Defective calcium binding to fibrillin-1: Consequence of an N2144S change for fibrillin-1 structure and function

Fibrllin-1 is a major structural component of 10-12 nm connective tissue mi crofibrils and has a modular organisation that includes 43 calcium binding epidermal growth factor-like (cbEGF) domains and seven transforming growth factor beta-binding protein-like (TB) domains. Mutations in the fibrillin-1 (FBN1) gene cause the Marfan syndrome (MFS) and related connective tissue disorders. We have previously investigated an N2144S change, identified in a MFS patient, which removes one of the key calcium binding ligands within cbEGF domain 32. In this study the structural consequences of the N2144S am ino acid change for the folding and calcium binding properties of mutant an d wild-type TB6-cbEGF32 and cbEGF32-33 domain pairs have been analysed by n uclear magnetic resonance. The presence of an N2144S substitution does not alter the native fold of either the TB6 domain, or cbEGF domains 32 and 33. Comparison of calcium dissociation constants measured for the wild-type an d mutant pairs shows that: (i) the affinity of cbEGF32 is weakly enhanced b y N-terminal linkage of TB6 relative to cbEGF32 in isolation; (ii) the affi nity of cbEGF32 is approximately ninefold decreased by the N2144S substitut ion in the TB-cbEGF pair; and (iii) reduced affinity of cbEGF32 does not re sult in lower affinity of cbEGF33 for calcium. Together, these data suggest that the TB6-cbEGF32 Linkage is flexible and the structural effect of the mutation is localised to the interdomain linkage. We have also investigated the effect of defective calcium binding to cbEGF3 2 on fibrillin-1 produced by N2144S MFS fibroblasts. S-35-pulse-chase analy sis shows that the N2144S substitution does not detectably affect fibrillin -1 biosynthesis, rate of secretion or processing. Deposition of reducible f ibrillin-1 into the extracellular matrix was also unaffected. The implicati ons of these results for the assembly and properties of the microfibril are discussed. (C) 1999 Academic Press.