Heterologous expression of the metal-binding domains of human copper-transporting ATPases (P1-ATPases).
Heterologous expression of the metal-binding domains of human copper-transporting ATPases (P1-ATPases).
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人铜转运 ATP 酶 (P1-ATP 酶) 金属结合域的异源表达。
DOI：
10.1111/j.1749-6632.1997.tb52246.x
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发表时间：
1997
影响因子：
5.2
通讯作者：
Kaplan,JH
中科院分区：
文献类型：
--
作者：
Lutsenko,S;Petrukhin,K;Gilliam,TC;Kaplan,JH
Human copper-transporting ATPases (Wilson and Menkes disease proteins) are members of the PI-subfamily of P-type ATPases.'ATPases in this group have unique structural features14 such as (1) a characteristic hydropathy profile with eight putative transmembrane segments, three pairs before the DKTG motif and only one transmembrane hairpin after the ATP-binding domain;(2) heavy-metal binding motif at the NH2-terminus, which is repeated one to six times depending on the species;(3) CPC/H sequence in the transmembrane segment; and (4) SEHPL motif, which seems to be characteristic of Cu-ATPases5 and is located after phosphorylation site DKTG. Based on the symptoms of the associated metabolic diseases, both Wilson (WND) and Menkes (MNK) disease gene products are presumed to be copper-specific proteins. However, the exact cation specificity of these two proteins remains to be determined. The presence of the GMTCxxC motif repeated six times at the NH2-terminus suggests that the NHz-terminal domain plays an important role in the selective binding andor transport of heavy metals by these proteins. Alignment of all six repeats of the MNK and WND proteins5 revealed that the overall level of identity between repeats varies between 15 and 42%, suggesting that affinity towards heavy metals as well as specificity may vary for the MNK and WND proteins. Here we report expression of the NH2-terminal domains of the WND and MNK proteins as fusions with maltose binding protein (MBP) and characterization of their metal-binding properties. Regions corresponding to the NH2-tenninal domain, including all six repeats, were incorporated into pMAL-c2 and pMAL-p2 vectors (New England Biolabs) at the 3'-end of coding sequence for MBP, after the Factor Xa cleavage site. To do so, segments corresponding to 64-1 868 bp of WND cDNA and 1-1836 bp of MNK cDNA were amplified by the polymerase chain reaction. Stop codons were engineered at the 3'-end and unique restriction sites (Hind1 at 5'-end and Hind11 at 3'-end) were added to create a 5'blunt end, 3'sticky end-cloning fragment.
