Calcium binding properties of an epidermal growth factor-like domain pair from human fibrillin-1

Knott, V.; Downing, A.K.; Cardy, C.M.; Handford, P.

Journal of Molecular Biology 255(1): 22-27


ISSN/ISBN: 0022-2836
PMID: 8568869
DOI: 10.1006/jmbi.1996.0003
Accession: 008255952

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Ca-2+ binding epidermal growth factor-like (EGF-like) domains are found in a large number of extracellular proteins with diverse functions, including those involved in blood coagulation, determination of cell fate, cell adhesion and connective tissue architecture. Their importance is emphasised by the identification of mutations in these domains in patients with haemophilia B (defective in coagulation factor IX) and the Marfan syndrome (defective in the connective tissue protein fibrillin-1). The X-ray crystal structure of a single Ca-2+ binding EGF-like domain from human coagulation factor IX has recently been solved. It shows that the Ca-2+ ligands form a pentagonal bipyramid, where one ligand is provided by an adjacent (N-terminal) EGF-like domain in the crystal. The N and C termini of the neighbouring domains are only apprx 4 ANG apart, hence the crystal packing has been proposed as a model for the association of contiguous EGF-like domains in proteins. Since the adjacent EGF-like domain in the crystal, although close, is not covalently linked to its neighbour, this model requires verification. In this study we have expressed and purified a Ca-2+ binding EGF-like domain pair from human fibrillin-1 and used an in vitro refolding system to obtain protein with the correct EGF fold. The Ca-2+ binding properties of the protein have been investigated by two-dimensional NMR. The affinity of the C-terminal domain for Ca-2+ is apprx 25-fold higher than that of the N-terminal domain, consistent with the two Ca-2+ binding sites having different local environments. In addition, these data provide the first direct experimental evidence that Ca-2+ plays a major role in defining the interdomain linkage in multiple repeats of Ca-2+ binding EGF-like domains.

Calcium binding properties of an epidermal growth factor-like domain pair from human fibrillin-1