CALCIUM-BINDING PROPERTIES OF AN EPIDERMAL GROWTH FACTOR-LIKE DOMAIN PAIR FROM HUMAN FIBRILLIN-1

Ca2+ binding epidermal growth factor-like (EGF-like) domains are found in a large number of extracellular proteins with diverse functions, i ncluding those involved in blood coagulation, determination of cell fa te, 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 t he Marfan syndrome (defective in the connective tissue protein fibrill in-1). The X-ray crystal structure of a single Ca2+ binding EGF-like d omain from human coagulation factor IX has recently been solved. It sh ows that the Ca2+ ligands form a pentagonal bipyramid, where one ligan d is provided by an adjacent (N-terminal) EGF-like domain in the cryst al. The N and C termini of the neighbouring domains are only similar t o 4 Angstrom apart, hence the crystal packing has been proposed as a m odel for the association of contiguous EGF-like domains in proteins. S ince the adjacent EGF-like domain in the crystal, although close, is n ot covalently linked to its neighbour, this model requires verificatio n. In this study we have expressed and purified a Ca2+ binding EGF-lik e domain pair from human fibrillin-1 and used an in vitro refolding sy stem to obtain protein with the correct EGF fold. The Ca2+ binding pro perties of the protein have been investigated by two-dimensional NMR. The affinity of the C-terminal domain for Ca2+ is similar to 25-fold h igher than that of the N-terminal domain, consistent with the two Ca2 binding sites having different local environments. In addition, these data provide the first direct experimental evidence that Ca2+ plays a major role in defining the interdomain linkage in multiple repeats of Ca2+ binding EGF-like domains. (C) 1996 Academic Press Limited