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Molecular effects of calcium binding mutations in Marfan syndrome depend on domain context
A J McGettrick1, V Knott, A Willis
1Department of Biochemistry and MRC Immunochemistry Unit, University of Oxford, UK.
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.
Insights
Marfan syndrome (MFS) mutations in fibrillin-1 (FBN-1) affect calcium binding. This study shows that the impact of these mutations depends on the specific protein domain context, influencing structural integrity and disease.
Area of Science:
- Biochemistry
- Genetics
- Molecular Biology
Background:
- Marfan syndrome (MFS) is an autosomal dominant connective tissue disorder caused by mutations in the human fibrillin-1 (FBN-1) gene.
- Fibrillin-1 is a crucial structural protein in microfibrils, composed of calcium-binding epidermal growth factor-like (cbEGF) and transforming growth factor beta1-binding protein-like (TB) domains.
- Many FBN-1 mutations are predicted to impair calcium binding to cbEGF domains, potentially disrupting fibrillin-1 structure and function.
Purpose of the Study:
- To investigate how mutations affecting calcium binding in fibrillin-1's cbEGF domains influence protein structure.
- To determine the role of domain context in the structural consequences of FBN-1 calcium-binding mutations.
Main Methods:
- Utilized three proteases to analyze structural changes in specific FBN-1 domain pairs (TB6-cbEGF32 and cbEGF32-33) with introduced N2144S and N2183S mutations.
- Performed N-terminal sequence analysis on digested domain pairs in the presence and absence of calcium.
Main Results:
- Domain interactions between TB6 and cbEGF32 were found to be calcium-independent.
- Interactions between adjacent cbEGF32 and cbEGF33 domains were calcium-dependent.
- N-to-S mutations increased proteolytic susceptibility only in the cbEGF33 domain, indicating calcium's role in rigidifying cbEGF linkages.
Conclusions:
- The structural impact of fibrillin-1 calcium-binding mutations is significantly influenced by their surrounding domain context.
- Calcium binding plays a critical role in stabilizing inter-cbEGF domain interactions within fibrillin-1.
- These findings provide new insights into the molecular mechanisms underlying Marfan syndrome pathogenesis.