METAL-ION DEPENDENCY OF MICROFIBRILS SUPPORTS A ROD-LIKE CONFORMATIONFOR FIBRILLIN-1 CALCIUM-BINDING EPIDERMAL GROWTH FACTOR-LIKE DOMAINS

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 wit hin the microfibril. The NMR structure of a covalently linked pair of cbEGF-like domains from fibrillin-1 recently identified a rigid rod-li ke conformation for the domain pair stabilised by interdomain calcium binding. This suggested that tandem arrays of fibrillin-1 cbEGF-like d omains may adopt an extended conformation within a microfibril. If cor rect, then removal of bound calcium from fibrillin-1 would be expected to increase the flexibility of each cbEGF-like interdomain Linkage, r esulting in a decrease in the length of the interbead region of the mi crofibril (and thus a decrease in bead to bead periodicity), a concomi tant increase in its diameter, and an overall increase in the flexibil ity of the microfibril. Our results show that removal of calcium by tr eatment with EGTA causes a large alteration of the microfibril structu re, resulting in microfibrils with a reduced beaded periodicity, a dis rupted interbead region and an increased overall flexibility. These ef fects are readily reversible by the readdition of calcium (in the form of CaCl2,), but not by the addition of magnesium (MgCl2,). This is co nsistent with conformational changes in cbEGF-like domains causing the major structural effects on the microfibril. These results provide th e first direct experimental evidence to support an extended rod-like c onformation for multiple tandem repeats of fibrillin-1 cbEGF-like doma ins within the microfibril, as predicted by the NMR structure of an is olated fibrillin-1 cbEGF-like domain pair. (C) 1998 Academic Press Lim ited.