosphoenzyme (absent in D1027N and C983A/C985A mutants) and continues at a slow rate involving alkali-resistant phosphoenzyme. If a copper chelator is added with the ATP chase, the initial burst is smaller, and further cleavage is totally inhibited. Analysis by proteolysis and mass spectrometry demonstrates that the alkali stable phosphoenzyme involves Ser478 and Ser481 (NMBD), Ser1121 (“N” domain) and Ser1453 (C terminus), and occurs with the same pattern ex vivo (COS-1) and in vitro (microsomes). The overall copper dependence of phosphorylation and hydrolytic cleavage suggests long range conformational effects, including interactions of NMBD and headpiece domains, with strong influence on catalytic turnover.