title: A Gly --> Ser change causes defective folding in vitro of calcium-binding epidermal growth factor-like domains from factor IX and fibrillin-1.
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, 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.
authors.affiliation: Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, United Kingdom.
authors.forename: P
authors.initials: P
authors.lastname: Whiteman
authors.forename: A K
authors.initials: AK
authors.lastname: Downing
authors.forename: R
authors.initials: R
authors.lastname: Smallridge
authors.forename: P R
authors.initials: PR
authors.lastname: Winship
authors.forename: P A
authors.initials: PA
authors.lastname: Handford
mesh_terms: D019943:Amino Acid Substitution; D004815:Epidermal Growth Factor / Q000737:chemistry* / Q000235:genetics; D005164:Factor IX / Q000737:chemistry* / Q000235:genetics; D000071838:Fibrillin-1; D000071837:Fibrillins; D005998:Glycine / Q000737:chemistry / Q000235:genetics; D006801:Humans; D008840:Microfilament Proteins / Q000737:chemistry* / Q000235:genetics; D009154:Mutation; D017510:Protein Folding; D012694:Serine / Q000737:chemistry / Q000235:genetics; D054392:Adipokines
chemical_list: D004815:Epidermal Growth Factor; D005164:Factor IX; D000071838:Fibrillin-1; D000071837:Fibrillins; D005998:Glycine; D008840:Microfilament Proteins; D012694:Serine; C000605722:FBN1 protein, human; D054392:Adipokines