X-ray diffraction analysis of scrapie prion: intermediate and folded structures in a peptide containing two putative alpha-helices.
X-ray diffraction analysis of scrapie prion: intermediate and folded structures in a peptide containing two putative alpha-helices.
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DOI：
10.1006/jmbi.1997.0949
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发表时间：
1997-05
影响因子：
5.6
通讯作者：
H. Inouye;D. Kirschner
中科院分区：
文献类型：
--
作者：
H. Inouye;D. Kirschner
被称为朊病毒的小蛋白质感染性颗粒会导致人类和动物的某些神经退行性疾病。感染性羊瘙痒病朊病毒PrP（Sc）的有限蛋白水解产生称为PrP 27-30的N截短多肽，其包括PrP（Sc）的残基90至231，并组装成100至200 A宽的淀粉样蛋白棒。据推测，感染性朊病毒是通过α-螺旋到β-折叠构象变化从其非感染性细胞形式（PrP（C））转化而来的。二级结构分析，计算机模拟和结构生物物理学方法支持这一假设。残基90至145的PrP，其中包含两个推定的α-螺旋结构域H1和H2，可能是特别相关的疾病的发病机制，在残基145处的C-末端截短被发现在一个遗传性朊病毒疾病的患者。此外，我们最近的X-射线衍射分析表明，由这些残基组成的肽（命名为SHa 90-145）密切模拟PrP的淀粉样β-片层核心。在本研究中，我们详细分析了SHa 90-145的X射线衍射图谱。检查了两个样品：一种在环境条件下同时在外部磁场中脱水（以诱导原纤维取向），另一种在部分干燥后密封。干燥的磁性取向样品显示出交叉β衍射图案，其中纤维轴（旋转轴）平行于β-片层的H-键合方向。主要的广角峰表明存在约40 A宽的β-微晶，其构成原丝。每个微晶由垂直于纤维（a轴）方向的几个正交晶胞组成，具有晶格常数a = 9.69 A，B = 6.54 A和c = 18.06 A。使用β-丝作为初始相模型通过迭代傅里叶合成计算电子密度图。密度分布表明，有两种类型的β-折叠，表明较大和较小的侧链定位于不同的片。这将由多肽的折叠引起，其中在H1和H2结构域的中间都有转角。发现单斜宏观晶格，a = 9.61 A，B = c = 52.99 A和α = 114.6度，索引所有的反射，包括那些在低角度区域。这表明β-微晶几乎是六边形堆积的。为了解释在电子显微镜中通过负染色可见的约100 A宽的纤维，β-微晶将以4聚体排列。部分干燥的样品显示出尖锐的4.7 A反射（来自H-键合）和叠加在单调递减的漫散射上的五个宽峰。这种类似溶液的散射是用一个不等轴矩形来模拟的，其厚度相当于一个单一的β链。在脱水过程中发生的结构，可能是一个短暂的α-螺旋β-折叠转换，这表明形成氢键之前的层间相互作用和组装成羊瘙痒症朊病毒的淀粉样蛋白。
Small proteinaceous infectious particles called prions cause certain neurodegenerative diseases in human and animals. Limited proteolysis of infectious scrapie prions PrP(Sc) yields an N-truncated polypeptide termed PrP 27-30, which encompasses residues 90 to 231 of PrP(Sc) and which assembles into 100 to 200 A wide amyloid rods. It has been hypothesized that the infectious prion is converted from its non-infectious cellular form (PrP(C)) by means of an alpha-helical to beta-sheet conformational change. Secondary structure analysis, computer modeling, and structural biophysics methods support this hypothesis. Residues 90 to 145 of PrP, which contain two putative alpha-helical domains H1 and H2, may be of particular relevance to the disease pathogenesis, as C-terminal truncation at residue 145 was found in a patient with an inherited prion disease. Moreover, our recent X-ray diffraction analysis suggests that the peptide consisting of these residues (designated SHa 90-145) closely models the amyloidogenic beta-sheet core of PrP. In the current study, we have analyzed in detail the X-ray diffraction patterns of SHa 90-145. Two samples were examined: one that was dehydrated under ambient conditions whilst in an external magnetic field (to induce fibril orientation), and another that was sealed after partial drying. The dried, magnetically oriented sample showed a cross-beta diffraction pattern in which the fiber axis (rotation axis) was parallel to the H-bonding direction of the beta-sheets. The major wide-angle peaks indicate the presence of approximately 40 A wide beta-crystallites, which constitute the protofilament. Each crystallite is composed of several orthogonal unit cells, normal to the fiber (a-axis) direction, having lattice constants a = 9.69 A, b = 6.54 A, and c = 18.06 A. Electron density maps were calculated by iterative Fourier synthesis using beta-silk as an initial phase model. The distribution of density indicated that there were two types of beta-sheet, suggesting that larger and smaller side-chains localized to different sheets. This would arise from folding of the polypeptide in which there are turns in the middle of both the H1 and H2 domains. A monoclinic macrolattice, with a = 9.61 A, b = c = 52.99 A and alpha = 114.6 degrees, was found to index all the reflections, including those in the low-angle region. This suggests that the beta-crystallites are nearly hexagonally packed. To account for the approximately 100 A wide fibers visualized by negative staining in the electron microscope, the beta-crystallites would be arranged in 4-mers. The partially dried sample showed a sharp 4.7 A reflection (from H-bonding) and five broad peaks superimposed on monotonically decreasing diffuse scattering. This solution-like scattering was modeled by an anisometric rectangle with a thickness comparable to a singe beta-chain. The structure, which occurred during dehydration, could be a transient in the alpha-helical to beta-sheet conversion, suggesting that formation of hydrogen bonding precedes the inter-sheet interaction and assembly into the amyloid of scrapie prion.