Three-dimensional structure of the nicotinic acetylcholine receptor and location of the major associated 43-kD cytoskeletal protein, determined at 22 A by low dose electron microscopy and x-ray diffraction to 12.5 A.
Three-dimensional structure of the nicotinic acetylcholine receptor and location of the major associated 43-kD cytoskeletal protein, determined at 22 A by low dose electron microscopy and x-ray diffraction to 12.5 A.
复制标题
烟碱乙酰胆碱受体的三维结构和主要相关 43-kD 细胞骨架蛋白的位置，通过低剂量电子显微镜和 12.5 A 的 X 射线衍射在 22 A 确定。
DOI：
10.1083/jcb.109.2.755
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发表时间：
1989
期刊：
影响因子：
--
通讯作者：
Stroud,RM
中科院分区：
文献类型：
--
作者：
Mitra,AK;McCarthy,MP;Stroud,RM
加利福尼亚鱼雷烟碱乙酰胆碱受体（AChR）的三维结构，在去除相关蛋白之前和之后结晶，最值得注意的是与AChR和肌动蛋白相互作用的主要43-kD细胞骨架蛋白，被确定为22 a的分辨率。这是第一次在结晶前从样品中除去43-kD蛋白的结构分析。因此，它提供了最小5个亚基AChR复合物结构构成的最可靠评估，并通过与天然膜的比较，确定了43-kD细胞骨架蛋白的位置。二维晶体的图像重建包括晶格化AChR的+/- 52度倾斜样本的电子图像信息。混合密度图包括垂直于膜的x射线衍射，分辨率为12.5 A，消除了仅基于电子显微镜分析的图中引入的一些扭曲。对比AChR与其正常补体相关蛋白之间的傅里叶密度差图，以及没有它们的对比表明，分配给肌动蛋白结合43-kD成分的主要密度与AChR的脂质双分子层以及细胞质结构域密切相关。它在acr旁边结合，而不是像其他人认为的那样在它下面结合（C. Toyoshima和N. Unwin 1988）。自然(Lond)。336:237 - 240)。结构部件的密度体积和基于其分子量的预期体积之间有很好的一致性。乙酰胆碱受体在没有任何细胞骨架蛋白的情况下聚集，这表明仅靠AChR就足以编码和稳定聚集，并且可能在突触发生过程中这样做。43-kD的主要成分可能与AChR的结合位置和速率有关。我们发现，在大约80%的AChR中，相邻五聚体的三角链交联的二硫键并不是稳定AChR晶格所必需的。网管结构是无限稳定的。这里描述的晶格的脂质体积比J. Kistler和R. M. Stroud（1981）最初获得和表征的晶格少20%。Proc。国家的。学会科学。美国。78:3678-3682)，或后来以A.布里森和P. N. T.昂温（1984）为特征的那些。J.细胞生物学。99:1202-1211)和A. Brisson和P. N. T. Unwin（1985）。自然(Lond)。315:474 - 477)。
The three-dimensional structure of the nicotinic acetylcholine receptor (AChR) from Torpedo californica, crystallized both before and after removal of associated proteins, most notably the main 43-kD cytoskeletal protein that interacts both with AChR and actin, is determined to a resolution of 22 A. This is the first structural analysis where the 43-kD protein has been removed from the sample before crystallization. Thus, it provides the most reliable assessment of what constitutes the structure of the minimal five subunit AChR complex, and, by comparison with the native membrane, of the location of the 43-kD cytoskeletal protein. Image reconstruction of two-dimensional crystals includes information from electron images of up to +/- 52 degrees tilted specimens of latticed AChR. Hybrid density maps that include x-ray diffraction perpendicular to the membrane to 12.5 A resolution were used and eliminate some of the distortions introduced in maps based only on electron microscopic analyses. Comparison of the difference Fourier density maps between AChR with its normal complement of associated proteins, and without them shows that the main density, assigned to the actin-binding 43-kD component is closely associated with the lipid bilayer as well as with the cytoplasmic domain of the AChR. It binds beside the AChR, not beneath it as suggested by others (C. Toyoshima and N. Unwin 1988. Nature [Lond.]. 336:237-240). There is good agreement between the volumes of density for structural components and expected volumes based on their molecular weight. Acetylcholine receptors aggregate in the absence of any cytoskeletal proteins, suggesting that the AChR alone is sufficient to encode and stabilize clustering, and perhaps to do so during synaptogenesis. The main 43-kD component may play a role in location and rate of association of AChR. We show that the disulfide bond that cross-links delta-delta chains of adjacent pentamers in about 80% of AChR, is not required to stabilize the lattice of AChR. Latticed tube structures are stable indefinitely. The lattices described here have 20% less volume of lipid than those originally obtained and characterized by J. Kistler and R. M. Stroud (1981. Proc. Natl. Acad. Sci. USA. 78:3678-3682), or those subsequently characterized by A. Brisson and P. N. T. Unwin (1984. J. Cell Biol. 99:1202-1211) and A. Brisson and P. N. T. Unwin (1985. Nature (Lond.). 315:474-477).