NDLI: A Gly → Ser Change Causes Defective Folding in Vitro of Calcium-binding Epidermal Growth Factor-like Domains from Factor IX and Fibrillin-1

Content Provider	Journal of Biological Chemistry (JBC)
Author	Whiteman, Pat Downing, A. Kristina Smallridge, Rachel Winship, Peter R. Handford, Penny A.
Abstract	The calcium-binding epidermal growth factor-like (cbEGF) domain is a common motif found in extracellular proteins. A mutation that changes a highly conserved Gly residue to Ser in this domain has been identified both in the factor IX (FIX) and fibrillin-1 genes, where it is associated with relatively mild variants of hemophilia B and Marfan syndrome, respectively. We have investigated the structural consequences in vitro of this amino acid change when introduced into single cbEGF domains from human FIX (G60S) and human fibrillin-1 (G1127S), and a covalently linked pair of cbEGF domains from fibrillin-1. High pressure liquid chromatography analysis, mass spectrometry, and 1H NMR analysis demonstrate that wild-type cbEGF domains purified in the reduced form and refoldedin vitro adopt the native fold. In contrast, the Gly → Ser change causes defective folding of FIX and fibrillin-1 cbEGF domains. However, in the case of the factor IX mutant domain, a Ca2+-dependent change in conformation, identified by NMR in a proportion of the refolded material, suggests that some material refolds to a native-like structure. This is consistent with enzyme-linked immunosorbent assay analysis of FIX G60S from a hemophilia B patient Oxford d2, which demonstrates that the mutant protein is partially recognized by a monoclonal antibody specific for this region of FIX. NMR analysis of a covalently linked pair of fibrillin cbEGF domains demonstrates that the C-terminal domain adopts the native epidermal growth factor fold, despite the fact that the adjacent mutant domain is misfolded. The implications of these results for disease pathogenesis are discussed.
Related Links	http://www.jbc.org/content/273/14/7807.abstract
Ending Page	7813
Starting Page	7807
Page Count	7
File Format	HTM / HTML PDF
ISSN	00219258
Journal	Journal of Biological Chemistry (JBC)
Issue Number	14
Volume Number	273
DOI	10.1074/jbc.273.14.7807
e-ISSN	1083351X
Language	English
Publisher	American Society for Biochemistry and Molecular Biology
Publisher Date	1998-04-03
Access Restriction	Open
Subject Keyword	PROTEIN CHEMISTRY AND STRUCTURE
Content Type	Text
Resource Type	Article
Subject	Cell Biology Biochemistry Molecular Biology

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