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J. Biol. Chem., Vol. 279, Issue 49, 51258-51265, December 3, 2004

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Structural Consequences of Cysteine Substitutions C1977Y and C1977R in Calcium-binding Epidermal Growth Factor-like Domain 30 of Human Fibrillin-1^*

Ji Young Suk, Sacha Jensen¶, Aileen McGettrick, Antony C. Willis||, Pat Whiteman, Christina Redfield**, and Penny A. Handford

From the Division of Molecular & Cellular Biochemistry and the ^||Medical Research Council Immunochemistry Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU and the ^**Department of Biochemistry and Oxford Centre for Molecular Sciences, Chemistry Research Laboratory, University of Oxford, Mansfield Road, Oxford OX1 3TA, United Kingdom

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.

Received for publication, July 19, 2004 , and in revised form, August 30, 2004.

^* This work was supported in part by the Medical Research Council, the Wellcome Trust, and the British Heart Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains a supplemental table.

Supported by scholarships from the Republic of Korea Government and the British Overseas Research Students Awards Scheme.

^¶ Supported by the EPA Cephalosporin Trust. Recipient of a Value in People Award from the University of Oxford.

To whom correspondence should be addressed: Dept. of Biochemistry, University of Oxford, OX1 3QU, UK. Tel.: 44-1865-285347; Fax: 44-1865-285327; E-mail: penny{at}bioch.ox.ac.uk.

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