Molecular features of the copper binding sites in the octarepeat domain of the prion protein
Molecular features of the copper binding sites in the octarepeat domain of the prion protein
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DOI：
10.1021/bi011922x
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发表时间：
2002-03-26
期刊：
影响因子：
2.9
通讯作者：
Millhauser, GL
中科院分区：
文献类型：
--
作者：
Burns, CS;Aronoff-Spencer, E;Millhauser, GL
最近的证据表明，PrP是一种铜结合蛋白。人PrP的N-末端区域包含高度保守的八重复序列PHGGGWGO的四个连续拷贝，跨越60-91个残基。该区域在体内选择性地与Cu2+结合。在先前使用肽设计、EPR和CD光谱的研究中，我们证明了每个八重复序列中的HGGGW片段包括基本的Cu2+结合单元[Aronoff-Spencer等人]。(2000年)生物化学40,13760-13771]。在这里，我们提出了第一个原子分辨率图的铜结合位置内的八个重复。HGGGW与Cu2+络合物的晶体结构表明，HGGGW由组氨酸咪唑、两个去质子化的甘氨酸酰胺和一个甘氨酸羰基组成的赤道配位，并有一个轴向的水桥连到色氨酸吲哚。在N-15标记多肽文库上进行的伴随S带EPR、X带ESEEM和HYSCORE实验表明，HGGGW和PHGGGWGQ中铜结合部位的结构与晶体结构一致。此外，对PrP(23-28，57-91)和N-15标记的类似物进行的EPR表明，所鉴定的结构保持在完整的PrP八重复结构域中。已有研究表明，铜能刺激PrP内吞作用。所确定的Gly-Cu键在pH约6.5以下是不稳定的，因此表明PrP检测细胞外基质中的Cu2+或在内体内释放PrP结合的Cu2+的分子机制依赖于pH。该结构还揭示了铜结构的HGGGW单元之间不寻常的互补相互作用，这可能有助于Pron蛋白之间的分子识别，从而暗示了一种跨膜信号转导机制，可能转化为致病形式。
Recent evidence suggests that the prion protein (PrP) is a copper binding protein. The N-terminal region of human PrP contains four sequential copies of the highly conserved octarepeat sequence PHGGGWGO spanning residues 60-91. This region selectively binds CU2+ in vivo. In a previous study using peptide design, EPR, and CD spectroscopy, we showed that the HGGGW segment within each octarepeat comprises the fundamental Cu2+ binding unit [Aronoff-Spencer et al. (2000) Biochemistry 40, 13760-13771]. Here we present the first atomic resolution view of the copper binding site within an octarepeat. The crystal structure of HGGGW in a complex with Cu2+ reveals equatorial coordination by the histidine imidazole, two deprotonated glycine amides, and a glycine carbonyl, along with an axial water bridging to the Trp indole. Companion S-band EPR, X-band ESEEM, and HYSCORE experiments performed on a library of N-15-labeled peptides indicate that the structure of the copper binding site in HGGGW and PHGGGWGQ in solution is consistent with that of the crystal structure. Moreover, EPR performed on PrP(23-28, 57-91) and an N-15-labeled analogue demonstrates that the identified structure is maintained in the full PrP octarepeat domain. It has been shown that copper stimulates PrP endocytosis. The identified Gly-Cu linkage is unstable below pH approximate to6.5 and thus suggests a pH-dependent molecular mechanism by which PrP detects Cu2+ in the extracellular matrix or releases PrP-bound CU2+ within the endosome. The structure also reveals an unusual complementary interaction between copper- structured HGGGW units that may facilitate molecular recognition between prion proteins, thereby suggesting a mechanism for transmembrane signaling and perhaps conversion to the pathogenic form.