FIBRILLIN DOMAIN FOLDING AND CALCIUM-BINDING - SIGNIFICANCE TO MARFAN-SYNDROME

Background: Marian syndrome is a heritable disorder of connective tiss ue which has been associated with mutations in a gene encoding fibrill in, a 350 kD glycoprotein found in microfibrils. This protein consists of similar to 60 domains, 47 of which have similarity to epidermal gr owth factor (EGF). The first mutations to be detected were found in tw o sporadic cases that had identical Arg to Pro changes within one EGF- like domain. Based on sequence features common to 43 of the EGF-like d omains, it was proposed that these domains might bind calcium. Through the synthesis and characterization of wild-type and mutated single do main peptides, we examined the structural and calcium-binding properti es of an isolated EGF-like domain from fibrillin and the effects of th e Arg to Pro sequence change. Results: A peptide corresponding to the thirteenth putative calcium-binding, EGF-like domain of fibrillin (the site of the first detected mutations) was synthesized. This peptide c ould be easily oxidized and refolded. The structure of this domain was probed using NMR methods, indicating features characteristic of the k nown structures of EGF-like domains. The domain bound to calcium with moderate affinity (K-d = 0.6 +/- 0.1 mM) with no major changes in stru cture induced upon calcium binding. A synthetic peptide containing the Arg to Pro mutation was found to be drastically impaired in its abili ty to fold in vitro. Conclusions: As predicted, a fibrillin domain for ms a calcium-binding, EGF-like module. As the putative calcium-binding sites are found at the amino-terminal end of the modules, we propose that calcium ions may bind at the interfaces between domains, affectin g the overal structure of the protein. The Arg to Pro mutation blocks domain folding in vitro, suggesting that lack of proper domain folding in vivo may contribute to the molecular defects responsible for Marfa n syndrome.