EGF-like domain calcium affinity modulated by N-terminal domain linkage inhuman fibrillin-1

Calcium binding epidermal growth factor-like domains (cbEGFs) are present i n many extracellular proteins, including fibrillin-1, Notch-3, protein S, f actor IX and the low density lipoprotein (LDL) receptor, which perform a di verse range of functions. Genetic mutations that cause amino acid changes w ithin these proteins have been linked to the Marfan syndrome (MFS), CADASIL , protein S deficiency, haemophilia B and familial hypercholesterolaemia, r espectively. A number of these mutations disrupt calcium binding to cbEGFs, emphasising the critical functional role of calcium in these proteins. We have determined the calcium binding affinity of two sites within a cbEGF pair (cbEGF12-13) from human fibrillin-1 using two-dimensional nuclear mag netic resonance (NMR) and fluorescence techniques. Fibrillin-1 is a mosaic protein containing 43 cbEGF domains, mainly arranged as tandem repeats. Our results show that the cbEGF13 site in the cbEGF12-13 pair possesses the hi ghest calcium affinity of any cbEGF investigated from fibrillin-1. A compar ative analysis of these and previously reported calcium binding data from f ibrillin-1 demonstrate that the affinity of cbEGF13 is enhanced more than 7 0-fold by the linkage of an N-terminal cbEGF domain. In contrast, compariso n of calcium binding by cbEGF32 in isolation relative to when linked to a t ransforming growth factor beta-binding protein-like domain (TB6-cbEGF32) re veals that the same enhancement is not observed for this heterologous domai n pair. Taken together, these results indicate that fibrillin-1 cbEGF Ca2affinity can be significantly modulated by the type of domain which is link ed to its N terminus. The cbEGF12-13 pair is located within the longest con tiguous section of cbEGFs in fibrillin-1, and a number of mutations in this region are associated with the most severe neonatal form of MFS. The affin ities of cbEGF domains 13 and 14 in this region are substantially higher th an in the C-terminal region of fibrillin-1. This increased affinity may be important for fibrillin assembly into 10-12 nm connective tissue microfibri ls and/or may contribute to the biomechanical properties of the microfibril lar network. (C) 1999 Academic Press.