THE CALCIUM-BINDING PROPERTIES AND MOLECULAR-ORGANIZATION OF EPIDERMAL GROWTH FACTOR-LIKE DOMAINS IN HUMAN FIBRILLIN-1

Human fibrillin-1 is a 350-kDa glycoprotein found in 10-nm connective tissue microfibrils. Mutations in the gene encoding this protein cause the Marfan syndrome, a disease characterized by cardiovascular, ocula r, and skeletal abnormalities. Fibrillin-1 has a modular structure tha t includes 47 epidermal growth factor-like (EGF-like) domains, 43 of w hich contain a consensus sequence associated with calcium binding. A m utation causing an Asn-2144 --> Ser amino acid change in one of the po tential calcium binding residues has been described in a patient with the Marfan syndrome. We have chemically synthesized a wild-type EGF-li ke domain (residues 2126-2165 of human fibrillin-1) and a mutant EGF-l ike domain containing the Asn-2144 --> Ser amino acid change and measu red calcium binding to each using H-1-NMR spectroscopy. The wild-type domain binds calcium with a similar affinity to isolated EGF-like doma ins from coagulation factors IX and X; however, the mutant domain exhi bits >5-fold reduction in affinity. Rotary shadowing of fibrillin-cont aining microfibrils, isolated from dermal fibroblast cultures obtained from the Marfan patient, shows that the mutation does not prevent ass embly of fibrillin into microfibrils but does alter the appearance of the interbead region. We have modeled a region of fibrillin-1 (residue s 2126-2331) encompassing five calcium binding FGF-like domains, using data derived from the recently determined crystal structure of a calc ium binding EGF-like domain from human factor IX. Our model suggests t hat these fibrillin-1 EGF-like domains adopt a helical arrangement sta bilized by calcium and that defective calcium binding to a single EGF- like domain results in distortion of the helix. We propose a mechanism for the interaction of contiguous arrays of calcium binding EGF-like domains within the microfibril.