A polymorphism in thrombospondin-1 associated with familial premature coronary heart disease causes a local change in conformation of the Ca2+-binding repeats

A single nucleotide polymorphism that substitutes a serine for an asparagine at residue 700 in the Ca2+ binding repeats of thrombospondin-1 is associated with familial premature coronary heart disease. We expressed the Ca2+-binding repeats alone (Ca) or with the third epidermal growth factor-like module (E3Ca), without (Asn-700) or with (Ser-700) the disease-associated polymorphism. The intrinsic fluorescence of a single tryptophan (Trp-698) adjacent to the polymorphic residue was quenched cooperatively by adding Ca2+. The third epidermal growth factor-like repeat dramatically altered the Ca2+ -dependent fluorescence transition for the Asn-700 constructs; the half-effective concentration (EC50) of Ca Asn-700 was 390 muM, and the EC50 of E3Ca Asn-700 was 70 muM. The Ser-700 polymorphism shifted the EC50 to higher Ca2+ concentrations (Ca Ser-700 EC50 of 950 pm and E3Ca Ser-700 EC50 of 110 pm). This destabilizing effect is due to local conformational changes, as the Ser-700 polymorphism did not influence the secondary structure of E3Ca or Ca as assessed by far UV circular dichroism. At 200 pm Ca2+, in which both E3Ca Asn-700 and Ser-700 are in the Ca2+-replete conformation at 37 degreesC, the fluorescence of E3Ca Ser-700 reverted to the Ca2+-depleted spectrum at 50degreesC compared with 65degreesC for E3Ca Asn-700. These findings indicate that the Ser-700 polymorphism subtly but significantly sensitizes the calcium-binding repeats to removal of Ca2+ and thermal denaturation.