title: Molecular effects of calcium binding mutations in Marfan syndrome depend on domain context.
abstract: Mutations in the human fibrillin-1 (FBN-1) gene cause Marfan syndrome (MFS), an autosomal dominant disease of connective tissue. Fibrillin-1, a 350 kDa extracellular calcium binding protein, is a major structural component of 10-12 nm microfibrils and consists predominantly of two repeated module types: the calcium binding epidermal growth factor-like (cbEGF) domain and the transforming growth factor beta1 binding protein-like (TB) domain. A group of reported FBN-1 mutations is predicted to reduce calcium binding to cbEGF domains by removal of a side chain ligand for calcium. These mutations occur in two protein domain contexts, either in a cbEGF preceded by a TB domain or in a cbEGF preceded by another cbEGF domain. In this study we have used three proteases to probe structural changes caused by an N2144S MFS calcium binding mutation in a TB6-cbEGF32 and a cbEGF32-33 domain pair, and an N2183S mutation in the cbEGF32-33 pair. N-terminal sequence analysis of domain pairs digested in the presence and absence of calcium show that: (i) domain interactions between TB6 and cbEGF32 are calcium independent, despite the presence of a calcium binding site in cbEGF32; (ii) domain interactions between cbEGF32 and cbEGF33 are calcium dependent; and (iii) an N-->S mutation causes increased proteolytic susceptibility only when located in cbEGF33, consistent with a key role for interdomain calcium binding in rigidifying cbEGF domain linkages. These data demonstrate for the first time that the structural consequences of calcium binding mutations in fibrillin-1 cbEGF domains can be influenced by domain context.
authors.affiliation: Department of Biochemistry and MRC Immunochemistry Unit, University of Oxford, UK.
authors.forename: A J
authors.initials: AJ
authors.lastname: McGettrick
authors.forename: V
authors.initials: V
authors.lastname: Knott
authors.forename: A
authors.initials: A
authors.lastname: Willis
authors.forename: P A
authors.initials: PA
authors.lastname: Handford
mesh_terms: D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000737:chemistry* / Q000378:metabolism; D000071838:Fibrillin-1; D000071837:Fibrillins; D005341:Fibrinolysin; D006801:Humans; D008382:Marfan Syndrome / Q000235:genetics / Q000378:metabolism*; D008840:Microfilament Proteins / Q000737:chemistry* / Q000378:metabolism; D020125:Mutation, Missense; D010196:Pancreatic Elastase; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D014357:Trypsin; D054392:Adipokines
chemical_list: D002118:Calcium; D002135:Calcium-Binding Proteins; D016326:Extracellular Matrix Proteins; D000071838:Fibrillin-1; D000071837:Fibrillins; D005341:Fibrinolysin; D008840:Microfilament Proteins; D010196:Pancreatic Elastase; D014357:Trypsin; C000605722:FBN1 protein, human; D054392:Adipokines