A Gly --> Ser change causes defective folding in vitro of calcium-binding epidermal growth factor-like domains from factor IX and fibrillin-1.

The Journal of Biological Chemistry
Pat WhitemanPenny A Handford

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 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 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, w...Continue Reading

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