Prion protein selectively binds copper(II) ions
Prion protein selectively binds copper(II) ions
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DOI：
10.1021/bi972827k
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发表时间：
1998-05-19
期刊：
影响因子：
2.9
通讯作者：
Prusiner, SB
中科院分区：
文献类型：
--
作者：
Stöckel, J;Safar, J;Prusiner, SB
朊病毒蛋白（PrPSc）的感染性同种型是由细胞PrP（PrPC）转化而来的，转化反应涉及二级和三级结构的剧烈重组。虽然我们对PrPSc的致病作用的理解有所增加，但PrPC的正常生理功能仍不清楚。使用重组叙利亚仓鼠朊蛋白[SHaPrP（29-231）]，我们研究了金属离子作为PrP的可能配体。近紫外圆二色光谱（CD）表明SHaPrP（29-231）的构象类似于从仓鼠脑中纯化的PrPC。在这里，我们通过CD和色氨酸（Trp）荧光光谱证明铜诱导SHaPrP的三级结构的变化（29-231）。铜的结合显著淬灭Trp荧光发射，使发射光谱向较短波长移动，并且还诱导SHaPrP的近UV CD光谱的变化（29-231）。结合位点对Cu 2+具有高度特异性，如通过缺乏与Ca 2+、Co 2+、Mg 2+、Mn 2+、Ni 2+和Zn 2+的Trp荧光发射的变化所指示的。Cu 2+的结合也促进了从主要的α-螺旋结构到P-折叠结构的构象转变。平衡透析实验表明，在生理相关浓度下，每个PrP分子的结合化学计量类似于2个铜分子，并且Cu 2+结合的pH滴定表明组氨酸作为螯合配体的作用。NMR光谱最近已经证明，在不存在Cu 2+的情况下，SHaPrP（29-231）中的八重复（PHGGGWGQ）缺乏二级或三级结构。我们的研究结果表明，每个Cu 2+结合到由两个octagrepeat（PHGGGWGQ）与一个组氨酸，也许是一个甘氨酸羰基螯合离子定义的结构。我们建议，两个铜离子的绑定到四个octrepeats诱导一个更明确的结构，这一地区。
The infectious isoform of the prion protein (PrPSc) is derived from cellular PrP (PrPC) in a conversion reaction involving a dramatic reorganization of secondary and tertiary structure. While our understanding of the pathogenic role of PrPSc has grown, the normal physiologic function of PrPC still remains unclear. Using recombinant Syrian hamster prion protein [SHaPrP(29-231)], we investigated metal ions as possible ligands of PrP. Near-UV circular dichroism spectroscopy (CD) indicates that the conformation of SHaPrP(29-231) resembles PrPC purified from hamster brain. Here we demonstrate by CD and tryptophan (Trp) fluorescence spectroscopy that copper induces changes to the tertiary structure of SHaPrP(29-231). Binding of copper quenches the Trp fluorescence emission significantly, shifts the emission spectrum to shorter wavelengths, and also induces changes in the near-UV CD spectrum of SHaPrP(29-231). The binding sites are highly specific for Cu2+, as indicated by the lack of a change in Trp fluorescence emission with Ca2+, Co2+, Mg2+, Mn2+, Ni2+, and Zn2+. Binding of Cu2+ also promotes the conformational shift from a predominantly a-helical to a P-sheet structure. Equilibrium dialysis experiments indicate a binding stoichiometry of similar to 2 copper molecules per PrP molecule at physiologically relevant concentrations, and pH titration of Cu2+ binding suggests a role for histidine as a chelating ligand. NMR spectroscopy has recently demonstrated that the octarepeats (PHGGGWGQ) in SHaPrP(29-231) lack secondary or tertiary structure in the absence of Cu2+. Our results suggest that each Cu2+ binds to a structure defined by two octarepeats (PHGGGWGQ) with one histidine and perhaps one glycine carbonyl chelating the ion. We propose that the binding of two copper ions to four octarepeats induces a more defined structure to this region.