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The largest group of disease-causing mutations affecting calcium-binding epidermal growth factor-like (cbEGF) domain function in a wide variety of extracellular and transmembrane proteins is that which results in cysteine substitutions. Although known to introduce proteolytic susceptibility, the detailed structural consequences of cysteine substitutions in cbEGF domains are unknown. Here, we studied pathogenic mutations C1977Y and C1977R, which affect cbEGF30 of human fibrillin-1, in a recombinant three cbEGF domain fragment (cbEGF29-31). Limited proteolysis, 1H NMR, and calcium chelation studies have been used to probe the effect of each substitution on cbEGF30 and its flanking domains. Analysis of the wild-type fragment identified two high affinity and one low affinity calcium-binding sites. Each substitution caused the loss of high affinity calcium binding to cbEGF30, consistent with intradomain misfolding, but the calcium binding properties of cbEGF29 and cbEGF31 were surprisingly unaffected. Further analysis of mutant fragments showed that domain packing of cbEGF29-30, but not cbEGF30-31, was disrupted. These data demonstrate that C1977Y and C1977R have localized structural effects, confined to the N-terminal end of the mutant domain, which disrupt domain packing. Cysteine substitutions affecting other cbEGF disulfide bonds are likely to have different effects. This proposed structural heterogeneity may underlie the observed differences in stability and cellular trafficking of proteins containing such changes.

Original publication

DOI

10.1074/jbc.M408156200

Type

Journal article

Journal

J Biol Chem

Publication Date

03/12/2004

Volume

279

Pages

51258 - 51265

Keywords

Amino Acid Sequence, Binding Sites, Calcium, Chelating Agents, Cloning, Molecular, Cysteine, DNA, Disulfides, Dose-Response Relationship, Drug, Egtazic Acid, Epidermal Growth Factor, Fibrillin-1, Fibrillins, Humans, Kinetics, Magnetic Resonance Spectroscopy, Microfilament Proteins, Models, Molecular, Molecular Sequence Data, Mutation, Protein Binding, Protein Conformation, Protein Folding, Protein Structure, Tertiary, Protein Transport, Recombinant Proteins, Spectrometry, Mass, Electrospray Ionization, Time Factors, Trypsin