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Human fibrillin-1, an extracellular matrix glycoprotein, has a modular organization that includes 43 calcium-binding epidermal growth factor-like (cbEGF) domains arranged as multiple tandem repeats. A missense mutation that changes a highly conserved glycine to serine (G1127S) has been identified in cbEGF13, which results in a variant of Marfan syndrome, a connective tissue disease. Previous experiments on isolated cbEGF13 and a cbEGF13-14 pair indicated that the G1127S mutation caused defective folding of cbEGF13 but not cbEGF14. We have used limited proteolysis methods and two-dimensional NMR spectroscopy to identify the structural consequences of this mutation in a covalently linked cbEGF12-13 pair and a cbEGF12-14 triple domain construct. Protease digestion studies of the cbEGF12-13 G1127S mutant pair indicated that both cbEGF12 and 13 retained similar calcium binding properties and thus tertiary structure to the normal domain pair, because all identified cleavage sites showed calcium-dependent protection from proteolysis. However, small changes in the conformation of cbEGF13 G1127S, revealed by the presence of a new protease-sensitive site and comparative two-dimensional NOESY data, suggested that the fold of the mutant domain was not identical to the wild-type, but was native-like. Additional cleavage sites identified in cbEGF12-14 G1127S indicated further subtle changes within the mutant domain but not the flanking domains. We have concluded the following in this study. (i) Covalent linkage of cbEGF12 preserves the native-like fold of cbEGF13 G1127S and (ii) conformational effects introduced by G1127S are localized to cbEGF13. This study demonstrates that missense mutations in fibrillin-1 cbEGF domains can cause short range structural effects in addition to long range effects previously observed with a E1073K mutation in cbEGF12.

Original publication

DOI

10.1074/jbc.M006547200

Type

Journal article

Journal

J Biol Chem

Publication Date

18/05/2001

Volume

276

Pages

17156 - 17162

Keywords

Amino Acid Sequence, Amino Acid Substitution, Binding Sites, Calcium, Cloning, Molecular, Conserved Sequence, Epidermal Growth Factor, Extracellular Matrix Proteins, Fibrillin-1, Fibrillins, Genetic Variation, Glycine, Humans, Marfan Syndrome, Microfilament Proteins, Models, Molecular, Molecular Sequence Data, Mutation, Missense, Nuclear Magnetic Resonance, Biomolecular, Protein Conformation, Protein Structure, Secondary, Recombinant Proteins, Serine