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Title: Molecular and structural characterisation of the human fibrillin-1 N-C terminal interaction
Author: Yadin, David
ISNI:       0000 0004 2746 8949
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2013
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Fibrillins are modular, disulphide-rich glycoproteins that assemble into microfibrils in the extracellular matrix (ECM). These microfibrils are critical structural elements of many non-elastic and elastic connective tissues. They also regulate the availability of transforming growth factor-β signalling molecules in the ECM. Defects in microfibrils are associated with acquired and inherited connective tissue disorders. In particular, mutations in the human FBN1 gene, which encodes fibrillin-1, are associated with a spectrum of diseases, including Marfan syndrome (MFS). One of the proposed initial steps in microfibril assembly is the interaction between the N- and C-terminal regions of fibrillin monomers. The minimal regions of human fibrillin-1 required for an interaction in vitro were previously identified: the four N-terminal domains, from the fibrillin unique N-terminal (FUN) domain to the third epidermal growth factor-like (EGF) domain (FUN-EGF3), and the three C-terminal calcium-binding EGF-like (cbEGF) domains (cbEGF41-43). Here, fragments corresponding to these regions were produced and shown to interact in pull-down and surface plasmon resonance assays. In addition, the structure of the FUN-EGF3 fragment was determined using nuclear magnetic resonance spectroscopy. This showed the novel structure of the FUN domain and the interdomain interfaces in this region of fibrillin. Combining structural and sequence conservation data may help to identify regions of FUN-EGF3 important for binding to cbEGF41-43. Here, the interaction was probed by site-directed mutagenesis. However, substituting individual residues in FUN-EGF3 with alanine did not abrogate binding to cbEGF41-43. Three MFS-associated residue substitutions were also introduced into the FUN-EGF3 fragment. While they did not abolish the interaction with cbEGF41-43, they did cause misfolding. Two of these substitutions, N57D and W71R, also resulted in the defective secretion of a larger N-terminal fragment by fibroblast cells, suggesting a potential mechanism of disease pathogenesis. Although specific residues involved in the N-C interaction were not identified here, the FUN-EGF3 structure will be vital for understanding the molecular surfaces involved in microfibril assembly and growth factor binding.
Supervisor: Handford, Penny; Redfield, Christina Sponsor: Wellcome Trust
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Molecular biophysics (biochemistry) ; Fibrillin ; N-terminus ; structure ; microfibril assembly ; elastic fibre ; extracellular matrix