Cardy C M, Handford P A
Sir William Dunn School of Pathology, University of Oxford, UK.
J Mol Biol. 1998 Mar 13;276(5):855-60. doi: 10.1006/jmbi.1997.1593.
The effects of the removal and replacement of divalent cations on the ultrastructure of 10 to 12 nm fibrillin-1-containing microfibrils have been studied, in order to investigate the conformation of fibrillin-1 calcium-binding epidermal growth factor-like (cbEGF-like) domains within the microfibril. The NMR structure of a covalently linked pair of cbEGF-like domains from fibrillin-1 recently identified a rigid rod-like conformation for the domain pair stabilised by interdomain calcium binding. This suggested that tandem arrays of fibrillin-1 cbEGF-like domains may adopt an extended conformation within a microfibril. If correct, then removal of bound calcium from fibrillin-1 would be expected to increase the flexibility of each cbEGF-like interdomain linkage, resulting in a decrease in the length of the interbead region of the microfibril (and thus a decrease in bead to bead periodicity), a concomitant increase in its diameter, and an overall increase in the flexibility of the microfibril. Our results show that removal of calcium by treatment with EGTA causes a large alteration of the microfibril structure, resulting in microfibrils with a reduced beaded periodicity, a disrupted interbead region and an increased overall flexibility. These effects are readily reversible by the re-addition of calcium (in the form of CaCl2), but not by the addition of magnesium (MgCl2). This is consistent with conformational changes in cbEGF-like domains causing the major structural effects on the microfibril. These results provide the first direct experimental evidence to support an extended rod-like conformation for multiple tandem repeats of fibrillin-1 cbEGF-like domains within the microfibril, as predicted by the NMR structure of an isolated fibrillin-1 cbEGF-like domain pair.
为了研究原纤维蛋白-1含微原纤维（直径10至12纳米）中二价阳离子的去除和替换对其超微结构的影响，我们对微原纤维中原纤维蛋白-1钙结合表皮生长因子样（cbEGF样）结构域的构象进行了研究。最近确定的原纤维蛋白-1中一对共价连接的cbEGF样结构域的核磁共振结构表明，通过结构域间的钙结合，该结构域对呈现出一种刚性的棒状构象。这表明原纤维蛋白-1的cbEGF样结构域串联阵列在微原纤维中可能呈伸展构象。如果这一推测正确，那么去除原纤维蛋白-1中结合的钙有望增加每个cbEGF样结构域间连接的灵活性，导致微原纤维珠间区域长度减小（从而珠间周期性降低），其直径随之增加，微原纤维的整体灵活性也增加。我们的结果表明，用乙二醇双四乙酸（EGTA）处理去除钙会导致微原纤维结构发生重大改变，产生珠状周期性降低、珠间区域破坏且整体灵活性增加的微原纤维。通过重新添加钙（以氯化钙形式），这些影响很容易逆转，但添加镁（氯化镁）则不能。这与cbEGF样结构域的构象变化对微原纤维产生主要结构影响是一致的。这些结果提供了首个直接实验证据，支持微原纤维中原纤维蛋白-1的cbEGF样结构域多个串联重复呈伸展棒状构象，这与分离的原纤维蛋白-1的cbEGF样结构域对的核磁共振结构预测一致。
