A GLY-]SER CHANGE CAUSES DEFECTIVE FOLDING IN-VITRO OF CALCIUM-BINDING EPIDERMAL GROWTH FACTOR-LIKE DOMAINS FROM FACTOR-IX AND FIBRILLIN-1

The calcium-binding epidermal growth factor-like (cbEGF) domain is a c ommon motif found in extracellular proteins. A mutation that changes a highly conserved Gly residue to Ser in this domain has been identifie d both in the factor IX (FIX) and fibrillin-1 genes, where it is assoc iated with relatively mild variants of hemophilia B and Marfan syndrom e, respectively. We have investigated the structural consequences in v itro of this amino acid change when introduced into single cbEGF domai ns from human FIX (G60S) and human fibrillin-1 (G1127S), and a covalen tly linked pair of cbEGF domains from fibrillin-1. High pressure liqui d chromatography analysis, mass spectrometry, and H-1 NMR analysis dem onstrate that wild-type cbEGF domains purified in the reduced form and refolded in 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 refo lded material, suggests that some material refolds to a native-like st ructure. This is consistent with enzyme-linked immunosorbent assay ana lysis of FIX G60S from a hemophilia B patient Oxford d2, which demonst rates 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-termin al 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.