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

Fibrillin-1 is a major structural component of 10-12 nm connective tissue microfibrils 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 amino acid change for the folding and calcium binding properties of mutant and wild-type TB6-cbEGF32 and cbEGF32-33 domain pairs have been analysed by nuclear 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 and mutant pairs shows that: (i) the affinity of cbEGF32 is weakly enhanced by N-terminal linkage of TB6 relative to cbEGF32 in isolation; (ii) the affinity of cbEGF32 is approximately ninefold decreased by the N2144S substitution in the TB-cbEGF pair; and (iii) reduced affinity of cbEGF32 does not result 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 cbEGF32 on fibrillin-1 produced by N2144S MFS fibroblasts. 35S-pulse-chase analysis shows that the N2144S substitution does not detectably affect fibrillin-1 biosynthesis, rate of secretion or processing. Deposition of reducible fibrillin-1 into the extracellular matrix was also unaffected. The implications of these results for the assembly and properties of the microfibril are discussed.