Qazi U, Gettins P G, Stoops J K
Department of Pathology and Laboratory Medicine, University of Texas Medical School, Houston, Texas 77030, USA.
J Biol Chem. 1998 Apr 10;273(15):8987-93. doi: 10.1074/jbc.273.15.8987.
The reconstructions of an intermediate form of human alpha2-macroglobulin (half-transformed alpha2M) in which two of its four bait regions and thiol ester sites were cleaved by chymotrypsin bound to Sepharose were obtained by three-dimensional electron microscopy from stain and frozen-hydrated specimens. The structures show excellent agreement and reveal a structure with approximate dimensions of 195 (length) x 135 (width) and 130 A (depth) with an internal funnel-shaped cavity. The structure shows that a chisel-shaped body is connected to a broad base at the opposing end by four stands. Four approximately 45 A diameter large openings in the body of the structure result in a central cavity that is more accessible to the proteinase than those associated with the native or fully transformed structures. The dissimilarity in the shapes between the two ends of alpha2M half-transformed and the similarity between its chisel-shaped body and that of native alpha2M indicate that the chymotrypsin has cleaved both bait regions in the bottom-half of the structure. Consequently, its functional division lies on the minor axis. The structural organization is in accord with biochemical studies, which show that the half-transformed alpha2M migrates on native polyacrylamide gels at a rate intermediate to the native and fully transformed alpha2M and is capable of trapping 1 mol of proteinase. Even though its upper portion is similar to the native molecule, significant differences in their shapes are apparent and these differences may be related to its slower reaction with a proteinase than the native structure. These structural comparisons further support the view that the transformation of alpha2M involves an untwisting of its strands with an opening of the cavity for entrance of the proteinase and a retwisting of the strands around the proteinase resulting in its encapsulation.
通过三维电子显微镜从染色和冷冻水合标本中获得了人α2-巨球蛋白中间形式（半转化α2M）的重建结构，其中其四个诱饵区域中的两个以及硫酯位点被结合到琼脂糖凝胶上的胰凝乳蛋白酶切割。这些结构显示出极好的一致性，并揭示了一个结构，其近似尺寸为195（长）×135（宽）和130埃（深），具有一个内部漏斗形腔。该结构表明，一个凿形主体通过四个支柱连接到相对端的宽基部。结构主体中有四个直径约45埃的大开口，形成一个中央腔，与天然或完全转化结构相比，蛋白酶更容易进入该中央腔。α2M半转化体两端形状的差异以及其凿形主体与天然α2M凿形主体的相似性表明，胰凝乳蛋白酶已切割了结构下半部分的两个诱饵区域。因此，其功能分区位于短轴上。这种结构组织与生化研究一致，生化研究表明，半转化α2M在天然聚丙烯酰胺凝胶上的迁移速率介于天然和完全转化α2M之间，并且能够捕获1摩尔蛋白酶。尽管其上部与天然分子相似，但其形状上的显著差异是明显的，这些差异可能与其与蛋白酶的反应比天然结构慢有关。这些结构比较进一步支持了这样一种观点，即α2M的转化涉及链的解捻，形成一个腔开口以供蛋白酶进入，以及围绕蛋白酶的链重新捻合，从而将其包裹。
