title: Metal ion dependency of microfibrils supports a rod-like conformation for fibrillin-1 calcium-binding epidermal growth factor-like domains.
abstract: 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.
authors.affiliation: Sir William Dunn School of Pathology, University of Oxford, UK.
authors.forename: C M
authors.initials: CM
authors.lastname: Cardy
authors.forename: P A
authors.initials: PA
authors.lastname: Handford
mesh_terms: D008841:Actin Cytoskeleton / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002413:Cations, Divalent / Q000378:metabolism; D002460:Cell Line; D002614:Chelating Agents; D004492:Edetic Acid; D004533:Egtazic Acid; D004815:Epidermal Growth Factor / Q000378:metabolism; D000071838:Fibrillin-1; D000071837:Fibrillins; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008382:Marfan Syndrome / Q000378:metabolism; D008840:Microfilament Proteins / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D008854:Microscopy, Electron; D011487:Protein Conformation; D054392:Adipokines
chemical_list: D002118:Calcium; D002413:Cations, Divalent; D002614:Chelating Agents; D004492:Edetic Acid; D004533:Egtazic Acid; D004815:Epidermal Growth Factor; D000071838:Fibrillin-1; D000071837:Fibrillins; D008840:Microfilament Proteins; C000605722:FBN1 protein, human; D054392:Adipokines