Ca(2+ )binding to complement-type repeat domains 5 and 6 from the low-density lipoprotein receptor-related protein

BACKGROUND: The binding of ligands to clusters of complement-type repeat (CR)-domains in proteins of the low-density lipoprotein receptor (LDLR) family is dependent on Ca(2+ )ions. One reason for this cation requirement was identified from the crystal structure data for a CR-domain from the prototypic LDLR, which showed the burial of a Ca(2+ )ion as a necessity for correct folding and stabilization of this protein module. Additional Ca(2+ )binding data to other CR-domains from both LDLR and the LDLR-related protein (LRP) have suggested the presence of a conserved Ca(2+ )cage within CR-domains from this family of receptors that function in endocytosis and signalling. RESULTS: We have previously described the binding of several ligands to a fragment comprising the fifth and the sixth CR-domain (CR56) from LRP, as well as qualitatively described the binding of Ca(2+ )ions to this CR-domain pair. In the present study we have applied the rate dialysis method to measure the affinity for Ca(2+), and show that CR56 binds 2 Ca(2+ )ions with an average affinity of K(D )= 10.6 microM, and there is no indication of additional Ca(2+ )binding sites within this receptor fragment. CONCLUSIONS: Both CR-domains of CR56 bind a single Ca(2+ )ion with an affinity of 10.6 microM within the range of affinities demonstrated for several other CR-domains.